HK1179652B - Production of dha and other lc-pufas in plants - Google Patents

Description

Production of DHA and other LC-PUFAs in plants

Technical Field

The present invention generally relates to recombinant host organisms (e.g., plants) that are genetically modified with a polyunsaturated fatty acid (PUFA) synthase system and one or more accessory proteins that permit and/or enhance production of PUFAs in the host organism. The invention also relates to methods of making and using such organisms (e.g., to obtain PUFAs) and products (e.g., oils and seeds) obtained from such organisms.

Background

Polyunsaturated fatty acids (PUFAs) are considered beneficial for nutritional applications, pharmaceutical applications, industrial applications, and other purposes. However, the existing supply of PUFAs from natural sources (such as fish oils) or from chemical synthesis is not sufficient for long-term commercial demand.

Vegetable oils derived from plants (such as oilseed crops) are less expensive and do not have the contamination issues associated with fish oils. However, PUFAs found in commercially developed plants and vegetable oils typically do not include more saturated or longer chain PUFAs, and only typically include fatty acids such as linoleic (eighteen carbons with 2 double bonds at the Δ 9 and 12 positions-18: 2 Δ 9,12) and linolenic (18:3 Δ 9,12,15) acids.

It has been demonstrated that less saturated or longer chain PUFAs can be produced in plants by modifying the fatty acids endogenously produced by the plant. For example, it has been described that genetic modification of plants with different individual genes encoding fatty acid elongases and/or desaturases results in leaves or seeds containing a large number of longer and less unsaturated PUFAs, such as eicosapentaenoic acid (EPA), but also a large number of mixed short and less unsaturated PUFAs (Qiatal, Nature Biotech.22:739 (2004); WO 04/071467; Abbadanetal, plant 16:1 (2004); NanpierandSayanova, proceedings Softstoutherology society64:387 393 (2005); Robertotal, functional plant biology32:473-479 (2005); U.S. Appl. Pub. No. 2004/0172682).

Brassica (Brassica) includes canola (canola), one of the most important oilseed crops in the world and the most important oilseed crops grown in temperate regions. Canola has traditionally been characterized by rape (Brassicanapus, a variety derived from interspecific crosses of turnip (brassicacanapa) and cabbage (Brassicaoleracea)), where erucic acid and glucosinolates have been removed or significantly reduced by conventional breeding. Most canola oils are produced in the form of vegetable oils for human consumption. There is also a growing market for the use of canola oil for industrial applications.

The quality of the edible and industrial oils derived from a particular species of canola is dependent upon their constituent fatty acids, since the type and amount of fatty acid unsaturation is contemplated for both dietary and industrial applications. Conventional canola oil contains about 60% oleic acid (C18:1), about 20% linoleic acid (C18:2), and about 10% linolenic acid (18: 3). The levels of polyunsaturated linolenic acid normally contained in conventional canola are undesirable because the oil is highly susceptible to oxidation, the rate of oxidation being affected by several factors, including the presence of oxygen, exposure to light and heat, the presence of intrinsic or extrinsic antioxidants and pro-oxidants within the oil. Oxidation by repeated frying (induced oxidation) or long term storage (spontaneous oxidation) leads to off-taste and spoilage. Oxidation also changes the lubricating and viscosity properties of canola oil.

Oils that exhibit reduced levels of polyunsaturated fatty acids and increased levels of monounsaturated oleic acid are associated with higher oxidative stability relative to conventional canola oils. The susceptibility of individual fatty acids to oxidation depends on their degree of unsaturation. Thus, linolenic acid, which possesses three carbon-carbon double bonds, oxidizes at a rate 25 times faster than oleic acid, which has only one double bond, and 2 times faster than linoleic acid, which has two double bonds. Linoleic and linolenic acids also have the greatest impact on taste and odor, since they readily form hydrogen peroxide.

High oleic oil (greater than or equal to 70% oleic acid) is less prone to oxidation during storage, frying and refining, and can be heated to higher temperatures without smoke making it more suitable as a cooking oil. An example of a commercially available canola variety having a fatty acid profile with more than 70% (by weight) oleic acid (C18:1) and less than 3.5% (by weight) linolenic acid (C18:3) in the seed oil is Nexera^TMThe variety, marketed by dow agro sciences llc (Indianapolis, IN), produces "omega-9 oil," a non-hydrogenated high oleic, low linolenic oil, and is currently used IN restaurants and catering for a wide variety of applications, including frying, quick frying, baking, spraying, and for salad dressings.

Summary of The Invention

There is a need in the art for a less expensive method of efficiently and effectively producing quantities, such as commercial quantities, of longer chain or less saturated PUFAs in plants, plant seeds or plant oils and quantities of lipids, such as Triglycerides (TAG) and Phospholipids (PL), enriched in such PUFAs in plants, plant seeds or plant oils. A system for providing and enhancing PUFA production in a host organism, such as a plant, by providing a recombinant host organism genetically engineered with a polyunsaturated fatty acid (PUFA) synthase and one or more accessory proteins as described herein is an important alternative to the practice of the art.

The present invention is directed to a genetically modified plant (such as a brassica), progeny, seed, cell, tissue or part thereof comprising: (i) a nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase system, such as an algal PUFA synthase system, that produces at least one PUFA; and (ii) a nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) that transfers a phosphopantetheinyl cofactor to an ACP domain of a PUFA synthase system, such as an algal PUFA synthase system. In certain embodiments, the genetically modified plant, progeny, seed, cell, tissue, or part thereof is from an economically important brassica species, such as oilseed rape or mustard (Brassicajuncea). In certain embodiments, the PUFA synthase system comprises an amino acid sequence that is at least 60% to 99% identical to the amino acid sequence of seq id No. 1 or comprises the amino acid sequence of seq id No. 1. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system comprises a nucleic acid sequence that is at least 60% to 99% identical to the nucleic acid sequence of seq id No. 6 or comprises the nucleic acid sequence of seq id No. 6. In certain embodiments, the PUFA synthase system comprises an amino acid sequence that is at least 60% to 99% identical to the amino acid sequence of seq id No. 2 or comprises the amino acid sequence of seq id No. 2. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system comprises a nucleic acid sequence that is at least 60% to 99% identical to the nucleic acid sequence of seq id No. 7 or a nucleic acid sequence comprising seq id No. 7. In certain embodiments, the PUFA synthase system comprises an amino acid sequence that is at least 60% to 99% identical to the amino acid sequence of seq id No. 3 or comprises the amino acid sequence of seq id No. 3. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system comprises a nucleic acid sequence that is at least 60% to 99% identical to the nucleic acid sequence of seq id No. 8 or comprises the nucleic acid sequence of seq id No. 8. In certain embodiments, the PUFA synthase system comprises seq id nos: 1. 2, or 3, or any combination thereof. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system comprises seq id nos: 6. 7 or 8 or any combination thereof.

In certain embodiments, the PPTase comprises an amino acid sequence that is at least 60% to 99% identical to SEQ ID NO. 5 or comprises the amino acid sequence of SEQ ID NO. 5. In certain embodiments, the nucleic acid sequence encoding a PPTase is at least 60% to 99% identical to or comprises the nucleic acid sequence of SEQ ID NO. 10.

In certain embodiments, the nucleic acid sequences of (i) and (ii) are contained in a single recombinant expression vector. In certain embodiments, the nucleic acid sequences of (i) and (ii) are operably linked to a seed-specific promoter. In certain embodiments, the nucleic acid sequences of (i) and (ii) are operably linked to a promoter selected from the group consisting of PvDlec2, Pv phaseolin, LfKCS3, and FAE 1.

In certain embodiments, the genetically modified plant (e.g., Brassica species producing canola oil), progeny, seed, cell, tissue or part thereof further comprises (iii) a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) that catalyzes the conversion of long chain PUFA Free Fatty Acids (FFA) to acyl-CoA. In certain embodiments, the ACoAS comprises an amino acid sequence that is at least 60% to 99% identical to seq id No. 4 or comprises the amino acid sequence of seq id No. 4. In certain embodiments, the ACoAS comprises a nucleic acid sequence at least 60% to 99% identical to the nucleic acid sequence of seq id No. 9 or comprises the nucleic acid sequence of seq id No. 9. In certain embodiments, the ACoAS-encoding nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO. 34. In certain embodiments, the nucleic acid sequences of (i), (ii), and/or (iii) are contained in a single recombinant expression vector. In certain embodiments, the nucleic acid sequences of (i), (ii), and/or (iii) are operably linked to a seed-specific promoter. In certain embodiments, the nucleic acid sequences of (i), (ii), and/or (iii) are operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1.

In certain embodiments, the genetically modified plant (e.g., brassica), progeny, cell, tissue or part thereof further comprises a nucleic acid sequence encoding an acetyl CoA carboxylase (ACCase) and/or a nucleic acid sequence encoding a type 2 diacylglycerol acyltransferase (DGAT 2).

The present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 6-10 and SEQ ID NO:34, recombinant expression vector pDAB7361, recombinant expression vector pDAB7362, recombinant expression vector pDAB7363, recombinant expression vector pDAB7365, recombinant expression vector pDAB7368, recombinant expression vector pDAB7369, recombinant expression vector pDAB7370, recombinant expression vector pDAB100518, recombinant expression vector pDAB101476, recombinant expression vector pDAB9166, recombinant expression vector pDAB9167, recombinant expression vector pDAB7379, recombinant expression vector pDAB7380, recombinant expression vector pDAB9323, recombinant expression vector pDAB9330, recombinant expression vector pDAB9337, recombinant expression vector pDAB9338, recombinant expression vector pDAB9344, recombinant expression vector pDAB9396, recombinant expression vector pDAB101412, recombinant expression vector pDAB7733, recombinant expression vector pDAB7734, recombinant expression vector pDAB101493, recombinant expression vector pDAB 109109507, recombinant expression vector pDAB109508, recombinant expression vector pDAB 10891207, recombinant expression vector pDAB108207, recombinant expression vector pDAB 109208, recombinant expression vector pDAB 91207, recombinant expression vector pDAB 109209, recombinant expression vector 207, recombinant expression vector, A recombinant expression vector pDAB9147, a recombinant expression vector pDAB108224, or a recombinant expression vector pDAB 108225.

In certain embodiments, the seed oil line obtained from the genetically modified plant, progeny, seed, cell, tissue, or part thereof comprises a detectable amount of DHA (docosahexaenoic acid (C22:6, n-3)) and/or EPA (eicosapentaenoic acid (C20:5, n-3)). In certain embodiments, the seed oil comprises 0.01% to 15% DHA, 0.05% to 10% DHA, or 0.05% to 5% DHA. In certain embodiments, the seed oil comprises 0.01% to 5% EPA, 0.05% to 5% EPA, or 0.05% to 1% EPA. In other embodiments, detectable amounts of DHA and/or EPA found in seed oil may also be found in food and/or diets derived from the genetically modified plant. In certain embodiments, detectable amounts of DHA and/or EPA are found in brassica species seed oil having a fatty acid content comprising-70% or more oleic acid (C18:1) and/or 4% or less linolenic acid (C18:3) by weight.

The invention relates to an oil or a seed obtained from a genetically modified plant (such as Brassica), progeny, cells, tissues or parts thereof as described herein. The present invention is directed to a food product comprising an oil obtained from a genetically modified plant, progeny, cell, tissue or part thereof as described herein. The present invention also relates to a functional food comprising an oil obtained from or a seed obtained from a genetically modified plant, progeny, cell, tissue or part thereof as described herein. The present invention relates to a pharmaceutical product comprising an oil obtained from a genetically modified plant, progeny, cell, tissue or part thereof as described herein.

The present invention relates to a method for producing an oil comprising at least one LC-PUFA, said method comprising recovering the oil from a genetically modified plant (such as brassica), progeny, cells, tissue or parts thereof as described herein, or from seeds of a genetically modified plant (such as brassica), progeny, cells, tissue or parts thereof as described herein. The invention also relates to a method for producing an oil comprising at least one LC-PUFA, which method comprises growing a genetically modified plant (such as brassica), progeny, cells, tissues or parts thereof as described herein. The invention also relates to a method for producing at least one LC-PUFA in seed oil, which method comprises recovering oil from seeds of genetically modified plants (such as brassica), their progeny, cells, tissues or parts.

The present invention relates to a method for producing at least one PUFA in seed oil, which comprises growing genetically modified plants (such as Brassica), their progeny, cells, tissues or parts. The invention also relates to a method of providing a supplement or therapeutic product containing at least one PUFA to a subject, the method comprising providing a genetically modified plant (such as brassica), progeny, cells, tissues or parts thereof, an oil as described herein, a seed as described herein, a food as described herein, a functional food as described herein, a pharmaceutical product as described herein to the subject. In certain embodiments, the PUFA contained in this embodiment is DHA and/or EPA.

The present invention is directed to a method of making a genetically modified plant (e.g., brassica), descendant, cell, tissue, or part thereof of the present invention comprising transforming a plant or plant cell with (i) a nucleic acid sequence encoding a PUFA synthase system (e.g., algal PUFA synthase system) that produces at least one polyunsaturated fatty acid (PUFA); and (ii) a nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) that transfers a phosphopantetheinyl cofactor to an ACP domain of a PUFA synthase system, such as an algal PUFA synthase system. In certain embodiments, the method further comprises transforming the plant or plant cell with: (iii) a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) that catalyzes the conversion of long chain PUFA Free Fatty Acids (FFA) to acyl-CoA.

Brief Description of Drawings

Various embodiments of the invention will be more fully understood from the following detailed description, drawings, and accompanying sequence descriptions.

FIG. 1 depicts ClustalW (alignment in VectorNTI) of the redesigned DNA sequence encoding each of the 9 repeat domains of PUFAOrfA.

Figure 2 shows a plasmid map of pDAB 7361.

Figure 3 shows a plasmid map of pDAB 7362.

Figure 4 shows a plasmid map of pDAB 7363.

FIG. 5 shows a sequence from canola event 5197[14]]T of-032.002₁The DHA content was analyzed for individual seeds.

FIG. 6 shows the results of SDS-PAGE Western blots of extracts of developing T1 seeds from canola event 5197[14] -032.002 at late (>30DAP) development with OrfA, OrfB and OrfC specific sera.

FIG. 7A shows lipid content of developing T2 seed samples collected 15, 20, 25, 30, 35 and 42 days post pollination from DHA producing canola event 5197[14] -032.002.Sx 002.

FIG. 7B shows by Western blotting that OrfA, OrfB and OrfC polypeptides are present in extracts of DHA-producing canola event 5197[14] -032.002.Sx 002.

FIG. 8 shows LC-PUFA content of homozygous T2 plants of greenhouse-grown T1 plants of canola event 5197[14] -032.002.

Fig. 9 shows a summary of single T2 seed analysis LC-PUFA for six homozygous lines.

FIG. 10 shows signals from two Ts₁DHA content of the resulting parent, which was crossed in the positive and negative (recipcalcoss) with untransformed omega-9 Nexera710, with F1 hybrid seed.

FIG. 11 shows the pat gene copy number of sixty individual T1 plants derived from canola event 5197[13] -010.001.

FIG. 12 shows the expression pattern of the gene of interest in the blank untransformed omega-9 Nexera710 line using the original intensity values at the respective 6 time points expressed as days post pollination (DAP).

FIG. 13 shows the expression pattern of the gene of interest in the blank untransformed ω -9Nexera710 line using normalized intensity values at the respective 6 time points indicated in DAP.

FIG. 14 shows the expression pattern of the gene of interest in the homozygous event 5197[14] -032.002 line using the original intensity values at the respective 6 time points indicated with DAP.

FIG. 15 shows the expression pattern of the gene of interest in the homozygous event 5197[14] -032.002 line using normalized intensity values at the respective 6 time points indicated as DAP.

FIG. 16 shows PUFA synthase activity of mature transgenic canola as measured by Thin Layer Chromatography (TLC).

FIG. 17 shows calculated ratios of reference peptides relative to each other from OrfA expressed in E.coli with and without co-expression HetI, and OrfA expressed in Arabidopsis events 5197[14] -032.002.

FIG. 18 shows the calculated ratio of apo2-9 peptide to each of the six reference peptides from OrfA expressed in E.coli with and without HetI, and OrfA expressed in transgenic canola events 5197[14] -032.002.

Figure 19 shows a plasmid map of pDAB 7365.

Figure 20 shows a plasmid map of pDAB 7368.

Figure 21 shows a plasmid map of pDAB 7369.

FIG. 22 shows a plasmid map of pDAB 7370.

Figure 23 shows a plasmid map of pDAB 100518.

FIG. 24 shows a plasmid map of pDAB 101476.

FIG. 25 shows a plasmid map of pDAB 101477.

FIG. 26 shows a plasmid map of pDAB 9166.

FIG. 27 shows a plasmid map of pDAB 9167.

Figure 28 shows a plasmid map of pDAB 7379.

Figure 29 shows a plasmid map of pDAB 7380.

FIG. 30 shows a plasmid map of pDAB 9323.

Figure 31 shows a plasmid map of pDAB 9330.

Figure 32 shows a plasmid map of pDAB 9337.

Figure 33 shows a plasmid map of pDAB 9338.

FIG. 34 shows a plasmid map of pDAB 9344.

FIG. 35 shows a plasmid map of pDAB 9396.

FIG. 36 shows a plasmid map of pDAB 101412.

FIG. 37 shows a plasmid map of pDAB 7733.

FIG. 38 shows a plasmid map of pDAB 7734.

FIG. 39 shows a plasmid map of pDAB 101493.

FIG. 40 shows a plasmid map of pDAB 109507.

FIG. 41 shows a plasmid map of pDAB 109508.

FIG. 42 shows a plasmid map of pDAB 109509.

FIG. 43 shows a plasmid map of pDAB 9151.

Figure 44 shows a plasmid map of pDAB 108207.

Figure 45 shows a plasmid map of pDAB 108208.

Figure 46 shows a plasmid map of pDAB 108209.

FIG. 47 shows a plasmid map of pDAB 9159.

FIG. 48 shows a plasmid map of pDAB 9147.

Figure 49 shows a plasmid map of pDAB 108224.

FIG. 50 shows a plasmid map of pDAB 108225.

FIG. 51 illustrates T of individual transgenic Arabidopsis events transformed with pDAB101493, pDAB7362, pDAB7369, pDAB101412 or pDAB7380₂DHA and LC-PUFA content of the seeds.

Detailed Description

As used herein, the term "polyunsaturated fatty acid" or "PUFA" refers to fatty acids having a carbon chain of at least 16 carbons, at least 18 carbons, at least 20 carbons, or 22 or more carbons and having at least 3 or more double bonds, 4 or more double bonds, 5 or more double bonds, or 6 or more double bonds, wherein the double bonds are all in the cis configuration.

As used herein, the term "long chain polyunsaturated fatty acids" or "LC-PUFAs" refers to fatty acids consisting of 18 and more carbon chains long, 20 and more carbon chains long, containing 3 or more double bonds, or 22 or more carbons with at least 3 or more double bonds, 4 or more double bonds, 5 or more double bonds, or 6 or more double bonds. The omega-6 series of LC-PUFAs include, but are not limited to, gamma-linolenic acid (C18:3), bis-l-gamma-linolenic acid (C20:3n-6), arachidonic acid (C20:4n-6), adrenic acid (also known as docosatetraenoic acid or DTA) (C22:4n-6), and docosapentaenoic acid (C22:5 n-6). The omega-3 series of LC-PUFAs include, but are not limited to, alpha-linolenic acid (C18:3), eicosatrienoic acid (C20:3n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid (C20:5n-3), docosapentaenoic acid (C22:5n-3), and docosahexaenoic acid (C22:6 n-3). LC-PUFAs also include fatty acids with greater than 22 carbons and 4 or more double bonds, including but not limited to C28:8 (n-3).

The terms "PUFA synthase" or "PUFA synthase system" or "SzPUFA" or "hSzThPUFA" or the like as used in the present application refer to enzyme systems which produce polyunsaturated fatty acids (PUFAs), and in particular long-chain PUFAs (LC-PUFAs), and also to any domain of the enzyme in the complex. The term PUFA synthase includes, but is not limited to, PUFAKS systems or PKS-like systems used to make PUFAs.

The term "phosphopantetheinyl transferase" or "PPTase" or "NoHetI" as used herein refers to an enzyme that activates a PUFA synthase system by transferring a cofactor, such as 4-phosphopantetheine, from coenzyme a (coa) to one or more ACP domains present in the PUFA synthase system.

As used herein, the term "acyl-CoA synthetase" or "ACoAS" or "SzACS-2" refers to an enzyme that catalyzes the conversion of long chain polyunsaturated Free Fatty Acids (FFA) to acyl-CoA.

As used herein, the term "plant" includes, but is not limited to, any progeny, cell, tissue, or part of a plant.

"nutraceutical" means a product isolated, purified, concentrated, or manufactured from plants that provides physiological assistance or provides protection against disease, including processed foods supplemented with such products, as well as foods manufactured from crops that have been genetically engineered to contain enhanced levels of such physiologically active ingredients.

By "functional food" is meant the following food: (a) a regular food product that is similar in appearance or can be consumed as part of a daily diet and (b) has enhanced nutritional value and/or specific dietary help by virtue of modifying the proportions of ingredients normally present in an unmodified food product.

The terms "polynucleotide" and "nucleic acid" are intended to encompass a single nucleic acid as well as a plurality of nucleic acids, nucleic acid molecules or fragments, variants or derivatives thereof, or constructs, such as messenger rna (mrna) or plasmid dna (pdna). The polynucleotide or nucleic acid may comprise the nucleotide sequence of the full-length cDNA sequence, or fragments thereof, including untranslated 5 'and 3' sequences and coding sequences. The polynucleotide or nucleic acid may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide or nucleic acid can be comprised of single/double stranded DNA, DNA mixed with single/double stranded regions, single/double stranded RNA, and RNA mixed with single/double stranded regions, hybrid molecules comprising DNA and RNA that can be single stranded or, more typically, double stranded or mixed with single/double stranded regions. The terms also encompass chemically, enzymatically, or metabolically modified forms of the polynucleotide or nucleic acid.

Polynucleotides or nucleic acid sequences may be referred to as "isolates" in which they are removed from their native environment. For example, a heterologous polynucleotide or nucleic acid encoding a polypeptide or polypeptide fragment having dihydroxy-acid dehydratase activity contained in a vector is considered isolated for the purposes of the present invention. Further examples of isolating a polynucleotide or nucleic acid include recombinant polynucleotides maintained within a heterologous host cell or (partially or substantially) purified polynucleotides or nucleic acids in solution. The isolated polynucleotides or nucleic acids according to the invention in turn comprise synthetically produced molecules of this type. An isolated polynucleotide or nucleic acid in the form of a polymer of DNA may comprise one or more cDNA fragments, genomic DNA, or synthetic DNA.

The term "gene" refers to a nucleic acid or fragment thereof capable of expressing a particular protein, optionally including regulatory sequences preceding (5 'no coding sequence) and following (3' no coding sequence) the coding sequence.

As used herein, the term "coding region" refers to a DNA sequence that encodes a particular amino acid sequence. "suitable control sequences" refer to nucleotide sequences located upstream (5 'no coding sequence), within, or downstream (3' no coding sequence) of a coding sequence that affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, poly a recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures.

As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and fragments thereof and refers to molecules composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a product of a particular length. Thus, any other term for a peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or for any chain or multiple chains consisting of two or more amino acids is included within the definition of "polypeptide", and the term "polypeptide" may be used interchangeably in place of any such term. Polypeptides may be derived from natural biological sources or made by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Can be produced in any manner, including chemical synthesis.

An "isolated" polypeptide or fragment, variant or derivative thereof means a polypeptide that is not in its natural environment. No particular degree of purification is required. For example, an isolated polypeptide may be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention, as are native or recombinant polypeptides isolated, cleaved, or otherwise partially or substantially purified by any suitable technique.

As used herein, "native" refers to the form of a polynucleotide, gene, or polypeptide found in nature-along with its own regulatory sequences, if present.

As used herein, "endogenous" refers to the native form of a polynucleotide, gene, or polypeptide at its natural location within an organism or genome of an organism. An "endogenous polynucleotide" is a polynucleotide that includes the native polynucleotide at its natural location within the genome of an organism. An "endogenous gene" line includes a native gene at its natural location within the genome of an organism. An "endogenous polypeptide" is one that includes a native polypeptide in its native location within an organism.

As used herein, "heterologous" refers to a polynucleotide, gene, or polypeptide that is not normally found in a host organism but is introduced into a host organism. A "heterologous polynucleotide" includes a native coding region, or a portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native polynucleotide. A "heterologous gene" includes a native coding region, or a portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is reintroduced into the native host as part of a chimeric gene that includes non-native regulatory regions. A "heterologous polypeptide" includes a native polypeptide that is reintroduced into the source organism in a form that is different from the corresponding native polypeptide.

As used herein, the term "modified" refers to changes in the polynucleotide disclosed herein that result in a reduction, substantial elimination, or elimination of the activity of a polypeptide encoded by the polynucleotide, and changes in the polypeptide disclosed herein that result in a reduction, substantial elimination, or elimination of the activity of the polypeptide. Such alterations may be made by methods well known in the art, including, but not limited to, deletions, mutations (e.g., spontaneous mutagenesis, random mutagenesis, mutagenesis resulting from addition of a gene by mutation, or transposon mutagenesis), substitutions, insertions, negative controls, altering cell location, altering polynucleotide or polypeptide states (e.g., methylation, phosphorylation, or ubiquitination), removal of cofactors, introduction of antisense RNA/DNA, introduction of interfering RNA/DNA, chemical modifications, covalent modifications, exposure to UV or X-ray, homologous recombination, mitotic recombination, promoter replacement, and/or combinations thereof. Guidance in determining which nucleotide or amino acid residue may be modified may be found in the following: the sequences of a particular polynucleotide or polypeptide and a homologous, e.g., yeast or bacterial, polynucleotide or polypeptide are aligned and the number of modifications made within the highly homologous regions (conserved regions) or common sequences is maximized.

The term "derivative" as used herein refers to a modification of the sequences disclosed herein. Such modifications are, for example, substitutions, insertions, and/or deletions of one or more bases associated with the nucleic acid sequence of the disclosed coding sequence, which retain, slightly alter, or otherwise enhance the function of the disclosed coding sequence in oilseed crop species. Such derivatives can be readily determined by those skilled in the art, for example, using computer simulation techniques to predict and optimize sequence structure. The term "derivative" thus also includes nucleic acid sequences having substantial sequence homology with the coding sequences disclosed herein, thereby enabling the disclosed functionality for making the LC-PUFAs of the present invention.

As used herein, the term "variant" refers to a polypeptide that differs from the specifically recited polypeptides of the invention by the insertion, deletion, mutation, and substitution of amino acids created using, for example, recombinant DNA techniques such as mutagenesis. Guidance in determining which amino acid residues can be substituted, added, or deleted without disrupting the activity of interest can be found in the following: the sequences of a particular polypeptide and a homologous polypeptide are aligned and amino acid sequence changes made within the highly homologous regions (conserved regions) are minimized or replaced with a common sequence.

Alternatively, recombinant polynucleotide variants encoding the same or similar polypeptides may be synthesized or selected using the "redundancy" of the gene code. Various codon substitutions may be introduced-for example to produce silent changes in various restriction sites-to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence may be reflected in the polypeptide or in other peptide domains added to the polypeptide to modify the properties of any portion of the polypeptide.

Amino acid "substitutions" may be the result of replacing an amino acid with another having similar structural and/or chemical properties, i.e., conservative amino acid substitutions, or they may be the result of replacing an amino acid with an amino acid having dissimilar structural and/or chemical properties, i.e., non-conservative amino acid substitutions. "conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues. For example, nonpolar (hydrophobic) amino acid systems include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids including aspartic acid and glutamic acid. Alternatively, "non-conservative" amino acid substitutions may be made by selecting differences in the polar, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of the amino acids. "insertions" or "deletions" may be within the scope of variations which are structurally or functionally tolerated in the recombinant protein. The permissible changes can be determined experimentally by systematically inserting, deleting, or substituting amino acids in the polypeptide molecule using recombinant DNA techniques and examining the activity of the resulting recombinant variants.

The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a coding sequence is located 3' to a promoter sequence. The promoter as a whole may be derived from the native gene, or consist of different units derived from different promoters present in nature, or even comprise synthetic DNA fragments. One skilled in the art will appreciate that different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which allow genes to be expressed in most cell types most of the time are commonly referred to as "constitutive promoters". It is also recognized that DNA fragments of different lengths may have equivalent promoter activity, since in most cases the exact boundaries of the regulatory sequences are not completely defined.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence if the promoter is capable of effecting expression of the coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). Coding sequences may be operably linked to regulatory sequences in either the synonymous or antisense orientation.

The term "expression" as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into polypeptide.

As used herein, the term "overexpression" refers to expression that is greater than the endogenous expression of the same or related gene. If the heterologous gene expression is higher than the expression of the analogous endogenous gene, the heterologous gene is overexpressed.

As used herein, the term "transformation" refers to the delivery of a nucleic acid or fragment into a host organism to produce genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The terms "plasmid" and "vector" as used herein refer to an extra chromosomal unit, often carrying genes that are not part of the central metabolism of the cell and usually in the form of circular double stranded DNA molecules. Such units may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, comprised of single-or double-stranded DNA or RNA, derived from any source in which a plurality of nucleotide sequences have been combined or recombined into a unique configuration capable of introducing into a cell a promoter fragment and DNA sequence for a selected gene product, along with appropriate 3' untranslated sequence.

As used herein, the term "codon degeneracy" refers to the fact that the genetic code allows for variations in the nucleotide sequence without affecting the nature of the amino acid sequence of the encoded polypeptide. The "codon-bias" exhibited by a particular host cell in specifying a given amino acid using a nucleotide codon is well known to those of skill in the art. Thus, when synthesizing a gene for enhanced expression in a host cell, it is desirable to design the gene so that the frequency of codon usage of the gene approaches the preferred frequency of codon usage of the host cell.

The term "codon-optimized," which refers to the coding region of a gene or nucleic acid molecule for transformation of a variety of hosts, refers to the alteration of codons in the coding region of the gene or nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a substantial number of codons with one or more codons that are more commonly used in genes of the organism.

Nucleotide sequences comprising codons encoding amino acids of any polypeptide chain tend to allow for variations in the sequence encoding the gene. Since each codon is composed of three nucleotides, and the DNA containing nucleotides is limited to four specific bases, there are 64 possible nucleotide combinations, of which 61 encode amino acids (the remaining three codons encode signals to end translation). The "gene code" showing which codon encodes which amino acid is reproduced in this case in Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by four triplets, serine and arginine are six, but tryptophan and methionine are encoded by only one triplet. This degeneracy allows for a wide variation in DNA base combinations without altering the amino acid sequence of the DNA-encoded protein.

TABLE 1 Standard Gene codes

Many organisms are biased towards using specific codons to encode specific amino acids for the peptide chain being extended. Codon bias, or codon bias-differences in codon usage between organisms-given by the degeneracy of the genetic code-has been documented in many organisms. Codon bias is often related to the efficiency of translation of messenger RNA (mRNA), which is believed to depend, inter alia, on the identity of the codons being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of selected tRNAs in cells roughly reflects the codons most frequently used for peptide synthesis. Thus, genes can be tailored to optimize gene expression in a given organism based on codon optimization.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequency of codon usage. Codon usage tables are readily available and can be adapted in a number of ways. See Nakamuraet. Nucl. acids sRs.28: 292 (2000). By using this or similar tables, one skilled in the art can apply frequency to any given polypeptide sequence and generate nucleic acid fragments encoding codon-optimized coding regions for that polypeptide using codons most suitable for the given species. The present invention relates to codon-optimized versions of OrfA, OrfB, chimeric OrfC, PPTase, and/or other accessory proteins of the invention, as further described herein.

The term "percent identity," as is known in the art, refers to the relatedness of two or more polypeptide sequences or two or more polynucleotide sequences as determined by the aligned sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, determined by the pairing between such sequence strings. "consistency" and "similarity" can be readily calculated by conventional methods, including but not limited to those disclosed in the following: 1) computationmolecular biology (Lesk, A.M., Ed.) Oxford university, NY (1988); 2) biocomputing, Informatical and genomic projects (Smith, D.W., Ed.) Academic, NY (1993); 3) computer analysis of sequence data, PartI (Griffin, A.M., and Griffin, H.G., Eds.) Humania: NJ (1994); 4) sequence analysis molecular biology (von heinje, G., Ed.) Academic (1987); and 5) sequence analysis primer (Gribskov, M.and Devereux, J., Eds.) Stockton: NY (1991).

The method for determining consistency isDesigned to give the best match to the tested sequences. Methods for determining consistency and similarity are incorporated into public computer programs. Sequence alignment and percent identity calculation can be performed using, for example, VectorAlignX program of the kit (Invitrogen, Carlsbad, Calif.) or MegAlign of the LASERGENE bioinformation calculation kit ^TMThe program (dnastar inc., Madison, WI). Multiple alignments of sequences were performed using the "Clustal alignment" which covers several algorithms, including the "Clustal V alignment" which corresponds to the alignment labeled Clustal V (disclosed by HigginsandSharp, CABIOS.5:151-153(1989); Higgins, D.G.et., Compout.Appl.biosci., 8:189-191 (1992)) and MegAlign of the LASERGENE bioinformation calculation suite^TMThe program (dnastar inc.). For multiple alignments, the default values correspond to GAPPENALTY =10 and GAPLENGTHPENALTY = 10. The preset parameters for pairwise alignment of protein sequences and calculation of percent identity using the Clustal method are KTUPLE =1, GAPPENALTY =3, WINDOW =5 and DIAGONALSSAVED = 5. For nucleic acids, these parameters are KTUPLE =2, GAPPENALTY =5, WINDOW =4 and diagonallssaved = 4. After aligning the sequences using the ClustalV program, it is possible to obtain the "percent identity" by looking at the "sequence distance" table in the same program. Furthermore, the "ClustalW alignment" is available and corresponds to the alignment labeled ClustalW (described by HigginsandSharp, CABIOS.5:151-153(1989); Higgins, D.G.et., Compout.appl.biosci.8: 189-191 (1992)) and MegAlign of the LASERGENE bioinformation calculation kit ^TMThe v6.1 program (DNASTAMIN.). Preset parameters for multiplex alignment (GAPPENALTY =10, GAPLENGTHPENALTY =0.2, delayed digegnseqs (%) =30, DNA transition weight =0.5, protein weighting matrix = Gonnet series, DNA weighting matrix = IUB). After aligning the sequences using the ClustalW program, it is possible to obtain "percent identity" by looking at the "sequence distance" table in the same program.

It is well understood by those skilled in the art that many levels of sequence identity can be used to identify polypeptides from other species, where such polypeptides have the same or similar function or activity. Useful examples of percent identity include, but are not limited to: 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage useful in describing the invention between 60% and 100%, such as 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Suitable nucleic acid fragments not only have the above-mentioned homology, but also typically encode polypeptides having at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, and at least 250 amino acids.

The term "sequence analysis software" refers to any computer algorithm or software program that can be used to analyze a nucleotide or amino acid sequence. "sequence analysis software" is commercially available or independently developed. Representative sequence analyses include, but are not limited to: 1.) GCG suite program (WisconsinPackage version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschultet al, J.mol.biol.,215: 403-; 3.) DNASTAR (DNASTAR, inc. madison, WI); 4.) Sequencher (GeneCodes corporation, AnnArbor, MI); and 5.) the FASTA program incorporated into the Smith-Waterman algorithm (W.R. Pearson, Compout.MethodsGenomeRes., [ Proc.int.Symp. ] (1994), MeetingDate1992, 111-20. Editor(s): suhai, Sandor. plenum: New York, NY). Within the context of the present application, it will be understood that when sequence analysis software is used for analysis, the analysis results will be based on the reference program "default" unless otherwise noted. As used herein, "default values" shall mean any set of values or parameters that would otherwise load software upon initial startup.

Standard recombinant DNA and molecular cloning techniques used in this case are well known in the art and are described, for example, in Sambrookettal, molecular cloning, Arabidopsis Manual, third edition, Cold spring harbor laboratory Press, Cold spring harbor, NY (2000); and Silhavyetal, Experimental switch Gene fusions, Cold spring harbor laboratory Press, Cold spring harbor, NY (1984); and Ausubeletal, Current protocols in molecular biology, published by Greene publishing Assoc. and Wiley-Interscience (1987 to date).

Genetic manipulation of recombinant hosts disclosed herein can be performed using genetic techniques and screening criteria and can be performed in any host cell suitable for genetic manipulation. In certain embodiments, the recombinant host cells disclosed herein can be any organism or microbial host useful for genetic modification and recombinant gene expression. In certain embodiments, the recombinant host can be, but is not limited to, any higher plant-including dicots and monocots-and edible plants, including crop plants and plants using oils thereof. Thus, any plant species or plant cell may be selected, as will be further described below.

The oil of the invention may be obtained from Brassica species canola seed producing DHA and/or EPA in the seed oil, wherein the oil has a fatty acid content comprising-70% or more oleic acid (C18:1) and/or 4% or less linolenic acid (C18:3) by weight. The oils are beneficial for heart health and have enhanced stability for food service and consumer packaging applications. Such oils also reduce hydrogenation requirements and provide nutritional advantages over soybean oil, palm oil and many other oils used in the food industry. The oxidative stability of such oils can be further increased by the addition of antioxidants and processing additives known in the art.

The oils of the present invention may also be used in non-cooking or dietary methods and combinations. Several of these uses may be industrial, cosmetic or medical. The oils of the present invention may also be used in any application for which the oils of the present invention are suitable. In general, the oils of the present invention are useful in the replacement of, for example, mineral oils, esters, fatty acids, or animal fats in a variety of applications, such as lubricants, lubricant additives, metal working fluids, hydraulic fluids, and flame retarded hydraulic fluids. The oils of the present invention are also useful as materials for processes for making modified oils. Examples of techniques for modifying the oils of the present invention include fractionation, hydrogenation, altering the oleic or linolenic acid content of the oil, and other modification techniques known to those skilled in the art.

Examples of cosmetic uses of the oils of the present invention include use as emollients in cosmetic compositions; as a replacement for petrolatum; as a component of soap, or as a material for a method of making soap; as part of an oral therapeutic solution; as part of an aging treatment composition; and as part of a skin or hair aerosol foam formulation.

In addition, the oils of the present invention may be used in medical applications. For example, the oils of the present invention can be used as a protective barrier against infection and oils rich in omega-9 fatty acids can be used to increase graft survival (U.S. Pat. No.6,210,700).

It should be understood that the foregoing is a non-limiting example of the non-culinary application for which the oil of the invention is suitable. As previously mentioned, the oils and modified oils of the present invention may be used to replace mineral oils, esters, fatty acids, or animal fats, for example, for all applications known to those skilled in the art.

PUFA synthase systems

The "standard" or "classical" pathway for the synthesis of long-chain PUFAs (LC-PUFAs) in eukaryotic organisms involves elongation and desaturation of medium-chain saturated or mono-unsaturated fatty acids and has been described. The pathway for the synthesis of long-chain PUFAs via a PUFA synthase system is also described and is very different from this "standard" pathway. In particular, PUFA synthases utilize malonyl-CoA as a carbon source and produce the final PUFA without releasing any significant amount of intermediates. Furthermore, with PUFA synthases, the appropriate cis double bond system is added during synthesis using an aerobic-free mechanism. In some embodiments, NADPH is used as a reducing agent in the synthesis cycle.

The present invention relates to host organisms (such as plants) genetically engineered to express PUFA synthase systems (either endogenously or by genetic manipulation). In certain embodiments, an organism genetically engineered to express a PUFA synthase system, wherein the organism does not naturally (endogenously, without genetic modification) express such system, or at least a particular PUFA synthase or portion thereof carried by the organism is genetically engineered, which may be referred to herein as a "heterologous" host organism, to the extent that the organism is modified with a PUFA synthase or with another protein that is not endogenously expressed by the organism. The genetic modifications of the invention are useful for enhancing PUFA production in a host organism endogenously expressing a PUFA synthase system, wherein the organism is not further modified with a different PUFA synthase or portion thereof.

The PUFA synthase systems according to the invention can comprise several multifunctional proteins (and can include single functional proteins, particularly PUFA synthase systems from marine bacteria) that can work together to perform iterative as well as non-iterative processing of fatty acid chains, including cis-trans isomerization and enoyl reduction reactions in selected cycles. These proteins may also be referred to herein as the core PUFA synthase enzyme complex or core PUFA synthase system. The general functions of the domains and motifs contained within these proteins are individually known in the art and have been described in detail in relation to various PUFA synthase systems from marine bacteria and eukaryotic organisms (see, e.g., U.S. Pat. No. 6,140,486; U.S. Pat. No. 6,566,583; Metzenal, Science293:290- & 293 (2001); U.S. Appl. Pub. No. 2002/0194641; U.S. Appl. Pub. No. 2004/0235127; U.S. Appl. Pub. No.2005/0100995 and WO 2006/135866). Domains may be found as a single protein (e.g. domains are synonymous with proteins) or as one of two or more (multiple) domains in a single protein, as mentioned above. The domain architecture of various PUFA synthetases from members of the marine bacteria and Thraustochytrium species, as well as the structural and functional characteristics of the genes and proteins comprising such PUFA synthetases, have been described (see, e.g., U.S. Pat. No. 6,140,486; U.S. Pat. No. 6,566,583; Metzetal, Science293: 290-.

Numerous examples of polynucleotides, genes and polypeptides having PUFA synthase activity are known in the art and can be used in the genetically modified hosts disclosed herein. PUFA synthase proteins or domains useful in the present invention can include bacterial and non-bacterial PUFA synthases. Non-bacterial PUFA synthases are derived or derived from systems that are not bacterial organisms, such as eukaryotes. Bacterial PUFA synthase systems are described, for example, in u.s.appl.pub.no. 2008/0050505. Genetically modified plants of the invention can be made by combining non-bacterial PUFA synthase domains with bacterial PUFA synthase domains, as well as PUFA synthase domains or proteins from other PKS systems (type I iterations or modules, type II, or type III) or FAS systems.

In certain embodiments, the PUFA synthase systems of the present invention comprise at least the following biologically active domains (a) at least one enoyl-ACP reductase (ER) domain, typically contained on three or more proteins; (b) (ii) multiple Acyl Carrier Protein (ACP) domains (e.g., at least one to four, preferably at least five ACP domains, in some embodiments up to six, seven, eight, nine, ten, or more than ten ACP domains); (c) at least two β -ketoacyl-ACP synthase (KS) domains; (d) AT least one Acyltransferase (AT) domain; (e) at least one β -ketoacyl-ACP reductase (KR) domain; (f) at least two FabA-like β -hydroxyacyl-ACP Dehydratase (DH) domains; (g) at least one Chain Length Factor (CLF) domain; (h) ACP acyltransferase (MAT) domain. In certain embodiments, the PUFA synthase systems according to the invention also include at least one region comprising a Dehydratase (DH) conserved active site motif.

In certain embodiments, the PUFA synthase system comprises at least the following biologically active domains (a) at least one enoyl-ACP reductase (ER) domain; (b) at least five Acyl Carrier Protein (ACP) domains; (c) at least two β -ketoacyl-ACP synthase (KS) domains; (d) AT least one Acyltransferase (AT) domain; (e) at least one β -ketoacyl-ACP reductase (KR) domain; (f) at least two FabA-like β -hydroxyacyl-ACP Dehydratase (DH) domains; (g) at least one Chain Length Factor (CLF) domain; and (h) at least one malonyl-CoA ACP acyltransferase (MAT) domain. In certain embodiments, the PUFA synthase systems according to the present invention also comprise at least one region or domain comprising a Dehydratase (DH) conserved active site motif that is not part of a FabA-like DH domain. The structural and functional features of each of these domains are described in detail in U.S. appl.pub.No. 2002/0194641; U.S. appl.pub.no. 2004/0235127; U.S. appl.pub.no. 2005/0100995; U.S. appl.pub.no.2007/0245431 and WO 2006/135866.

There are three open reading frame regions that form the core Schizochytrium (Schizochytrium) PUFA synthase system and have been previously described in, for example, u.s.appl.pub.no. 2007/0245431. The domain structure of each open reading code region is as follows.

Schizochytrium limacinum open reading area A (OrfA or Pfa 1): OrfA is a 8730 nucleotide sequence (excluding the stop codon) encoding a 2910 amino acid sequence. There are twelve domains within OrfA (a) a β -ketoacyl-ACP synthase (KS) domain; (b) an ACP acyltransferase (MAT) domain; (c) nine Acyl Carrier Protein (ACP) domains; and (d) a Ketoreductase (KR) domain. Genomic DNA clones (plasmids) encoding OrfA from both schizochytrium ATCC20888 and ATCC20888 sub-strains, referred to as schizochytrium N230D strain, have been isolated and sequenced.

Genomic clone pJK1126 (designated pJK1126OrfA genomic clone in the form of an E.coli plasmid vector containing the "OrfA" gene from Schizochytrium ATCC 20888) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA, and assigned ATCC accession number PTA-7648.

Genomic clone pJK306 (designated pJK306OrfA genomic clone, in the form of an E.coli plasmid containing the 5' portion of the OrfA gene from Schizochytrium N230D (overlapping 2.2kB with pJK 320) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA, and assigned ATCC accession number PTA-7641.

Genomic clone pJK320 (designated pJK320OrfA genomic clone, in the form of an E.coli plasmid containing the 3' portion of the OrfA gene from Schizochytrium N230D (2.2 kB overlap with pJK 306)) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA, and assigned ATCC accession number PTA-7644.

Schizochytrium limacinum open reading area B (OrfB or Pfa 2): OrfB is a 6177 nucleotide sequence (excluding the stop codon) that encodes a 2059 amino acid sequence. Within OrfB there are four domains: (a) a-ketoacyl-ACP synthase (KS) domain; (b) a Chain Length Factor (CLF) domain; (c) an Acyltransferase (AT) domain; and (d) an enoyl ACP-reductase (ER) domain. Genomic DNA clones (plasmids) encoding OrfB from both the schizochytrium ATCC20888 and ATCC20888 sub-strains, referred to as the schizochytrium N230D strain, have been isolated and sequenced.

Genomic clone pJK1129 (designated pJK1129OrfB genomic clone, in the form of an E.coli plasmid vector containing the "OrfB" gene from Schizochytrium ATCC 20888) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA, and assigned ATCC accession number PTA-7649.

Genomic clone pJK324 (designated pJK324OrfB genomic clone in the form of an E.coli plasmid containing the OrfB gene sequence from Schizochytrium N230D) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA, and assigned ATCC accession number PTA-7643.

Schizochytrium limacinum open reading area C (OrfC or Pfa 3): OrfC is a 4506 nucleotide sequence (excluding the stop codon) that encodes a 1502 amino acid sequence. Within OrfC are three domains: (a) two FabA-like-hydroxyacyl-ACP Dehydratase (DH) domains; and (b) an enoyl ACP-reductase (ER) domain. Genomic DNA clones (plasmids) encoding OrfC from both the schizochytrium ATCC20888 and ATCC20888 sub-strains, referred to as the schizochytrium N230D strain, have been isolated and sequenced.

Genomic clone pJK1131 (designated pJK1131OrfC genomic clone in the form of an E.coli plasmid vector containing the "OrfC" gene from Schizochytrium ATCC 20888) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA and assigned ATCC accession number PTA-7650.

Genomic clone pBR002 (designated pBR002OrfC genomic clone, in the form of an E.coli plasmid vector containing the OrfC gene sequence from Schizochytrium N230D) was deposited at Jun.8,2006 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA, and assigned ATCC accession number PTA-7642.

In addition, there are three open reading frames forming the core thraustochytrid PUFA synthase and have been previously described. The domain structure of each open reading code region is as follows.

Thraustochytrid 23B open reading region a (orfa): OrfA is a sequence of 8433 nucleotides (excluding the stop codon) which encodes a sequence of 2811 amino acids. The following domains are present in th.23borfa (a) a β -ketoacyl-ACP synthase (KS) domain; (b) an ACP acyltransferase (MAT) domain; (c) eight Acyl Carrier Protein (ACP) domains; and (d) a β -ketoacyl-ACP reductase (KR) domain.

The genomic clone Th23BOrfA _ pBR812.1 (designated Th23BOrfA _ pBR812.1 genomic clone in the form of an E.coli plasmid vector containing the OrfA gene sequence from Thraustochytrium 23B) was deposited at Mar.1,2007 in the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA and assigned ATCC accession number PTA-8232. The genomic clone Th23BOrfA _ pBR811 (designated Th23BOrfA _ pBR811 genomic clone in the form of an E.coli plasmid vector containing the OrfA gene sequence from Thraustochytrium 23B) was deposited at Mar.1,2007 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA and assigned ATCC accession number PTA-8231.

Thraustochytrid 23B open reading region B (orfb): OrfB is a sequence of 5805 nucleotides (excluding the stop codon) that encodes a 1935 amino acid sequence. The following domains are present in Th.23BOrfB (a) a β -ketoacyl-ACP synthase (KS) domain; (b) a Chain Length Factor (CLF) domain; (c) an Acyltransferase (AT) domain; and (d) an enoyl-ACP reductase (ER) domain. The genomic clone Th23BOrfB _ pBR800 (designated Th23BOrfB _ pBR800 genomic clone in the form of an E.coli plasmid vector containing the OrfB gene sequence from Thraustochytrium 23B) was deposited at Mar.1,2007 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA and assigned ATCC accession No. PTA-8227.

Thraustochytrid 23B open reading region c (orfc): OrfC is a sequence of 4410 nucleotides (excluding the stop codon) that encodes a 1470 amino acid sequence. The following domains appear at th.23borfc: (a) two FabA-like β -hydroxyacyl-ACP Dehydratase (DH) domains, each having FabA protein (an enzyme that catalyzes the synthesis of trans-2-decenoyl-ACP and reversibly isomerizes this product to cis-3-decenoyl-ACP) homology; and (b) an enoyl-ACP reductase (ER) domain having high homology to the ER domain of OrfB of Schizochytrium sp. The genomic clone Th23BOrfC _ pBR709A (designated Th23BOrfC _ pBR709A genomic clone in the form of an E.coli plasmid vector containing the OrfC gene sequence from Thraustochytrium 23B) was deposited at Mar.1,2007 at the American Type Culture Collection (ATCC), 10801 Universal boulevard, Manassas, Va.20110-2209USA and assigned ATCC accession number PTA-8228.

Chimeric or hybrid PUFA synthase systems: in certain embodiments, the PUFA synthase system comprises domains selected from any of those described herein, wherein the domains are combined (e.g., mixed and paired) to form a complete PUFA synthase system that meets minimum requirements described herein. In certain embodiments, a genetically engineered organism of the invention may be further modified with at least one domain of another PUFA synthase system or a biologically active fragment thereof. In certain embodiments, any domain of a PUFA synthase system can be modified from its native structure to modify or enhance the function of that domain in the PUFA synthase system (e.g., to modify the types of PUFAs produced by the system or the ratio thereof). Such combinations of domains for making chimeric PUFA synthase systems are described in the patents and publications referenced herein.

In certain embodiments, the PUFA synthase system comprises a schizochytrium PUFA synthase system, wherein the OrfC line from the schizochytrium PUFA synthase system is replaced with OrfC from thraustochytrium 23B. In certain embodiments, such chimeric OrfC lines from Thraustochytrium 23B encode nucleic acid sequences optimized for Schizochytrium codon usage. As a non-limiting example of such a chimeric OrfC, the plasmid pThOrfC-synPS (referred to as pThOrfC-synPS, in the form of an e.coli plasmid vector containing "perfect-needle" synthetic thraustochytrium 23BPUFAPKSOrfC, codon optimized for expression in schizochytrium or other heterologous hosts) was deposited at mar.1,2007 at American Type Culture Collection (ATCC), 10801 veruniversity boulevard, Manassas, va.20110-2209USA, and assigned ATCC accession No. PTA-8229 (see also u.s.appl.pub.no. 2008/0022422).

Other examples of PUFA synthase genes and polypeptides that can be used in the genetically modified organisms of the invention include, but are not limited to, the following codon-optimized sequences generated by the methods further described herein: SEQ ID NO:1(SzPUFAOrfAv3 protein); SEQ ID NO. 2(SzPUFAOrfBv3 protein); SEQ ID NO. 3(hSzThPUFAOrfCv3 protein); SEQ ID NO. 6(SzPUFAOrfA gene); SEQ ID NO. 7(SzPUFAOrfBv3 gene); and SEQ ID NO 8(hSzThPUFAOrfCv3 gene), as well as active variants, portions, fragments, or derivatives of such sequences, wherein the gene encodes-or the polypeptide or protein has-PUFA synthase activity. The invention includes isolated polynucleotides or polypeptides comprising or consisting of one or more of these sequences.

Other examples of PUFA synthase genes and polypeptides that can be used in the genetically modified organisms of the invention include, but are not limited to, PUFA synthase genes or polypeptides having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the PUFA synthase enzymes or sequences described herein. Useful ranges may be chosen between any of these values (e.g., 60% to 99%, 65% to 95%, 70% to 95%, 75% to 95%, 80% to 95%, 85% to 95%, or 90% to 99%). Still other examples of PUFA synthase genes and polypeptides that can be used in the genetically modified organisms of the invention include, but are not limited to, active variants, portions, fragments, or derivatives of any of the PUFA synthase enzymes or sequences described herein, wherein such genes encode-or such polypeptides possess-PUFA synthase activity.

In certain embodiments, the PUFA synthase system can be an algal PUFA synthase. In certain embodiments, the PUFA synthase system can comprise an amino acid sequence that is at least 60% to 99% identical to the amino acid sequence of seq id No. 1. In certain embodiments, the PUFA synthase system can comprise the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system can comprise a nucleic acid sequence that is at least 60% to 99% identical to the nucleic acid sequence of seq id No. 6. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system can comprise the nucleic acid sequence of SEQ ID NO. 6. In certain embodiments, the PUFA synthase system can comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of seq id No. 2. In certain embodiments, the PUFA synthase system can comprise the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system can comprise a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO. 7. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system can comprise the nucleic acid sequence of SEQ ID NO. 7. In certain embodiments, the PUFA synthase system can comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of seq id No. 3. In certain embodiments, the PUFA synthase system comprises the amino acid sequence of SEQ ID NO. 3. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO. 8. In certain embodiments, the nucleic acid sequence encoding the PUFA synthase system comprises the nucleic acid sequence of SEQ ID NO. 8.

In certain embodiments, the PUFA synthase system comprises seq id no: 1. 2, or 3, or any combination thereof. In certain embodiments, the PUFA synthase system comprises seq id no: 6. 7 or 8 or any combination thereof.

In certain embodiments, the sequences of other PUFA synthase genes and/or polypeptides can be identified in the literature and in databases of biological information well known to those skilled in the art using the sequences disclosed herein and available in the art. For example, such sequences can be identified by BLAST searching public databases for known PUFA synthase genes or polypeptide sequences. In this class of methods, consistency can be based on ClustalW alignment using preset parameters for protein weighting matrices of the GAPPENALTY =10, GAPLENGTHPENALTY =0.1, and Gonnet250 series.

In addition, the PUFA synthase genes or polypeptide sequences disclosed herein or known in the art can be used to identify other PUFA synthase homologs in nature. For example, the individual PUFA synthase nucleic acid fragments disclosed herein can be used to isolate genes encoding homologous proteins. The use of sequence-related procedures to isolate homologous genes is well known in the art. Examples of sequence-related procedures include, but are not limited to, (1) nucleic acid hybridization methods; (2) DNA and RNA amplification methods, for example, are used in a variety of applications of nucleic acid amplification techniques [ e.g., Polymerase Chain Reaction (PCR), mulliserial, U.S. Pat. nos. 4,683,202; the Ligase Chain Reaction (LCR), Tabor, S.et al, Proc.Acad.Sci.USA82:1074 (1985); or Strand Displacement Amplification (SDA), walker et al, proc.natl.acad.sci.u.s.a.,89:392 (1992); and (3) constructing a gene library and carrying out complementation screening.

All of these methods can be readily performed by those skilled in the art using known or recognized sequences encoding the proteins of interest. In certain embodiments, DNA sequences located around the coding sequence of the target PUFA synthase are also useful in certain modification procedures and can be readily found in public databases by those skilled in the art. Methods for creating mutations in genes are common and well known in the art and can be applied to the work of creating mutants.

Phosphopantetheinyl transferases

Phosphopantetheinyl transferases (PPTases) are a family of enzymes characterized by fatty acid synthesis, polyketide synthesis, and nonribosomal peptide synthesis. In particular, ACP domains present in PUFA synthase enzymes require activation by attachment of a cofactor from coenzyme a (4-phosphopantetheine) to an Acyl Carrier Protein (ACP). Attachment of this cofactor is performed by PPTases. If endogenous PPTases of the host organism are unable to activate the PUFA synthase ACP domain, it is necessary to provide PPTases capable of performing this function. The sequences of many PPtases are known and the crystal structures have been determined (e.g.Reuteret al, EMBO J.18:6823-31(1999)) and also mutational analysis of amino acid residues important for activity (Mofidet al, Biochemistry43:4128-36 (2004)).

An example of a heterologous PPTase that has previously been validated to identify the orfa accp domain in this case as a substrate is the HetI protein of candida (Nostocsp.) PCC7120 (formerly Anabaenasp PCC 7120). HetI is present in the Candida gene group which is known to be responsible for the synthesis of long-chain hydroxy-fatty acids, which are components of the glycolipid layer present in heterotypic cells of this organism (BlackandWolk, J.Bacteriol.176: 2282-. HetI appears to activate the ACP domain of the HglE protein present in this group. The two ACP domains of HglE have high sequence homology to the ACP domains found in schizochytrium OrfA and other PUFA synthases.

In certain embodiments, a PUFA synthase can be considered to include at least one 4' -phosphopantetheinyl transferase (PPTase) domain, or such domains can be considered as accessory domains or proteins of a PUFA synthase. PPTases structural and functional features have been described in detail, for example, in u.s.appl.pub.no. 2002/0194641; U.S. appl.pub.no. 2004/0235127; and U.S. appl.pub.no. 2005/0100995.

Numerous examples of genes and polypeptides having PPTase activity are known in the art and can be used in the genetically engineered organisms of the present invention, provided that they are capable of activating the ACP domain of the particular PUFA synthase being used. Examples of genes and polypeptides that may be used in genetically modified organisms of the invention may include, but are not limited to, the following codon-optimized sequences as further described herein: SEQ ID NO:5(NoHetiv3 protein) and SEQ ID NO:10(NoHetiv3 gene).

Other examples of PPTase genes and polypeptides that may be used in genetically modified organisms of the invention include, but are not limited to, PPTase genes or polypeptides having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the PPTases or sequences described herein. Useful ranges may be chosen between any of these values (e.g., 60% to 99%, 65% to 95%, 70% to 95%, 75% to 95%, 80% to 95%, 85% to 95%, 90% to 99%). Still other examples of PPTase genes and polypeptides that may be used in the genetically modified organisms of the invention include, but are not limited to, active variants, fragments, portions, or derivatives of any of the PPTase sequences described herein, wherein such genes encode-or such polypeptides have-PPTase activity.

In certain embodiments, the PPTase may be an algal PPTase. In certain embodiments, the PPTase can comprise an amino acid sequence that is at least 60% to 99% identical to the amino acid sequence of SEQ ID NO. 5. In certain embodiments, the PPTase can comprise the amino acid sequence of SEQ ID NO. 5. In certain embodiments, the nucleic acid sequence encoding an algal PPTase can comprise a nucleic acid sequence that is at least 60% to 99% identical to the nucleic acid sequence of SEQ ID NO. 10. In certain embodiments, the nucleic acid sequence encoding an algal PPTase can comprise the nucleic acid sequence of SEQ ID NO. 10.

In certain embodiments of the invention, PPTase is provided for the production and/or accumulation of PPTase in a heterologous host.

In certain embodiments, a gene and/or polypeptide encoding a PPTase may be used to identify another PPTase gene and/or polypeptide sequence and/or may be used to identify a PPTase homolog of another cell. Such PPTase coding sequences may be identified, for example, in the literature and/or in biological information databases well known to those skilled in the art. For example, using bioinformatics to identify PPTase coding sequences in another cell species can be achieved by searching public databases with BLAST (as disclosed above) with conventional PPTase coding DNA and polypeptide sequences, e.g., any provider of the present case. Identity may be based on ClustalW alignment using preset parameters for protein weighting matrices of the GAPPENALTY =10, GAPLENGTHPENALTY =0.1, and Gonnet250 series.

In certain embodiments, the genetically modified plant (e.g., Brassica), progeny, cells, tissues, or parts thereof comprise the nucleic acid sequences of (i) and (ii) in a single recombinant expression vector.

acyl-CoA synthetase

The present invention provides acyl-CoA synthetase (ACoAS) proteins that catalyze the conversion of long chain PUFA Free Fatty Acids (FFA) to acyl-CoA. Endogenous producers of PUFAs, PUFA synthase-Schizochytrium-possess one or more AcoASs that convert the free fatty acid products of their PUFA synthase into acyl-CoA. This is evident in light of the fact that large amounts of PUFAs are accumulated in these parts of the organism. Thus, Schizochytrium, but also other organisms that endogenously contain PUFA synthase (such as other Thraustochytrium), or other organisms that convert PUFAFFAs into acyl-CoAs (such as Thalashiopsis pseudonana or Crypthecodinium cohnii) may represent a source of genes encoding enzymes that allow or increase the accumulation of PUFA synthase products expressed in heterologous hosts. Other ACoAS sequences have been described in u.s.appl.pub.no. 2007/0245431.

Numerous examples of genes and polypeptides having ACoAS activity are known in the art and can be used in the genetically modified organisms of the invention. Examples of genes and polypeptides that may be used in genetically modified organisms of the invention may include, but are not limited to, the following codon-optimized sequences as further described herein: SEQ ID NO. 4(SzACS-2v3 protein) and SEQ ID NO. 9(hSzThACS-2v3 gene).

Other examples of ACoAS genes and polypeptides that can be used in genetically modified organisms of the invention include, but are not limited to, ACoAS genes or polypeptides having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the ACoAS or sequences described herein. Useful ranges may be chosen between any of these values (e.g., 60% to 99%, 65% to 95%, 70% to 95%, 75% to 95%, 80% to 95%, 85% to 95%, 90% to 99%). Still other examples of ACoAS genes and polypeptides that can be used in genetically modified organisms of the invention include, but are not limited to, active variants, fragments, portions, or derivatives of any of the ACoAS sequences described herein, wherein the gene encodes-or the polypeptide has-ACoAS activity.

In certain embodiments, the ACoAS can be an algal ACoAS. In certain embodiments, the ACoAS can comprise an amino acid sequence that is at least 60% to 99% identical to the amino acid sequence of seq id No. 4. In certain embodiments, the ACoAS can comprise the amino acid sequence of SEQ ID NO. 4. In certain embodiments, the nucleic acid sequence encoding algal ACoAS can comprise a nucleic acid sequence that is at least 60% to 99% identical to the nucleic acid sequence of seq id No. 9. In certain embodiments, the nucleic acid sequence encoding algal ACoAS may comprise the nucleic acid sequence of SEQ ID NO. 9. In certain embodiments, the nucleic acid sequence encoding AcoAS comprises the nucleic acid sequence of SEQ ID NO. 34.

In certain embodiments of the invention, ACoAS may be provided for the production and/or accumulation of ACoAS in a heterologous host, as well as the production and/or accumulation of ACoAS in a homologous host.

In certain embodiments, the gene and/or polypeptide encoding ACoAS can be used to identify another ACoAS gene and/or polypeptide sequence and/or can be used to identify an ACoAS homolog of another cell. Such ACoAS coding sequences can be identified, for example, in the literature and/or in biological information databases well known to those skilled in the art. For example, using bioinformatics to identify an ACoAS coding sequence in another cell species can be achieved via BLAST (as disclosed above) with conventional ACoAS coding DNA and polypeptide sequences-e.g., any provider searching public databases. Identity may be based on ClustalW alignment using preset parameters for protein weighting matrices of the GAPPENALTY =10, GAPLENGTHPENALTY =0.1, and Gonnet250 series.

In certain embodiments, the genetically modified plant (e.g., brassica), progeny, cells, tissues, or parts thereof comprise the (i), (ii), or (iii) nucleic acid sequence contained within a single recombinant expression vector, or any combination thereof. In certain embodiments, the (i), (ii), or (iii) nucleic acid sequence, or any combination thereof, is under the control of one or more seed-specific promoters and/or is contained within a single recombinant expression vector.

Method for producing genetically modified organisms

To produce significantly high yields of one or more desired polyunsaturated fatty acids, an organism (e.g., a plant) can be genetically engineered to introduce PUFA synthases into the plant. The invention also relates to methods of improving or enhancing the efficiency of such genetic modifications, in particular, methods of improving or enhancing the production and/or accumulation of a PUFA synthase end product, such as PUFA(s).

Methods for expressing genes in genetically engineered organisms, including but not limited to plants, are well known in the art. In certain embodiments, the coding region of the PUFA synthase gene to be expressed can be codon optimized for the target host cell as described below. Expression of a gene in a recombinant host cell, including but not limited to a plant cell, may require a promoter, and/or a transcription terminator, operably linked to the coding region of interest. Numerous promoters can be used to construct genetic vectors, including but not limited to seed-specific promoters (such as PvDlec2, LfKCS3, and FAE 1). Other non-limiting examples of promoters useful in the present invention include the acyl carrier protein promoter disclosed in WO 1992/18634; the beta-leguminous globulin promoter and truncated versions of kidney bean are disclosed in Slightomeral (Proc. Natl. Acad. Sci. U.S.A.80:1897-1901; 1983); Sengutta-Gopalatant (Proc. Nat. Acad. Sci.82:3320-3324; 1985); vanderGeestet (plantaMol. biol.33:553-557;1997), and Butosetal (EMBOJ.10:1469-1479; 1991).

In certain embodiments of the invention, a recombinant vector is a genetically engineered (e.g., manufactured) nucleic acid molecule that is used as a tool for manipulating a selected nucleic acid sequence and for introducing such nucleic acid sequence into a host cell. The recombinant vector is thus suitable for cloning, sequencing, and/or manipulating the nucleic acid sequence of choice, e.g., by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such vectors typically contain a heterologous nucleic acid sequence that is not adjacent to the nucleic acid sequence to be cloned or delivered, as is naturally found, although the vector may also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) adjacent to the nucleic acid molecule of the invention that are naturally found or that are beneficial for expression of the nucleic acid molecule of the invention. The vector may be RNA or DNA, prokaryotic or eukaryotic, and is typically a plasmid. The vector may be maintained as an extrachromosomal element (e.g., a plasmid) or may be integrated into the chromosome of a recombinant organism (e.g., a microorganism or a plant). The entire vector may be left in situ in the host cell, or under certain conditions, the plasmid DNA may be deleted, leaving the nucleic acid molecule of the invention. The integrating nucleic acid molecule may be under the control of a chromosomal promoter, under the control of a native or plasmid promoter, or under the control of a combination of several promoters. Single or multiple copies of a nucleic acid molecule can be integrated into a chromosome. The recombinant vectors of the invention may contain at least one selectable marker.

In certain embodiments, the recombinant vector for the recombinant nucleic acid molecules of the invention is an expression vector. In such embodiments, the nucleic acid sequence encoding the product to be produced (e.g., a PUFA synthase) is inserted into a recombinant vector to produce a recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operably links the nucleic acid sequence to regulatory sequences within the vector, which enable transcription and translation of the nucleic acid sequence in the recombinant host cell.

Vectors useful for transforming a wide variety of host organisms and cells are common and disclosed in the literature. Typically, the vector contains a selectable marker and sequences that permit autonomous replication or chromosomal integration in the intended host. In addition, suitable vectors may contain a promoter region containing transcriptional initiation controls and a transcriptional termination control region between which a DNA segment of the coding region may be inserted to provide for expression of the coding region both of which may be derived from homologous genes of the transformed host cell, although it will be appreciated that such control regions may also be derived from genes which are not native to the particular species selected as the production host.

The present invention encompasses the expression of one or more acyl-CoA synthetases as described and exemplified herein, together with a PUFA synthase as described herein, together with an exogenous PPTase, alone or in combination with any one or more of the strategies described herein, such as any one, two, three, or four of codon optimization, cell targeting, enhancing PUFA synthase competition for malonyl CoA (e.g., inhibition of FAS), and/or expression of one or more acyltransferases or related enzymes, to increase PUFA production and/or accumulation in a heterologous host.

Certain embodiments of the invention relate to targeted expression of synthetase enzymes, PPTase, and/or any one or more accessory proteins and/or targeted genetic modifications to one or more organelles of a host. For example, in certain embodiments, expression of the PUFA synthase system and PPTase can be targeted to the plastid of a plant. In certain embodiments, expression of the PUFA synthase and PPTase is targeted to the cytoplasm. In certain embodiments, expression of PUFA synthases and pptases is targeted to both the plastid and the cytoplasm of the plant. In any of these embodiments, the other target may refer to an involved plastid or cytoplasm.

In certain embodiments, the acyl-CoA synthetase is expressed cytoplasmic to convert DHA and/or other LC-PUFA free fatty acids to acyl-CoAs, which in turn can be utilized by acyltransferases.

An example of a plastid targeting sequence is derived from a brassinoyl-ACP thioesterase and is described in u.s.appl.pub.no. 2007/0245431. Various other plastid targeting sequences are known in the art and are useful in heterologous hosts that are plants or plant cells and wherein targeting plastids is a desired embodiment.

The present invention includes the use of organelle targeting (such as plastids or chloroplasts of plants) in conjunction with expression of the PUFA synthetases described herein and exogenous PPTases, alone or in combination with any one or more of the strategies described herein (such as any one, two, three, or four of codon optimization, enhancement of PUFA synthetase competition for malonyl CoA (such as inhibition of FAS), expression of one or more acyl-CoA synthetases, and/or expression of one or more acyltransferases or related enzymes) to increase PUFA production and/or accumulation in a heterologous host.

Targeting of gene products to plastids or chloroplasts is controlled by the information sequences found at the codon termini of the various proteins and excised during transport to produce the mature protein (see, e.g., comefinal, j. biol. chem.263:15104-15109 (1988)). The message sequences can be fused to a heterologous gene product to allow the heterologous product to be transported into the chloroplast (vandenBroecketal. Nature313:358-363 (1985)). DNA encoding the appropriate information sequences can be isolated from cDNAs encoding the RUBISCO proteins, CAB proteins, EPSP synthase enzymes, GS2 proteins and many other proteins known to localize to chloroplasts.

In certain embodiments of the invention, localization of a protein employed in the invention is directed to the subcellular compartment, for example, to the plastid or chloroplast. The protein can be directed to the chloroplast by including a Chloroplast Transit Peptide (CTP) at its amino terminus. Similarly, proteins can be directed to plastids by including a plastid transit or pheromone peptide at their N-terminus.

Naturally occurring chloroplast-targeting proteins-synthesized as larger precursor proteins, containing an amino-terminal chloroplast-targeting peptide that directs the precursor to the chloroplast transit machinery-are well known in the art. Chloroplast targeting peptides are typically cleaved by specific endoproteases located in the chloroplast organelle, thus releasing the targeted mature and preferably active enzyme from the precursor into the chloroplast environment. Examples of sequences encoding peptides suitable for directing a gene or gene product to the chloroplast or plastid of a plant cell include petunia epspssctp, arabidopsis epssctp 2, and introns, among others known to those skilled in the art. Such targeting sequences are provided for delivery of the protein to be expressed to the most effective cellular structure or by delivery of the protein to be expressed to the cellular region of the cellular process necessary for aggregation of the desired phenotypic function. Specific examples of chloroplast targeting peptides are well known in the art and include the arabidopsis thaliana rubisco small subunit ats1A transit peptide, the arabidopsis thaliana EPSPS transit peptide, and the maize (zeaize) rubisco small subunit transit peptide.

Optimized transit peptides are described, for example, in vandenBroecketal, Nature 313:358-363 (1985). Prokaryotic and eukaryotic information sequences are disclosed, for example, in Michaelissetal, Ann. Rev. Microbiol.36:425 (1982). Additional examples of transit peptides that can be used in the present invention include chloroplast transit peptides, such as those described in VonHeijneetal, plant mol, biol, Rep.9:104-126 (1991); mazuret et al, plantaphysiol.85: 1110 (1987); vorstetal, Gene65:59 (1988). Chen & Jagendorf (J.biol.chem.268:2363-2367(1993)) has described the use of chloroplast transit peptides to deliver heterologous transgenes. The peptide used was a transit peptide from the rbcS gene of Nicotiana rugosa (Poulseneet al. mol. Gen. Genet.205: 193-200 (1986)). One CTP that functions in this case to localize heterologous proteins to the chloroplast is derived from the brassinoyl-ACP thioesterase.

Alternative ways of localizing genes to the chloroplast or plastid include chloroplast or plastid transformation. Recombinant plants can be made in which only the chloroplast DNA has been altered to incorporate the molecules envisioned by the present application. Promoters which act in chloroplasts are known in the art (Hanley-Bowdeneet, trends Biochemical sciences12:67-70 (1987)). Methods and combinations for obtaining cells containing chloroplasts with inserted heterologous DNA are described, for example, in danielet al (U.S. patent No. 5,693,507) and maligaet al (U.S. patent No. 5,451,513).

Policy combination

In accordance with the present invention, any one or more (any combination of) of the strategies described herein for improving the production and/or accumulation of PUFAs in a host may be used in the production of a heterologous host for producing and accumulating one or more target PUFAs. Indeed, it is contemplated that various combinations of strategies will add or enhance and provide improved production and/or accumulation of PUFAs, as compared to the absence of one or more of such strategies. Indeed, the examples provide exemplary strategies for producing PUFAs in a host organism.

Any plant or plant cell line using these combinations of modifications, or any other modification or combination of modifications, described herein is encompassed by the present invention. In certain embodiments, such plants are further genetically engineered to express the helper proteins described herein to improve the production and/or accumulation of PUFAs (or other biologically active products of PUFA synthases) by a host, such as AcoAS, GPAT, LPAAT, DAGAT, or acetyl CoA carboxylase (ACCase). Furthermore, any host cell or biological system using any modification or combination of modifications described herein is encompassed by the present invention, and any product derived from such cells or organisms-including seeds or oils comprising the target PUFAs-are also encompassed by the present invention.

In certain embodiments, plants (e.g., plant host cells) genetically engineered according to the present invention include, but are not limited to, any higher plant, including dicotyledonous and monocotyledonous plants, and particularly edible plants, including crop plants and particularly plants using oils thereof. Such plants may include, but are not limited to, for example: canola, soybean, rapeseed, linseed, corn, safflower, sunflower and tobacco. Thus, any plant species or plant cell may be selected. In particular embodiments, plant cells and plants grown or derived therefrom for use herein include, but are not limited to, cells obtained from: canola (Brassicanapus); rape (Brassicanapus); brassica napobrassica (Brassicajuncea); chlamydomonas (Brassicacarinata); turnip (Brassicapa); cabbage (Brassicaoleracea); soybean (glycine max); flaxseed/flax (linumusittissimum); maize (corn) (zeays); safflower (carthamustocincotius); sunflower (helianthus shannunus); tobacco (Nicotianatabacum); arabidopsis, brazilian walnut (betholettiaxelsa); castor bean (ricinuscolimus); coconut (cocussnucifera); coriander (coriandems); cotton (gossypumspp.); peanuts (arachis pygaea); jojoba (Simmondsiachinensis); oil palm (Elaeisguineeis); olive (oleaeurpa); rice (Oryzasativa); pumpkin (Cucurbitamaxima); barley (hordeum vulgare); wheat (Triticumaestivum); and duckweed (lemnaceae sp.). In some embodiments, the genetic background of the plant species may vary.

As used herein, "plant part" includes any part of a plant, including, but not limited to, seeds (including both mature and immature seeds), pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, and the like. In certain embodiments, genetically modified plants have genomes modified (e.g., mutated or altered) from their normal (e.g., wild-type or naturally occurring) form such that a desired result (e.g., increased or modified PUFA synthase and/or production and/or accumulation of a desired product using PUFA synthase) is achieved. In certain embodiments, plant genetic modification can be accomplished using classical breeding and/or molecular genetic techniques. Methods of making transgenic plants in which a recombinant nucleic acid molecule encoding a desired amino acid sequence is incorporated into the genome of the plant are well known in the art. In certain embodiments, plants genetically modified according to the invention are plants suitable for consumption by animals, including humans.

Plant lines from these plants-optimized for specific desired characteristics, such as disease resistance, ease of plant transformation, oil content or disposition, and the like-can be made, selected, or identified. In certain embodiments, the plant lines may be selected by plant breeding, or by methods such as marker assisted breeding and farming. In certain embodiments, plant cell cultures may be used, for example, instead of growing into differentiated plants and growing using common agricultural practices, growth is maintained in liquid media.

In certain embodiments, the plant may be an oilseed plant, wherein the oilseed, and/or the oil within the oilseed, comprises PUFAs produced by a PUFA synthase. In certain embodiments, such oils can contain detectable amounts of at least one target or primary PUFA, which is a product of a PUFA synthase. In certain embodiments, such oils may be substantially free of non-target or major PUFA products and are not intermediates or by-products that are naturally produced by the FAS system endogenous to the wild-type plant (e.g., wild-type plants produce certain shorter or medium chain PUFAs, e.g., 18 carbon PUFAs, via the FAS system, but will have new or additional fatty acids produced by the plant as a result of genetic modification with the PUFA synthase system).

In terms of making genetically modified plants, plant genetic engineering methods are well known in the art. For example, a number of plant transformation protocols have been developed, including procedures for the biological and physical transformation of dicotyledonous and also monocotyledonous plants (such as Goto-Fumiyuki et al, Nature Biotech17:282-286 (1999); Mikietal, MethodsinPlatymolecular biology and Biotechnology, Glick, B.R. andMethampson, J.E.Eds., CRCPres, Inc., BocaRaton, pp.67-88 (1993). furthermore, vectors for plant cell or tissue transformation and in vitro culture methods and plant regeneration can be obtained, for example, in Gruberal, MethodsinPlantMoltmolecular biology and Biotechnology, Glick, B.R.dTimpson, J.E.Eds, CRCPres, Raton, Inc. 89-119 (1993)).

The present invention relates to compositions comprising a polypeptide selected from the group consisting of seq id nos: 6-10 and isolated nucleic acid molecules comprising modifications or mutations of such sequences as described herein. The present invention relates to compositions comprising a polypeptide selected from the group consisting of seq id nos: 1-5 and isolated polypeptides comprising modifications or mutations of such sequences as described herein.

The present invention includes recombinant expression vector pDAB 7361. The present invention includes recombinant expression vector pDAB 7362. The present invention includes recombinant expression vector pDAB 7363. The present invention includes recombinant expression vector pDAB 7365. The present invention includes recombinant expression vector pDAB 7368. The present invention includes recombinant expression vector pDAB 7369. The present invention includes recombinant expression vector pDAB 7370. The present invention includes recombinant expression vector pDAB 100518. The present invention includes recombinant expression vector pDAB 101476. The present invention includes recombinant expression vector pDAB 9166. The present invention includes recombinant expression vector pDAB 9167. The present invention includes recombinant expression vector pDAB 7379. The present invention includes recombinant expression vector pDAB 7380. The present invention includes recombinant expression vector pDAB 9323. The invention includes recombinant expression vector pDAB 9330. The invention includes recombinant expression vector pDAB 9337. The invention includes recombinant expression vector pDAB 9338. The present invention includes recombinant expression vector pDAB 9344. The present invention includes recombinant expression vector pDAB 9396. The present invention includes recombinant expression vector pDAB 101412. The present invention includes recombinant expression vector pDAB 7733. The present invention includes recombinant expression vector pDAB 7734. The present invention includes recombinant expression vector pDAB 101493. The present invention includes recombinant expression vector pDAB 109507. The present invention includes recombinant expression vector pDAB 109508. The present invention includes recombinant expression vector pDAB 109509. The present invention includes recombinant expression vector pDAB 9151. The present invention includes recombinant expression vector pDAB 108207. The present invention includes recombinant expression vector pDAB 108208. The present invention includes a recombinant expression vector pDAB 108209. The present invention includes recombinant expression vector pDAB 9159. The present invention includes recombinant expression vector pDAB 9147. The present invention includes recombinant expression vector pDAB 108224. The present invention includes recombinant expression vector pDAB 108225.

As used herein, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (e.g., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" is used interchangeably with the term "transfection", and when the term is used to refer to the introduction of a nucleic acid molecule into a microbial cell, such as algae, bacteria and yeast, or into a plant cell. In microbial and plant systems, the term "transformation" is used to describe genetic alterations resulting from the acquisition of exogenous nucleic acids by a microorganism or plant and is essentially synonymous with the term "transfection". In certain embodiments, transfection techniques include, but are not limited to, transformation, particle bombardment, diffusion, active transport, channel sonication, electroporation, microinjection, lipofection, adsorption, infection, and protoplast fusion.

Widely used methods for introducing expression vectors into plants are based on the natural transformation system of Agrobacterium. Horschel, Science227:1229 (1985). Agrobacterium tumefaciens and Agrobacterium rhizogenes are known plant pathogenic soil bacteria that can be used for the genetic transformation of plant cells. The Ti and Ri plasmids of Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively, carry genes responsible for plant gene transformation. Kado, C.I., Crit.Rev.plant.Sci.10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene delivery are also available, for example, Gruberet, supra, Mikieet, supra, Moloney, plantaCellReports 8:238(1989) and U.S. Pat. Nos. 4,940,838 and 5,464,763.

Another well-known method of plant transformation is microprojectile-mediated transformation, in which DNA is carried on the surface of microprojectiles. In this method, expression vectors are introduced into plant tissue by a gene gun device that accelerates microprojectiles to a velocity sufficient to penetrate the plant cell wall and membrane. Sanfordet al, part.Sci.Techol.5: 27(1987), Sanford, J.C., Trends Biotech.6:299(1988), Sanford, J.C., Physiol.plant79:206(1990), Kleinet al, Biotechnology10:268 (1992).

Yet another method for physical transport of DNA to plants is ultrasonication of target cells. Zhangegel, Bio/Technology9:996 (1991). Also, liposome or spheroplast fusion has been used to introduce expression vectors into plants. Deshayetotal, EMBOJ.,4:2731(1985), Christouetal, ProcNatl.Acad.Sci.USA84:3962(1987). Using CaCl₂Precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported to allow protoplasts to directly take up DNA. Hainettal, mol.Gen.Genet.199:161(1985) and Draperetal, plantaCellPhysiol.23: 451 (1982). Electroporation of protoplasts and whole cells and tissues is also described. Donnetal, Absactsof VIIth International Congresson PlantlandSuzureaPCTC, A2-38, p.53 (1990); d' Halluinetal, plant cell4:1495-1505(1992) and Spenceretal, plant mol. biol.24:51-61 (1994). In addition, silicon carbide whiskers (Kaeplerenal, 1990, plantaCellReports) can also be used with, for example, plant transformation using the floral dip method (CloughandBi, planta J.16:735-743 (1998)). The actual plant transformation method can be seen as transforming a selected plant species and a slight variation of the selected plant cell type, such as the type of cell derived from seedling, e.g. hypocotyls and cotyledons or embryonic tissue.

After introduction of the gene construct into a plant cell, the plant cell can be grown and a mature plant can be produced after the appearance of differentiated tissues such as shoots and roots. In certain embodiments, a plurality of plants may be produced. Methodologies for plant regeneration are well known to those skilled in the art and can be found, for example, in plantacelllandtestueculture, 1994, vasilandtorpeeeds, kluweracademy publishers and in plantalcellcultureprotocols (methods molecular biology111,1999 halledstemmanaapress).

In certain embodiments, the genetically modified plants described herein can be cultured in a fermentation medium or grown in a suitable medium, such as soil. In certain embodiments, suitable growth medium for higher plants may include any plant growth medium, including but not limited to soil, sand, any other granular medium that supports root growth (such as vermiculite, perlite, etc.) or hydroponics, as well as suitable light, water and nutritional supplements to optimize higher plant growth.

One skilled in the art will appreciate that the control of expression of transfected nucleic acid molecules can be improved using recombinant DNA techniques by manipulating, for example, the number of copies of the nucleic acid molecules in the host cell, the efficiency of transcription of the nucleic acid molecules, the efficiency of translation of the resulting transcripts, and the efficiency of post-translational modifications. In addition, the promoter sequence may be genetically engineered to improve expression levels compared to the native promoter. Recombinant techniques that can be used to control the expression of a nucleic acid molecule include, but are not limited to, integration of the nucleic acid molecule into one or more host cell chromosomes, addition of vector stabilizing sequences to plasmids, substitution or modification of transcriptional control signals (e.g., promoters, operators, enhancers), substitution or modification of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of the nucleic acid molecule to correspond to the codon usage of the host cell, and deletion of sequences that destabilize transcripts.

In certain embodiments, plants may include those plants conventionally produced or genetically engineered to produce compounds useful as pharmaceuticals, flavors, nutraceuticals, functional food ingredients, or cosmetically active agents.

These embodiments of the invention are all applicable to the discussion of any genetically engineered organism described herein and methods of making and using such organisms.

Products of genetically modified organisms

In certain embodiments, the genetically engineered biological systems of the invention produce one or more polyunsaturated fatty acids, including, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), ALA (C18:3, n-3), and/or SDA (C18:4, n-3)), while in certain embodiments, one or more longer chain PUFAs include, but are not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), or DTA (C22:4, n-6), or any combination thereof. In certain embodiments, the genetically modified plants of the invention produce one or more polyunsaturated fatty acids, including, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), and/or DPA (C22:5, n-6 or n-3), or any combination thereof.

In certain embodiments, the genetically engineered biological system is genetically engineered to recombinantly express a PUFA synthase system and a PPTase in a plant, as described herein. In certain embodiments, such plants are further genetically engineered to express the helper proteins described herein to improve the production and/or accumulation of PUFAs (or other biologically active products of PUFA synthases) by a host (e.g., AcoAS, GPAT, LPAAT, DAGAT, or ACCase).

Certain embodiments of the invention include the production of polyunsaturated fatty acids of desired chain length and with the desired number of double bonds and-broadly-oilseeds comprising such PUFAs and oils obtained from genetically modified plants as described herein (e.g., oils or seeds obtained from such plants). Examples of PUFAs that can be produced by the present invention include, but are not limited to, DHA (docosahexaenoic acid (C22:6, n-3)), ARA (arachidonic acid or arachidonic acid (C20:4, n-6)), DPA (docosapentaenoic acid (C22:5, n-6 or n-3)), and EPA (eicosapentaenoic acid (C20:5, n-3)), and any combination thereof. By genetically modifying plants developed by the present inventors through the use of PUFA synthase systems for the production of PUFAs, the present invention allows the production of commercially valuable lipids enriched in one or more desired (target or primary) PUFAs.

In certain embodiments, a given PUFA synthase system derived from a particular organism produces a particular PUFA(s), such that selection of a PUFA synthase system from a particular organism results in production of a specified target or primary PUFAs. In certain embodiments, the ratio of PUFAs may vary depending on the choice of a particular PUFA synthase system and how the system responds to particular conditions of expression. For example, the use of PUFA synthase enzymes from thraustochytrium 23B (atccno.20892) may also result in the production of DHA and DPAn-6 as target or primary PUFAs; however, in the case of thraustochytrium 23B, the ratio of DHA to DPAn-6 is about 10:1 (and may range from about 8:1 to about 40:1), but in schizochytrium, the ratio is typically about 2.5: 1. In certain embodiments, a given PUFA synthase can be modified by mixing proteins and domains from different PUFA synthases, or one can modify a domain or protein of a given PUFA synthase to alter the target PUFA product and/or ratio.

In certain embodiments, reference to an "intermediate" or "byproduct" of an enzyme system for the production of PUFAs refers to any product, particularly a fatty acid product, produced by the enzyme system as a result of the target or primary PUFA(s) of the production system, but which are not the main or target PUFA(s). In certain embodiments, intermediates or by-products can include non-target fatty acids that are naturally produced by wild-type plants, or that the parent plants are used as recipients for a given genetic modification, but are now classified as intermediates or by-products because they are produced as a result of the genetic modification-at greater levels than the levels at which the wild-type plants naturally produce, or that the parent plants are used as recipients for a given genetic modification. In certain embodiments, the primary or target PUFA of one enzyme system may be an intermediate of a different enzyme system, wherein the primary or target product is a different PUFA. For example, fatty acids such as GLA, DGLA and SDA are produced in large quantities as intermediates when EPA is manufactured using standard routes (e.g. u.s.appl.pub.no. 2004/0172682). Similarly, also exemplified by u.s.appl.pub.no.2004/0172682, in the case of DHA production using the standard route, in addition to the fatty acids described above, ETA and EPA (note that the target PUFA of the first example above) are produced in large quantities and can occur in larger quantities relative to the total fatty acid product than the target PUFA itself.

In certain embodiments, plants can be genetically engineered to introduce PUFA synthase systems into the plant in order to produce significantly high yields of one or more desired polyunsaturated fatty acids. Plants do not conventionally contain PUFA synthase enzymes endogenously, and therefore, the present invention represents an opportunity to produce plants with unique fatty acid production capabilities. The present invention provides genetically engineered plants to produce one or more PUFAs in the same plant, including but not limited to EPA, DHA, DPA (n3 or n6), ARA, GLA, SDA, and the like, including any combination thereof. The present invention offers the ability to create any of a wide variety of "tailored oils" in various proportions and forms. In certain embodiments, the use of a PUFA synthase system from a particular marine organism as described herein extends the range of PUFA production and successfully produces such PUFAs over a temperature range useful for growing a majority of crop plants.

In certain embodiments, a system for synthesizing PUFAs being "substantially free" of intermediates or byproducts, or having no intermediates or byproducts present in substantial parts, means that genetically modified plants (and/or plant parts and/or seed oil fractions) can produce less than about 10% by weight of total fatty acids produced by the plant, more preferably less than about 9%, more preferably less than about 8%, more preferably less than about 7%, more preferably less than about 6%, of any intermediate or byproduct fatty acids (non-target PUFAs) as a result of the introduction or presence of an enzyme system that produces PUFAs, such as is not produced by a wild-type plant or used by a parent plant as a recipient for a given genetic modification, more preferably less than about 5%, more preferably less than about 4%, more preferably less than about 3%, more preferably less than about 2%, more preferably less than about 1% by weight of the total fatty acids produced by the plant, and more preferably less than about 0.5% by weight of the total fatty acids produced by the plant.

In certain embodiments, the genetically modified plants of the invention or the oil or seed lines obtained from the genetically modified plants of the invention comprise a detectable amount of DHA (docosahexaenoic acid (C22:6, n-3)) or EPA (eicosapentaenoic acid (C20:5, n-3)). In certain embodiments, the genetically modified plant of the invention or the oil or seed line obtained from the genetically modified plant of the invention comprises 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or 15% DHA. Useful ranges can be selected between any of these values, for example, 0.01-15%, 0.05-10%, and 1-5% DHA.

In certain embodiments, the genetically modified plant of the invention or the oil or seed line obtained from the genetically modified plant of the invention comprises 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% EPA. Useful ranges may be chosen between any of these values, for example, 0.01-10%, 0.05-5% and 0.1-5% EPA.

In certain embodiments, when the target product of a PUFA synthase system is a long chain PUFA, such as DHA, DPA (n-6 or n-3), or EPA, intermediates or by-products that are not present in substantial amounts in the total lipid of a plant genetically engineered with such PUFA synthase systems can include, but are not limited to: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and various other intermediates or by-products, such as 20: 0; 20:1(Δ 5); 20:1(Δ 11); 20:2(Δ 8, 11); 20:2(Δ 11, 14); 20:3(Δ 5,11, 14); 20:3(Δ 11,14, 17); melittic acid (20: 3; Δ 5,8, 11); or 20:4 (. DELTA.5, 1,14, 17).

Genetic modification of a plant according to the invention may result in the plant producing one or more PUFAs. In some embodiments, the ratio of PUFAs produced by the plant to PUFAs is not necessarily the same as the ratio of PUFAs produced by the organism from which the PUFA synthase is derived.

In certain embodiments, the genetically modified plants of the invention can be genetically engineered to produce PUFAs via the activity of PUFA synthase. In certain embodiments, PUFAs can be recovered via purification processes that extract compounds from the plant. In some embodiments, PUFAs may be recovered by harvesting the plant. In certain embodiments, PUFAs can be recovered by collecting oil from the plant (e.g., from oilseeds) or collecting seeds from the plant. In some embodiments, the plant may also be consumed in its native state or further processed into a comestible product.

In certain embodiments, the genetically modified plants of the invention can produce one or more polyunsaturated fatty acids. In certain embodiments, the plant can produce (e.g., in its mature seed, in the case of an oilseed plant, or seed oil of an oilseed plant) at least one PUFA (target PUFA), wherein the total fatty acid profile of the plant or plant part that accumulates PUFAs (e.g., in the mature seed, in the case of the plant being an oilseed plant, or seed oil of an oilseed plant) comprises a detectable amount of this PUFA or PUFAs. In certain embodiments, the target PUFA is a PUFA of at least 20 carbons and includes at least 3 double bonds, more preferably at least 4 double bonds, and even more preferably at least 5 double bonds. In certain embodiments, the target PUFA may be a PUFA that is not naturally produced by the plant. In certain embodiments, the total fatty acid profile of a plant or plant part (including plant seed oil) that accumulates PUFAs comprises at least 0.1% by weight of total fatty acids of target PUFA(s), at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, more than 75% by weight of total fatty acids of the plant part(s) that accumulates PUFAs produced by the plant, or any percentage from 0.1% to 75%, or greater than 75% (up to 100% or 100%) -in 0.1% increments-of the target PUFA(s).

As used generally herein, reference to the percentage parts of PUFA production is based on the total fatty acids produced by the organism (plant), unless otherwise indicated. In certain embodiments, the total fatty acids produced by the plant are present as a weight percentage of the Fatty Acid Methyl Ester (FAME) preparation as determined by Gas Chromatography (GC) analysis, but the determination of total fatty acids is not limited to this method.

In certain embodiments, the total fatty acids within a plant (and/or plant parts and/or seed oil fraction) of the invention may contain less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% by weight of the total fatty acids produced by the plant of any one or more fatty acids selected from the group consisting of: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and various other fatty acids, e.g., 20: 0; 20:1(Δ 5); 20:1(Δ 11); 20:2(Δ 8, 11); 20:2(Δ 11, 14); 20:3(Δ 5,11, 14); 20:3(Δ 11,14, 17); melittic acid (20: 3; Δ 5,8, 11); or 20:4 (. DELTA.5, 1,14, 17).

The present invention includes any seed made from the plants described herein, as well as any oil made from the plants or seeds of the present invention. The invention also includes any product made using the plant, seed or oil described herein.

The invention relates to the use and products of genetically modified organisms

The present invention includes a method of producing PUFAs by growing or breeding the genetically modified organisms (e.g., plants) of the present invention as detailed above. In certain embodiments, such methods comprise, for example, the step of planting a plant previously described herein and having genetic modification according to the present invention in a suitable environment, such as soil.

The present invention includes a process for producing an oil comprising at least one PUFA, comprising recovering the oil from a genetically modified plant of the invention or from the seed of a genetically modified plant of the invention. The invention includes a method of producing an oil comprising at least one PUFA, comprising growing a genetically modified plant of the invention. The present invention includes a process for producing at least one PUFA in a seed oil comprising recovering the oil from the seed of a genetically modified plant of the present invention. The invention includes a method of producing at least one PUFA in a seed oil, comprising growing a genetically modified plant of the invention.

The invention includes a method of providing a supplement or therapeutic product containing at least one PUFA to a subject, comprising providing to the subject a genetically modified plant of the invention, an oil of the invention, a seed of the invention, a food of the invention, a functional food of the invention, or a pharmaceutical product of the invention. The invention also includes a method of making a genetically modified plant of the invention, comprising transforming a plant or plant cell with: (i) a nucleic acid sequence encoding an algal PUFA synthase system that produces at least one polyunsaturated fatty acid (PUFA); and (ii) a nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) that transfers a phosphopantetheinyl cofactor to an ACP domain of an algal PUFA synthase system. In certain embodiments, the method further comprises transforming the plant or plant cell with: (iii) a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) that catalyzes the conversion of long chain PUFA Free Fatty Acids (FFA) to acyl-CoA.

In certain embodiments, the PUFA of such processes is DHA or EPA.

The invention further includes any of the organisms or parts thereof described herein (such as plants, plant parts (such as oilseeds), or preparations or fractions thereof), as well as any oils made by the organisms described herein. The invention also includes any product made using the organisms, parts thereof, or oils described herein.

The present invention relates to a method of modifying a product containing at least one fatty acid, the method comprising adding to the product an organism, a part thereof, or an oil produced according to the invention and as described herein by a genetically modified organism, such as a genetically modified plant as described herein. Any product made by this method or containing substantially any organism, part thereof, or oil from an organism as described herein is also encompassed by the present invention.

In some embodiments, the product is selected from the group consisting of: food, dietary supplement, pharmaceutical formula, human milk, infant formula and health functional food. Suitable pharmaceutical formulations include, but are not limited to, anti-inflammatory formulations, chemotherapeutic agents, active excipients, osteoporosis agents, antidepressants, anticonvulsants, anti-helicobacter pylori agents, agents for the treatment of neurodegenerative diseases, agents for the treatment of degenerative liver diseases, antibiotics, and cholesterol lowering formulations. In certain embodiments, the product is for use in treating a condition selected from the group consisting of: chronic inflammation, acute inflammation, gastrointestinal distress, cancer, cachexia, cardiovascular restenosis, neurodegenerative disease, liver degeneration disorder, blood lipid disorder, osteoporosis, osteoarthritis, autoimmune disease, preeclampsia, preterm labor, age-related macular degeneration, pulmonary disorder, and peroxisomal disorder.

In certain embodiments, the product is a food or functional food. Suitable food products include, but are not limited to, fine bakery products, bread and rolls, breakfast cereals, processed and unprocessed cheeses, dressings (tomato sauce, mayonnaise, etc.), dairy products (milk, yogurt), puddings and gelatin snacks, carbonated beverages, tea, powdered drink mixes, processed fish products, fruit-based beverages, chewing gum, hard candies, frozen dairy products, processed meats, nut and nut-based undercoats, pasta, processed poultry products, gravies and sauces, potato chips and other flakes or crisps, chocolate and other confections, soups and soup mixes, soy-based products (such as milk, beverages, creams, whiteners), vegetable oil-based undercoats, and vegetable-based beverages.

In certain embodiments of the invention, the product is a feed or dietary composition for animals, or an additive to a feed or dietary composition. The term animal includes all animals, including humans. Non-limiting examples of animals are non-ruminants (such as pigs, livestock, or fish), and ruminants (such as cattle, sheep, and horses feed or feed compositions means any compound, preparation, mixture, or composition suitable or intended for ingestion by an animal.

In certain embodiments, the genetically modified plant, seed, or oil (e.g., canola) comprises reduced levels of polyunsaturated fatty acids and increased levels of monounsaturated oleic acid as compared to conventional oils. Such plants, seeds or oils may exhibit, for example, a high degree of oxidative stability. In certain embodiments, the genetically modified plant, seed, or oil comprises a high oleic oil background (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% oleic acid). Such plants, seeds or oils may, for example, be less susceptible to oxidation during storage, frying and/or refining, and/or may be heated to higher temperatures without smoke making them more suitable as cooking oils. In certain embodiments, the genetically modified plant, seed, or oil comprises a DHA portion and a high oleic acid oil background as described herein (e.g., greater than or equal to 70% portion including 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% oleic acid and any ranges thereof). In certain embodiments, the genetically modified plant, seed, or oil comprises a DHA portion and a low linolenic acid background as described herein (e.g., an amount less than or equal to 10% including 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.05%, 0.02%, or 0.01% linolenic acid and any ranges thereof). In certain embodiments, the genetically modified plant, seed, or oil comprises a DHA portion as described herein, a high oleic oil background (e.g., present in an amount of greater than or equal to 70% of the portion, including 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% oleic acid and any ranges thereof), and a low linolenic acid background (e.g., an amount of less than or equal to 10% of the portion, including 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.05%, 0.02%, or 0.01% linolenic acid and any ranges thereof). In certain embodiments, such genetically modified plants, seeds, or oils (such as canola) can be incorporated into the products described herein.

Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting.

Examples

Example 1

Codon optimization of PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase OrfC, acyl-CoA synthase and 4' -phosphopantetheinyl transferase HetI

DNA sequence analysis of the coding regions encoding PUFAOrfA from Schizochytrium ATCC 20888 (GenBank ID: AF378327, GI:158518688), PUFAOrfB from Schizochytrium ATCC 20888 (GenBank ID: AF378328, GI:158518690), chimeric PUFAOrfC from Schizochytrium ATCC 20888 and Thraustochytrium (U.S. Appl. Pub.No.2008/0022422) ("chimeric OrfC" or "hybrid OrfC"), acyl-CoA synthetase from Schizochytrium ATCC 20888 (U.S. Appl. Pub.No.2007/0245431), and 4' phosphopantetheinyl transferase HetI from Candida 7120 (GenBank ID: P37695, GI:20141367) showed that several sequence motifs contain non-optimized codon combinations that may be detrimental to optimized plant expression. The gene design(s) encoding the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI proteins are optimized to produce DNA sequences that are more "plant-like" in nature, wherein the sequence modifications do not prevent translation or destabilize mRNA due to non-optimized codon combinations.

Due to the plasticity conferred by the redundancy/degeneracy of the genetic code (e.g., certain amino acids are designated by more than one codon), the genomic evolution of different organisms or organism species results in the differential usage of synonymous codons. This "codon bias" is reflected in the average base combination of the protein coding regions. For example, a genome with a relatively low G + C content utilizes more codons with a or T than the third position of the synonymous codon, while those with a higher G + C content utilize more codons with G or C than the third position. Furthermore, it is believed that a "small number" of codons present in an mRNA may reduce the absolute translation rate of the mRNA, particularly when the relative abundance of charged trnas corresponding to the small number of codons is extremely low. The extension of the inference is that the reduction in translation rate due to individual small numbers of codons at least adds up at multiple small numbers of codons. Thus, mRNAs with a relatively high content of a small number of codons will have a correspondingly low translation rate. This rate is reflected by a correspondingly low level of encoded protein.

In genetically engineering genes encoding PUFA synthase OrfA, PUFA synthase OrfB, chimeric PUFA synthase OrfC, acyl-CoA synthase, and 4' phosphopantetheinyl transferase HetI proteins for expression in Arabidopsis (or other plants, e.g., rice, tobacco, maize, cotton, or soybean), the codon usage of Arabidopsis was obtained in public databases (Table 2).

TABLE 2 expression of synonymous codons in the coding region of the Brassica napus (canola) gene (columns C and G). The values for the set of balanced bias codon progeny tables for plant optimized synthetic gene design are in columns D and H.

DNU = no use

To balance the distribution of remaining codon usage for an amino acid, the weighted average expression for each codon was calculated using the following formula (Table 2):

weighted average of C1% =1/(% C1+% C2+% C3+, etc.) x% C1x100, where C1 is the codon involved and% C2,% C3, etc. represent the average of the% values of the remaining synonymous codons of the Arabidopsis thaliana of Table 2 (the average% values of the relevant codons are taken from columns C and G).

The weighted average% values for each codon are provided in table 2 in columns D and H.

In designing coding regions for plant expression, the major ("preferred") codon preferred by the plant is determined, along with the second, third, fourth, etc. selections that favor codons, if multiple selections exist. New DNA sequences encoding substantially the same amino acid sequence of PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase OrfC, acyl-CoA synthase, and 4 'phosphopantetheinyl transferase HetI were then designed, but differing from the original DNA sequence (encoding PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase, and 4' phosphopantetheinyl transferase HetI) by substituting plant (first, second, third, or fourth, etc.) codons to specify each site amino acid within the amino acid sequence.

The new sequence is then analyzed with restriction enzyme sites created by the sequence modification. The recognition site is then modified with a first, second, third, or fourth selected codon usage. The sequence was then further analyzed and modified to reduce the frequency of TA or GC doublets.

Analysis of these sequences showed that the novel DNA sequences were amino acid sequences that substantially encoded the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI proteins, but optimized expression in canola was designed using a balanced codon distribution of common codons found in the canola gene, respectively. In particular, the new DNA sequence differs from the original DNA sequence encoding PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI by codon substitution in the plant (first, second, third, or fourth preference) to specify the appropriate amino acid at each position within the amino acid sequence of the protein.

Plant optimized DNA sequence design was initiated by reverse translation of the protein sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 using the canola codon bias tables constructed in tables 2, D and H. The protein sequence of acyl-CoA synthetase (SEQ ID NO:4) is modified from the original sequence; wherein the second amino acid alanine is removed from the protein. The initial sequence is then modified by compensating for codon changes (while maintaining overall weighted average codon expression) to remove or add restriction enzyme recognition sites, remove highly stable intrachain secondary structures, and remove other sequences that may be detrimental to the cloning operation of the plant or to modify gene expression. The DNA sequence is then analyzed again with the restriction enzyme recognition sites that these modifications may create. The recognition site is further modified by replacing the relevant codon with the first, second, third, or fourth selected favored codon. Other sites within the sequence that may affect transcription or translation of the gene of interest include the exons: intron junctions (5 'or 3'), poly a addition signal, or RNA polymerase termination signal. The modified sequence was further analyzed and further modified to decrease the frequency of TA or CG doublets and increase the frequency of TG or CT doublets. In addition to these duplexes, sequence motifs having more than about six [ G + C ] or [ A + T ] contiguous residues may affect sequence transcription or translation. Thus, the sequence motifs are also obtained by replacing the preferred codons of the other selection with codons of the first or second selection etc. Rare codons are not included to a substantial extent in gene design, but are only used when-compared to the codon combinations themselves-it is necessary to move about different design criteria (such as the addition or deletion of restriction enzyme recognition sites).

The protein encoded by PUFA synthase OrfA contains 10 repetitive "proline-alanine" domains ranging in size from 17 to 29 amino acids. Interspersed between the proline-alanine repeats are 9 longer repeat domains comprising 87 amino acids. The repeated amino acid sequences varied only at 4 sites, with only two amino acid selections at each site of variation. Analysis of the amino acid sequence of the 9 repeats using the ClustalW computer program produced homology values of 100% and identity values of 95.4%. At the DNA level, the sequences encoding the 9 repeats were 100% homologous, 89.7% identical, and varied at only 27 sites out of 261 bases encoding each repeat (23 of the 27 changes were "silent" differences, in which synonymous codons for the same amino acid were interchanged).

Standard gene design methods cannot be easily adapted to develop new codon-biased DNA sequences for multiple repeats of this size, because one must continually balance all codon usage in individual repeats with the codon usage at the same sites in the remaining 8 repeats to avoid generating highly related DNA sequences. For each of the 87 residue repeats, there is more than 4.5x10 ⁴³The possible DNA sequences encoding the same amino acid sequence (calculated by multiplying the number of synonymous codons for each amino acid in the sequence). Thus, there is a tremendous computational space available for generating a consistent coding DNA sequence. The following flow describes a method for generating (in a computer) a multiple sequence design for each of the individual repeats, followed by a batch alignment of all sequence versions to identify the set of highly differentiated sequences representing the code for the repeats:

step 1: the native DNA sequence encoding each repetitive amino acid domain was taken as a separate sequence.

Step 2: introduction of individual repetitive DNA sequences as individual sequences into a gene design program (e.g., OPTGENE)^TMOcimum biosolutions, Hyderabad, India). Steps 3-5 are separated in each sequenceAnd (6) executing.

And step 3: the DNA sequence is translated using the standard gene code.

And 4, step 4: the translated protein sequence was reverse translated using the standard gene code and appropriate codon bias tables. In this example, a biased codon table was used which pools 530 canola protein coding regions, each generating sequence having the code number "nap" (referred to as "napus") plus the number of versions. Thus, the first untranslated codon-biased sequence of repeat 1 is referred to as "rpt1 napl". In this example, the method is performed 10 times to generate 10 versions of the DNA sequence encoding the protein sequence of repeat 1.

And 5: the 10 sequence versions are output to a corresponding number of text files.

Step 6: repeating steps 3-5 for each of the remaining repeat sequence domains. In this example, a total of 90 "nap" sequence versions (10 for each repeat unit) are generated.

And 7: the 90 sequence files were imported into ClustalW program mega3.1 (available from Megasoftware) and multiple sequence alignments were performed using all 90 sequences as input. Since the sequences are fragments of the protein coding region, the alignment is performed at impermissible intervals. After ClustalW alignment, the evolutionary tree was composed and visualized to visually pick one of ten codon-optimized sequences for each of the nine repeat domains in the protein. Each selected sequence version is selected from the most deeply divergent part of the tree.

And 8: the selected sequence for each repeat domain is incorporated into the codon-optimized DNA sequence encoding the entire protein at the correct site for each particular repeat segment.

And step 9: final analysis of the entire codon-optimized sequence, including the individually designed differentiating repeats, was performed to ensure that no undesired motifs, restriction enzyme recognition sites, etc., were present.

The newly designed canola optimized PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetIDNA sequences are set forth in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, respectively. The codon-optimized sequence is identified throughout the specification as version 3(v 3). The sequence labeled version 2(v2) describes the original non-codon optimized sequence.

The resulting DNA sequence has a higher codon diversity, a desired base combination, contains strategically placed restriction enzyme recognition sites, and is free of sequences that may interfere with gene transcription or translation of mRNA products. Tables 3, 4, 5, 6 and 7 present the codon combinations of PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI protein coding regions found in the original genes, plant optimized versions and proposed alignments of plant optimized sequence codon combinations calculated from columns D and H of table 2.

TABLE 3 codon combinations of PUFAOrfA

TABLE 4 codon combinations for PUFAOrfB

TABLE 5 PUFA chimeric OrfC codon combinations

TABLE 6 acyl-CoA synthetase codon combinations

TABLE 7 phosphopantetheinyl transferase HetI codon combinations

After codon optimization of the coding region sequence is completed, additional nucleotide sequences are added to the optimized coding region sequence. Restriction sites that will facilitate cloning; the Kozak sequence was added to the plant optimized coding sequence with an additional stop codon. Furthermore, the second series of PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and phosphopantetheinyl transferase HetI coding sequence was designed to contain chloroplast targeting sequences from Arabidopsis thaliana ribulose bisphosphate carboxylase small chain 1A (GenBank ID: NM-202369.2). This sequence of SEQ ID NO:11 was added to the previously described coding sequence for PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC and phosphopantetheinyl transferase HetI. The initial methionine removal of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 10 and replacement into chloroplast targeting sequences. The sequence containing this chloroplast-targeting sequence was identified throughout the specification as version 4(v 4).

A second chloroplast transit peptide was added to the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and phosphopantetheinyl transferase HetI coding sequences. The coding sequences are designed to contain chloroplast targeting sequences from acyl-ACP-thioesterases (GenBank ID: X73849.1). This sequence-SEQ ID NO: 38-was added to the previously described coding sequence for PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC and phosphopantetheinyl transferase HetI. The initial methionine removal and replacement of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10 into chloroplast targeting sequences. The sequence containing this chloroplast-targeting sequence was identified throughout the specification as version 5(v 5).

Once the plant-optimized DNA sequence is designed on paper or in silico, the actual DNA molecule, whose sequence corresponds exactly to the designed sequence, can be synthesized in the laboratory. Such synthetic DNA molecules can be cloned or manipulated as if they were derived from natural or native sources. The synthesis of DNA fragments comprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 containing the above additional sequences was performed by commercial suppliers (GeneartAG, Regensburg, Germany). The synthetic DNA clones were then placed into expression vectors and transformed into Agrobacterium and Arabidopsis as described in examples 2, 3, and 4.

Using this method in conjunction with codon optimization of the PUFA synthase OrfA coding sequence leads to the selection of repetitive proline-alanine sequences which are sufficiently differentiated to avoid repetitive sequence instability. The sequences are selected from the deepest branch of the evolutionary tree (i.e., farthest from another of the set of sequences). The Smith-Wasserman global alignment was performed on all pairwise combinations and the homology ranges were 74-81%, with possible parity 76-77% (Table 8).

TABLE 8 Smith-Wasserman homology of selected codon-optimized sequences of the PUFAOrfA repeats

The ClustalW alignment (VectorNTI, Invitrogen, Carlsbad, CA) of selected 9 newly designed coding regions of the 9 repeat domains is shown in figure 1. Overall, the sequences were 93.1% homologous, 61.7% identical compared to the original sequences which were 100% homologous and 89.7% identical. Greater sequence diversity can be achieved by using more than 10 sequence iterations and choosing from them (rather than visually selecting sequences) using computer programs or mathematical algorithms. However, the exemplified sequences are highly differentiated and result in stable polynucleotide fragment-containing nucleotides.

Example 2

plasmid construction of pDAB7361, pDAB7362, pDAB7363 and additional constructs

Construction of pDAB7361

The pDAB7361 plasmid (FIG. 2; SEQ ID NO:35) was constructed using the multiple site Gateway (Gateway) recombinant L-R reaction (Invitrogen, Carlsbad, Calif.). pDAB7361 contains three PUFA synthase Plant Transcription Units (PTUs) as follows, an acyl-CoA synthetase PTU, a phosphopantetheinyl transferase PTU, and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains a truncated phaseolin-L gene promoter (PvDlec2 promoter v 2; GenBank accession number X06336), the 5' untranslated region of the Arabidopsis AT2S3 gene (2S5' UTR; GenBank accession number NM-118850), the Schizochytrium polyunsaturated fatty acid synthase open reading region A (SzPUFAOrfAv2), and the Arabidopsis thaliana 2S albumin gene 3' untranslated region terminator v1(At2SSSP terminator v 1; GenBank accession number M22035). The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, schizochytrium polyunsaturated fatty acid synthase open reading frame B (SzPUFAOrfBv3), and At2SSSP terminator v 1. The third PUFA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, Schizochytrium and Thraustochytrium polyunsaturated fatty acid synthetase open reading region C (hSzThPUFAOrfCv3) and At2SSSP terminator v 1. acyl-CoA synthetase PTUPvDlec2 promoter v2, 2S5' UTR, Schizochytrium acyl-CoA synthetase (SzACS-2v3) and At2SSSP terminator v 1. The phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, Nohetiv3 HetI (NohetIV3) and At2SSSP terminator v 1.

Plasmids pDAB7355, pDAB7335, pDAB7336, pDAB7339 were recombined with pDAB7333 to form pDAB 7361. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. pDAB 7333-which contains, among other regulatory units, for example, overdrive sequences (Toroetal, PNAS85 (22): 8558-8562; 1988) and T-strand boundary sequences (T-DNA boundary A and T-DNA boundary B; Gardnereal, Science231: 725-727; 1986 and WO2001/025459A1) -also contains phosphinothricin acetyltransferase PTU: Tapioca vein mosaic virus promoter (CsVMV promoter v 2; Verdagueret, plant molecular biology31: 1129-1139; 1996), phosphinothricin acetyltransferase (PATv 5; Wolebenet, Gene70: 25-37; 1988) and the untranslated region of Agrobacterium tumefaciens ORF13' (Atu3625 ' ORF13' V4; Anutrgetegal, J.tuberculosis.172: Hu4-1822; 1990). Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7362

The pDAB7362 plasmid (FIG. 3; SEQ ID NO:36) was constructed using a multiple site gateway recombinant L-R reaction. pDAB7362 contains three PUFA synthase PTUs, an acyl-CoA synthase PTU, a phosphopantetheinyl transferase PTU sequence and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzACS-2v3 gene and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Recombining plastids pDAB7334, pDAB7335, pDAB7336, pDAB7339 with pDAB7333 to form pDAB 7362. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7363

pDAB7363 (FIG. 4; SEQ ID NO:37) was constructed using a multiple site gateway L-R recombination reaction. pDAB7363 contained three PUFA synthase PTUs, an acyl-CoA synthase PTU and a phosphopantetheinyl transferase PTU sequence. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 4, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv4 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 4 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzACS-2v3 gene and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv4 and At2SSSP terminator v 1. Furthermore, all PTUs also contain an arabidopsis thaliana ribulose bisphosphate carboxylase small chain 1A chloroplast targeting sequence, indicated by the label "v 4".

Plasmids pDAB7340, pDAB7341, pDAB7342, pDAB7344 were recombined with pDAB7333 to form pDAB 7363. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7365

pDAB7365 was a binary plasmid constructed to contain the native, non-codon optimized versions of SzPUFAOrfAv2, SzPUFAOrfBv2, hSzThPUFAOrfCv2, SzACS-2v2, and NoHetIv 2. The pDAB7365 plasmid (FIG. 19; SEQ ID NO:39) was constructed using a multiple site gateway L-R recombination reaction. pDAB7365 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 2, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv2 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfCv2 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzACS-2v2 gene and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv2 and At2SSSP terminator v 1.

Plasmids pDAB7355, pDAB7356, pDAB7357, pDAB7360 were recombined with pDAB7333 to form pDAB 7365. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv2, SzPUFAOrfBv2, SzPUFAOrfCv2, SzACS-2v2, NoHetIv 2. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of six PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7368

pDAB7368 is a binary plasmid constructed to contain the native, non-codon optimized versions of SzPUFAOrfAv2, SzPUFAOrfBv2, hSzThPUFAOrfCv2, and NoHetIv 2. This construct does not contain the SzACS-2 coding sequence. The pDAB7368 plasmid (FIG. 20; SEQ ID NO:40) was constructed using a multiple site gateway L-R recombination reaction. pDAB7368 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 2, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv2 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfCv2 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv2 and At2SSSP terminator v 1.

Plasmids pDAB7355, pDAB7356, pDAB7357, pDAB7359 were recombined with pDAB7333 to form pDAB 7368. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv2, SzPUFAOrfBv2, SzPUFAOrfCv2, NoHetIv 2. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7369

pDAB7369 was a binary plasmid constructed to contain a reconstituted, codon-optimized version of SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, and NoHetIv 3. This construct does not contain the SzACS-2 coding sequence PTU. The pDAB7369 plasmid (FIG. 21; SEQ ID NO:41) was constructed using a multiple site gateway L-R recombination reaction. pDAB7369 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB7338 were recombined with pDAB7333 to form pDAB 7369. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7370

pDAB7370 is a binary plasmid constructed to contain a reconstituted, codon-optimized version of SzPUFAOrfAv4, SzPUFAOrfBv4, hSzThPUFAOrfCv4, and NoHetIv4, which contain ribulose bisphosphate carboxylase small chain 1A (labeled SSU-TPv1) ligated to the amino terminus of the coding sequence. This construct does not contain the SzACS-2 coding sequence PTU. The pDAB7370 plasmid (FIG. 22; SEQ ID NO:42) was constructed using the multiple site gateway L-R recombination reaction. pDAB7370 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU, and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 4, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv4 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 4 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv4 and At2SSSP terminator v 1.

Plasmids pDAB7340, pDAB7341, pDAB7342, pDAB7343 were recombined with pDAB7333 to form pDAB 7370. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv4, SzPUFAOrfBv4, hsztthpufaorfcv 4, NoHetIv 4. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB100518

pDAB100518 is a binary plasmid constructed to contain reconstructed, codon-optimized versions of SzPUFAOrfAv5, SzPUFAOrfBv5, hSzThPUFAOrfCv5, and NoHetIv5, which contain chloroplast transit peptides (labeled thioesterase transit peptides) from acyl-ACP-thioesterases joined to the amino terminus of the coding sequence. In addition, the plasmid contains the SzACS-2v3 coding sequence PTU without chloroplast transit peptide. The pDAB100518 plasmid (FIG. 23; SEQ ID NO:43) was constructed using a multiple site gateway L-R recombination reaction. pDAB100518 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 5, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv5 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 5 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzACS-2v3 gene and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv5 and At2SSSP terminator v 1.

Plasmids pDAB100517, pDAB100514, pDAB100511, pDAB100515 were recombined with pDAB7333 to form pDAB 100518. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv5, SzPUFAOrfBv5, hSzThPUFAOrfCv5, SzACS-2v3, NoHetIv 5. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing six PTUs were then isolated and tested for incorporation of the six PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB101476

pDAB101476 is a binary plasmid constructed to contain a reconstituted, codon-optimized version of SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, and NoHetIv 3. The SzACS-2v2 gene sequence is a native, non-codon optimized version. The pDAB101476 plasmid (FIG. 24; SEQ ID NO:44) was constructed using a multiple site gateway L-R recombination reaction. pDAB101476 contains three PUFA synthase PTUs, an acyl-CoA synthase PTU, a phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzACS-2v2 gene and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB101471 and pDAB7333 were recombined to form pDAB 101476. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, SzACS-2v2, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing six PTUs were then isolated and tested for incorporation of the six PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB101477

pDAB101477 is a binary plasmid constructed to contain a reconstituted, codon-optimized version of SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, and NoHetIv 3. The pDAB101477 plasmid (FIG. 25; SEQ ID NO:45) was constructed using a multiple site gateway L-R recombination reaction. pDAB101477 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzACS-2v4 gene and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB101472 were recombined with pDAB7333 to form pDAB 101477. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, SzACS-2v4, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing six PTUs were then isolated and tested for incorporation of the six PTUs by restriction enzyme digestion and DNA sequencing.

Example 3

Agrobacterium strains producing plasmids pDAB7361, pDAB7362, pDAB7363

The pDAB7361, pDAB7362 and pDAB7363 plasmids were transformed into agrobacterium tumefaciens using standard electroporation techniques. Specifically, Agrobacterium tumefaciens strain Z707S (Hepburn. J. Gen. Microbiol.131:2961-2969(1985)) was electroporated with pDAB7361, pDAB7362 or pDAB7363 plasmids. Transformed colonies of Agrobacterium containing these plasmids were picked and confirmed using restriction enzyme digestion. Agrobacterium strains containing pDAB7361, pDAB7362, or pDAB7363 were stored at-80 ℃ as glycerol stocks.

Example 4

Agrobacterium-mediated transformation of canola

Preparation of Agrobacterium

A loop of glycerol stock of Agrobacterium strains containing pDAB7361, pDAB7362 or pDAB7363 was blotted to blot on YEP (Bacto brand peptone 20.0gm/L and yeast extract 10.0gm/L) plates containing streptomycin (100mg/ml) and spectinomycin (50mg/ml) and incubated at 28 ℃ for 2 days. A2 day dip was then applied to a ring of plates, which were incubated to 150mL modified YEP with streptomycin (100mg/mL) and spectinomycin (50mg/mL), placed in sterile 500mL shake flasks(s) and shaken at 28 ℃ at 200 rpm. The culture broth was resuspended in M-medium (LS salts, 3% glucose, modified B5 vitamins, 1. mu.M merozoin, 1. mu.M 2,4-D, pH5.8) and diluted to the appropriate density (50Klett units) prior to canola hypocotyl transformation.

Canola transformation

Seed germination: canola seeds (variety Nexera710) were surface sterilized in 10% Clorox for 10 minutes and rinsed three times with sterile distilled water (during this process, the seeds were placed in steel filters). Planting seeds in 1/2MS canola culture medium placed in a Phytatray-brand shallow tray, waiting for germination (1/2MS, 2% sucrose, 0.8% agar), placing 25 seeds in each Phytatray-brand shallow tray into a Percival-brand constant temperature and humidity box, adjusting the growth mechanism to 25 ℃, and lighting for 16 hours and dark for 8 hours in the light cycle; and waiting for 5 days for germination.

Pretreatment: on day 5, -3 mm hypocotyl fragments were aseptically excised and the root bud fractions discarded (these were placed in 10ml sterile milliQ water during the excision process to avoid drying out the hypocotyls). The hypocotyl fragments were horizontally placed on sterile filter paper and callus induction medium MSK1D1(MS, 1mg/l merozoin, 1mg/l2,4-D, 3% sucrose, 0.7% Phytagar brand agar) for 3 days, and pre-treated in a Percival brand constant temperature and humidity cabinet to adjust the growth mechanism to 22-23 ℃ (16 hours photoperiod, 8 hours dark).

And (3) co-culturing with agrobacterium: one day prior to Agrobacterium treatment, flasks containing YEP medium containing the appropriate antibiotic were grown. The embryonic axis fragments were transferred from the filter paper to a 100x25mm empty petri dish containing 10ml of liquid M medium to avoid drying out the embryonic axis fragments. At this stage the section is scooped up with a spatula and transported. Liquid M medium was removed with a micropipette and 40ml of Agrobacterium suspension was added to the dish (500 fragments with 40ml of Agrobacterium solution). The dish was periodically rotated and the fragments were processed for 30 minutes to maintain the embryonic axis submerged in the Agrobacterium solution. At the end of the treatment period, the agrobacterium solution is transferred to a waste beaker with a micropipette, sterilized and discarded (the agrobacterium solution is completely removed to avoid overgrowth of agrobacterium). The treated embryonic axis were transferred back to the original tray with filter paper containing MSK1D1 using forceps (care was taken to ensure that the fragments did not dry out). The hypocotyl fragment together with the control fragment were returned to Percival brand constant temperature and humidity chamber, the light intensity was reduced (the plates were covered with aluminum foil), and the finished hypocotyls and Agrobacterium were co-cultured for 3 days.

Callus induction was performed on selective medium: after 3 days of co-cultivation, the hypocotyl fragments were transferred individually with forceps onto callus induction medium MSK1D1H1(MS,1mg/l merozoite, 1mg/l2,4-D, 0.5gm/l MES, 5mg/l AgNO3, 300mg/l Tenctinom, 200mg/l penicillin, 1mg/l haobisi, 3% sucrose, 0.7% Phytagar brand agar). The embryonic axis segments are anchored to the medium rather than embedded in the medium.

Selection and bud regeneration: 7 days after callus induction medium, the hypocotyl fragments producing callus were transferred to shoot regeneration medium # 1(MS, 3mg/l BAP, 1mg/l Zeatin, 0.5gm/l MES, 5mg/l AgNO3, 300mg/l Tenctinia (Timentin), 200mg/l Carbenicillin (Carbenicillin), 1mg/l Hairbi (Herbiace), 3% sucrose, 0.7% Phytagar brand agar) with optional MSB3Z1H 1. After 14 days, the germinated embryonic axis were transferred to regeneration medium No. 2 (MS, 3mg/l BAP, 1mg/l Zeatin, 0.5gm/l MES, 5mg/l AgNO3, 300mg/l Terminalia, 200mg/l penicillin Carbenicillin, 3mg/l nicera, 3% sucrose, 0.7% Phytagar brand agar) with enhanced selectivity MSB3Z1H 3.

Bud elongation: after 14 days, the budded fragments were transferred to bud extension medium MSMESH5(MS, 300mg/l Terminactimina, 5mg/l Haobisi, 2% sucrose, 0.7% TC agar). The extended shoots were isolated and transferred to MSMESH 5. After 14 days, the first round of non-extended remaining shoots was placed on MSMESH5 and transferred to the same combination of fresh selective media. All remaining hypocotyl segments are discarded at this stage.

After 2 weeks the shoots elongated on MSB3Z1H3 medium were isolated and transferred to MSMESH5 medium. The first round of non-extended remaining shoots on MSMESH5 were isolated and transferred to the same combination of fresh selection medium. All remaining hypocotyl segments are discarded at this stage.

Root induction: after 14 days, the shoots are transferred to MSMEST medium (MS, 0.5g/l MES, 300mg/l Terminactimina, 2% sucrose, 0.7% TC agar to induce roots.the ungerminated shoots transferred first back to MSMESST medium are transferred in a second or third back to MSMESST medium until rooted plants are obtained.

PCR analysis: the PCR samples were isolated after at least 14 days of shoot culture on MSMESH5 medium. Leaf tissue from green shoots was tested for the presence of the PAT selectable marker gene by PCR. All the chloroses were discarded without being subjected to the PAT test. Samples positive for the PCR reaction were retained and shoots were left on msmesst medium to extend the developing roots. Shoots that were negative according to the PCR test were discarded.

Plants rooted on MSMESH5 or MSEST and PCR-positive were transplanted to soil. After being strengthened, T ₀Canola plants were further analyzed for events containing all transgenic PTU cassettes, after which plants were transferred to the greenhouse, planted to maturity and seeds harvested for additional analysis.

Example 5

Copy number analysis and coding region detection of transgenic canola

T selected from example 4₀Plants were further analyzed to identify plants containing each transgenic PTU expression cassette. Invader and hydrolysis Probe assays were performed to initially screen for putatively transformed T₀Plant samples to identify events containing PAT expression cassettes. Subsequent PCR analysis of the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI gene expression cassettes was performed to further identify plants containing the respective gene expression cassette PTU from the binary vector used to transform plants. Selecting events containing all PTUs to progress to T₁A plant.

Tissue samples were collected in 96-well collection trays and freeze-dried for 2 days. Tissue dissociation was performed with a Kleco brand tissue disruptor and tungsten beads (Kleco, Visalia, Calif.). After tissue dissociation, genomic DNA was isolated in a high-throughput format using the DNeasy96 plant kit (Qiagen, Germantown, MD) according to the manufacturer's suggested protocol.

gDNA was quantified using the Quant-ITPicoGreenDNA assay kit (molecular probes, Invitrogen, Carlsbad, Calif.). The quantified gDNA was adjusted to 10 ng/. mu.l for use in a Biorobot3000 automated liquid processor (Qiagen, Germantown, Md.)Tested or adjusted to 2 ng/. mu.l for hydrolysis probe testing.

Routine for canola pat analysisThe assay was developed by ThirdWave technologies (Madison, Wis.). gDNA samples (7.5. mu.l, 10 ng/. mu.l gDNA) placed in a 96-well plate format were first denatured by incubation at 95 ℃ for 10 minutes, followed by cooling to ambient temperature. Next, 7.5. mu.l of premix (3. mu.l of probe mixture for pat with HMG internal reference gene (Weng,2005) WengH.et., (2005) J.AOACInt.88(2):577-84., 3.5. mu.l of Cleavase XIFRET mixture, and 1. mu.l of Cleavase XI enzyme/MgCl₂Solution) was added to each well and the sample was covered with mineral oil. The discs were sealed and mounted on BioIncubate at 63 ℃ for 1 hour in a RadTetrad thermocycler. The disks were allowed to cool to ambient temperature prior to reading with a fluorescent disk reader. All discs contained 1 copy, 2 copy and 4 copy standards as well as wild type control samples and blank wells without samples.

Readings are collected for both FAM (λ 485-528nm) and RED (λ 560-620nm) channels and the multiple (fold) of each channel from which each sample exceeds zero (i.e., background value) is determined by dividing the sample raw signal by the template-free raw signal. From this data, a standard curve was constructed and a best fit curve was determined using linear regression analysis. Using the parameters identified from this fit line, the apparent pat copy number for each sample is then estimated.

Test with hydrolysis Probe-analogously toTest-determination of transgene copy number line useThe 480 system (Roche applied science, Indianapolis, IN) was performed by real-time PCR. The test was conducted usingProbe design software version 2.0 was designed for pat and internal reference gene HMG. In order to carry out the amplification,480 Probe premix (Roche applied science, Indianapolis, IN) was prepared at 1 Xfinal concentration IN 10. mu.L volume multiplex reactions containing 0.4. mu.M of each primer and 0.2. mu.M of each probe (Table 8). The two-step amplification reaction was performed with extension at 60 ℃ for 35 seconds, while fluorescence was collected. All samples were run in triplicate and the mean cycle threshold (Ct) value was used for each sample analysis.

Real-time PCR data analysis SystemSoftware version 1.5, relative usage determinationThe quantity module is executed on the basis of the Δ Δ Ct method. To this end, gDNA samples from single copy calibrators and conventional 2 copy controls were included in each run (as those used in the Invader assay described above).

Is contained in T₀The presence of the remaining gene expression cassettes within the plant event is detected by a separate PCR reaction. The five PTU coding region-specific primer pairs (Table 9) were used for detection.

TABLE 9 primer and Probe information for hydrolysis Probe assay of pat and internal reference (HMG)

The PUFA synthase OrfAPCR reaction requires two separate PCR reactions and different conditions (e.g., PCR primers and cycling conditions) to amplify the open reading frame of the gene sequence. All PCR reactions were completed using the conditions described in table 10 using the EX-TAQPCR kit (taka rabiotechnologies inc. otsu, Shiga, Japan) for 35 cycles according to the manufacturer's instructions. The PCR product was resolved and identified by TAE agarose gel electrophoresis. The expected PCR product gel fragment size indicating the presence of full-length PTU is illustrated in table 10 under the "expected size" column.

Table 10 PCR primers and conditions.

A total of 197 canola events were identified as pat positive by the Invader and hydrolysis probe experiments. Fifteen of these events produced PCR amplification products for transformation of all five gene expression cassettes (PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI) contained within the binary of the plant. Table 11 provides the fifteen events, which were further analyzed for docosahexaenoic acid (DHA) production. The T' s₀Canola plants grow to maturity in the greenhouse and are then self-pollinated. Collection of mature T₁Seeds were analyzed for DHA via GC-FAME analysis.

TABLE 11 PCR detection of docosahexaenoic acid (DHA) -producing genes in transgenic canola plants

Example 6

Detection of DHA in transgenic canola lipids

Canola samples (single seed or batch samples) were homogenized in heptane containing triacylglycerol (Nu-Chekprep) as a triglyceride standard using a steel ball mill. Freshly prepared sodium methoxide (Sigma-Aldrich, st. louis, MO) dissolved in 0.25M methanol was added before homogenization. The extraction was carried out by shaking at 40 ℃. Recovery was verified by recovery of the methylated substitute C17 fatty acid. The extraction of FAMEs (fatty acid methyl esters) was repeated three times and the heptane layers were pooled before analysis. The presence of endogenous FAMEs was checked at the fourth extraction/derivatization to verify reaction completion. The resulting FAMEs were analyzed by GC-FID using a capillary column BPX70(15 mx0.25mmx0.25. mu.M) from SGE. Each FAME was identified by retention time and quantified by injection of a rapeseed oil reference mixture from MatreyaLLC (PleasantGap, PA) as a calibration standard and addition of the appropriate long chain polyunsaturated fatty acid (Nu-chekpep, elsian).

At T₁After GC-FAME analysis of seeds, FAMEs extracts of seeds corresponding to seven events were found to contain peaks corresponding to DHA and DPA (n-6) (listed below in table 12). Table 12 shows that the number of DHA-containing seeds varied (as expected from the insertion of the various copies of the segregating transgene into the canola genome), as did the maximum amount of DHA observed on a single seed.

Table 12: LC-PUFA content of seven transgenic canola event T1 seeds containing PUFA synthase genes, SzACS-2 and HetI genes

a. From the T1 batch analysis, number of seeds/total number of seeds containing detectable DHA.

b. Average DHA content (% total lipid) of all DHA-positive seeds.

c. Average PUFA content (% total lipid) of all DHA-positive seeds.

Average% ratio of DHAn-3/total LC-PUFA (DHA + DPAn-6).

e. The highest DHA content observed on a single seed.

Developing seeds from additional events were analyzed and found to contain DHA, but T produced by mature plants₁The seeds were not sufficient for further analysis. Identification of peak of long chain polyunsaturated fatty acid (LC-PUFA) by mass spectrometryAnalyzed and compared with a real standard (Nu-ChekPrep, Elysian nMN).

From an event (event 5197[14 ]]-032.002) of pairs T₁A single seed analysis of the DHA content of the seeds is shown in figure 5. A single seed contains up to 1% DHA (% total FAMEs). DHA levels appear to separate into three families (0, — 0.4% and-0.9% DHA), reflecting the separation of single loci containing DHA-producing genes.

The data indicate that DHA is produced in plants transformed with plasmids pDAB7361, pDAB7362 and pDAB 7363. The pDAB7362 plasmid contains plant-optimized versions of all five genes (encoding PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI) driven by the phaseolus vulgaris lectin-L gene promoter. In pDAB7361, the native gene sequence of PUFA synthase OrfA (SzOrfAv2) replaced the plant-optimized version (SzOrfAv 3). pDAB7363 is also similar to pDAB7362 except that the arabidopsis thaliana ribulose bisphosphate carboxylase small chain 1A chloroplast transit peptide was added to the N-terminus of PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, and 4' phosphopantetheinyl transferase HetI to target the polypeptides to the plastid.

Example 7

Detection of PUFA synthase protein from canola

The PUFA synthase polypeptides from mature transgenic seed samples were detected by western blotting. Analytical seeds were prepared by milling the dried seeds with 2 stainless steel balls due to a Kleco bead beater (Garcia machine, Visali, Calif.). Extraction buffer (50mM Tris,10mM EDTA,2% SDS) was added and the sample tube was gently shaken for 30 minutes. The samples were centrifuged at 3000rcf for 30 minutes. The supernatant was collected for analysis. The total soluble protein content in the seed extracts was determined by Lowry assay (BioRad, Hercules, Calif.). Samples were normalized to 1.55mg/ml total soluble protein and prepared in LDS sample buffer (Invitrogen, Carlsbad, Calif.) containing 40mM DTT for a normalized loading of 20. mu.g total soluble protein per lane. Samples were electrophoresed in 3-8% Tris acetate gels (Invitrogen, Carlsbad, Calif.) and transferred to nitrocellulose membranes. Blots were blocked in blocking buffer and probed with antibodies against different PUFA synthase OrfA, OrfB and OrfC polypeptides. Rabbit anti-A2-A directed to the A2 region of Schizochytrium PUFA synthase subunit A (SzPUFS-A) and rabbit anti-B3-A directed to the B3 region of Schizochytrium PUFA synthase subunit B (SzPUFS-B) were used. The B3 region is the Enoyl Reductase (ER) region. Subunit C also has an ER region, so in Western blotting, this antiserum will recognize both subunits B and C. Anti-rabbit fluorescently labeled secondary antibody (goat anti-rabbit AF633(Invitrogen, Carlsbad, CA)) was used for detection. The blot was viewed on a typhoon trioplus fluorescence imager (GEHealthcare, new brunswick nj).

SDS-PAGE Western blots of T1 seed extracts from late (>30DAP) development of event 5197[14] -032.002 showed appropriately sized bands probed with OrfA, OrfB and OrfC specific sera (FIG. 6). The bands can also be viewed with coomassie blue direct stain. OrfA, OrfB and OrfC were also detected in seed samples from DHA production events 5197[13] -010.001, 5197[21] -052.001, 5197[21] -053.001 and 5217[6] -065.002.

A set of developing T2 seed samples from DHA-producing canola events 5197[14] -032.002.Sx002, days post pollination (DAP)15, 20, 25, 30, 35, and 42 were collected and analyzed for lipid content (FIG. 7A) and for the presence of OrfA, OrfB, and OrfC polypeptides by Western blotting (FIG. 7B).

Expression of all three polypeptides was detected in developing seeds at 30 and 35 days post-pollination, especially at 42 days post-pollination and mature seeds (FIGS. 7A and 7B).

Example 8

T₂DHA, DPA and EPA levels of canola

Will come from event 5197[14]]T of-032.002₁The seeds were grown in a greenhouse and leaf samples for DNA analysis were taken from the 4-5 leaf stage of 96 plants to determine individual plantsT₁Transgene copy number in isolated plants. This was performed using the above procedure with a hydrolysis probe test of pat gene and identifying three different classes of isolates; 21 homozygous, 45 crossed and 30 blank plants. All homozygous and 31 blank plants were grown in the greenhouse to maturity and seeds were harvested. Average T per plant for homozygous and null plants ₂The seed yields were 7.36gm and 8.61gm, respectively.

As described previously, batch extractions of 8-12 seeds were determined from event 5197[14 ] by GC-FAME analysis]-032.002 greenhouse cultivation T₁T of plants₂Long chain polyunsaturated fatty acid (LC-PUFA) content of the seeds. 21 blank isolates were also grown to maturity as controls. The LC-PUFA content of homozygous plants is shown in FIG. 8. No LC-PUFAs could be detected in the seeds of any of the blank isolates. Twenty transgenic lines produced between 0.28% and 0.90% DHA in batch seed analysis while one line did not produce any LC-PUFA. DHA containing seeds also contain between 0.09 and 0.34% DPA (n-6). The average proportion of DHA in the total PUFA (DHA + DPA) is 77%.

The fatty acid composition of seeds from four lines producing more than 0.7% DHA is shown in table 13, compared to that obtained for four blank segregating lines.

For T from these homozygotes₁Plant 48T of six lines (4, 35, 63, 96, 50, and 106)₂Seeds were subjected to single seed analysis. Detailed analysis of the GC-FAME pattern showed that additional peaks continued for seeds containing DHA and DPA. This was identified as C20:5(n-3) EPA by comparison with the authentic standard (NU-Chek). The retention times correspond to the retention times of authentic EPA (C20:5(n-3)) and the nominal molecular weights determined by GC-MS of PolarisQ are the same.

Single T from six lines₂LC-PUFA trim for seed analysis is shown in figure 9. Mono-isomers with DHA content up to 1.6% were foundOne seed. In addition, plants with EPA content up to 0.27% were identified.

At two T₁Positive and negative crosses were made between lines and untransformed Nexera 710. The resultant parental and F1 hybrid seeds were analyzed for DHA content (FIG. 10). In FIG. 10, the diamonds represent the mean ANOVA of the classes on the X-axis. The vertical bars represent the class mean and the distance between the vertices of the diamond is the 95% confidence interval. F1 seeds accumulated half as much DHA (0.29% and 0.28%) as the transgenic parent seeds (0.51% and 0.47%). Quantitative association of phenotype to degree of conjugation can be inferred from this result.

In summary, the data show that the DHA profile conferred by the five transgenes is heritable and maintained to the second generation.

Example 9

DHA production in T2 seed of canola event-10

Arabidopsis thaliana event 5197[13 ]]Sixty T of-010.001 (containing two copies of the pat Gene, as shown in FIG. 11)₁The seeds were grown in a greenhouse. Hydrolysis probe testing of the pat gene identified five different isolates, corresponding to 0-4 copies of the pat gene.

The two loci corresponding to the transgene insert can be distinguished by southern blot analysis (referred to as loci A and B). DNA from all plants containing two pat copies can be analyzed by southern blotting to determine the genotypes of these (homozygous for either locus A or locus B, or hemizygous for both loci). Four single copies and two blank control plants were also analyzed as control groups. Let all T ₁Plants were grown to maturity in the greenhouse. Seeds were harvested and analyzed for LC-PUFA content in a batch seed analysis (table 14).

TABLE 14 from event 5197[13]]T of-010.001₁T of isolate₂LC-PUFA content of the seeds (levels on average compared with Tukey-KramerHSD test and not linked with the same letter are significantly different).

The data show that event 5197[13] -010.001 locus A is homozygous for the plant line directing LC-PUFA production, and that locus B is homozygous for the seed. Furthermore, locus B interferes with the production of LC-PUFA, as four copies of the double homozygote produce very low levels of DHA, as do three copies of the plant. Similarly, plants half-in-one single copy locus a produced 0.47% LC-PUFA, but half-in-one single copy locus B produced very low levels of LC-PUFA (0.02%).

The complete fat combinations from batches of T2 seeds derived from event 5197[13] -010.001 plants homozygous for locus A (locus B blank) as determined by GC-FAME analysis are shown in Table 15.

Example 10

Production of canola DHA in field

Collecting 5197[14 ] with the highest level of DHA]-032.002T of ten homozygous lines₂Seed, 60gm seed was generated. Seeds from 10 blank isolates were also collected to yield 47gm seeds as a negative control. In May 2009 the seeds were grown in two regions of North Dakota, 8 plots of the seeds containing the transgene, 6 plots of the seeds of the blank isolate and two commercial controls of the seeds (Nexera845 CL). All transgenic plant fields and blank isolates four of them were covered with spacer cages during flowering. The remaining two blank plants and the field of Nexera845CL were not covered. The fields were harvested in September according to routine practice. At locus 1, 0.95kg of seeds were obtained from transgenic plants and 0.99kg from blank plants, on average, in one plot. At position 2, the mean square root 0.64kg was obtained for the plants and 0.73kg from the blank. GC-FAME lipid analysis was performed on seeds from each field to determine LC-PUFAs levels for seeds planted in the field (table 16).

The results in Table 16 represent the analysis of three samples per block. The seeds from fields 1-11 contained lower levels of 18:1(65.5%) and higher levels of 18:3(7.6%) compared to the fields at other site 1 (average 76.7%18:1 and 2.9%18:3) and were therefore considered to be widely contaminated with conventional canola. This was excluded from subsequent analysis. Analysis of T from transgenic plants in 10-seed lots₃The average DHA content of the seeds was 0.19% (site 1) and 0.26% (site 2). The maximum DHA content was 0.38% (together with 0.03% EPA). The average% n-3 LC-PUFA/total PUFAs ratio is 73%.

Each T used in the field test₂The line samples were planted in the greenhouse. Analysis of T with 10-seed batches₃The average DHA content of greenhouse seeds was 0.22%, with individual plants having up to 0.8% DHA. This correlates with the amount of DHA produced in the field.

These data show that the target PUFA synthase gene set can direct the production of DHA in field conditions.

TABLE 16 analysis from 5197[14 ] with 10-seed batches]-032.002 field planting T₂DHA content of T3 seeds of plants.

Example 11

DHA gene expression analysis using microarray technology

Developing canola seeds from transformed homozygous event 5197[14] -032.002 lines and untransformed null plants were harvested on post-pollination Days (DAP)15, 20, 25, 30, 35 and 42. A monochromatic global gene expression pattern design was used to determine the expression level of each gene of each newly introduced homozygous transformed line compared to the untransformed blank line at each given time point during seed development. Three identical industrial replicates of an oligo array of 60-monomers (agilent technologies inc., santa clara, CA) were hybridized with amplified, Cy 3-labeled cRNA of each sample. The foregoing hybridization was performed using a 60-mer full-transcript canola oligonucleotide array of custom design (eArray, Agilent technologies Inc., Santa Clara, Calif.). This array contained over 37,000 different canola transcripts obtained from published data sources (agilent technologies inc., santa clara, CA). To efficiently measure the expression level of each transcript, the oligomers present in the array are designed to be unique and specific to each target to efficiently hybridize to the desired target sequence. Oligomers that form a biploid with more than one transcript are removed from the array. Each oligomer also satisfies the chemical and physical properties required for optimal performance throughput microarray processing. In addition, proprietary and unique oligomers representing newly introduced genes and several other genes of interest are also presented in custom designed canola oligo arrays. 60-monomer oligomers were synthesized in situ using the Sure-Print technology of the manufacturer.

RNA isolation and purification

Developing seed samples from event 5197[14] -032.002 and the blank control were frozen and pooled for use as starting material for RNA isolation and purification. A total of 500mgs of seed tissue per pooled sample was spiked with liquid nitrogen using a pestle and mortar and approximately 50mgs of the milled tissue was resuspended in 450. mu.L of extraction buffer RLT in RNeasy kit for RNA extraction (Qiagen, Valencia, Calif.). The sample is rapidly shaken to destroy the tissue before continuing with the extraction protocol. Total RNA was purified according to the instructions of RNeasy kit (Qiagen, Valencia, CA) for RNA extraction. Purified total RNA was then quantified using a nanosquant (TECAN, research and brand name, NC) spectrometer and visualized by standard 1% agar gel electrophoresis.

For labeling, a total of 1.0. mu.g of purified total RNA from each sample was reverse transcribed, amplified, and labeled with Cy3-CTP according to the Agilent (Santa Clara, CA) Monochromatic microarray Gene expression QuickAmp labeling protocol. Since each canola array contained over 1300 internal incorporation controls, the single color RNA incorporation kits (Agilent, santa clara, CA) were also labeled according to manufacturer instructions. The sample was reverse transcribed using MMLV reverse transcriptase and amplified using T7RNA polymerase. After amplification, the cRNA line was purified using Qiagen's Rneasy mini centrifuge column and quantified using a nanostar spectrometer (TECAN, research tiangregork, NC). The specific activity of Cy3 was determined by the following formula: (Cy3 concentration/(cRNA concentration). 1000= Cy3 amount in pmol per μ g cRNA. the hybridized samples were normalized to 1.65 μ g s, specific activity was >9.0pmol Cy3 per μ g cRNA.

Hybridization, scanning and feature extraction

The oligomeric gene expression arrays were hybridized using the gene expression hybridization kit from Agilent technologies (Santa Clara, Calif.) with a wash buffer kit. The hybridization was carried out in a fully automated TECANHS4800PRO (TECAN, research and slide, NC) hybridization workstation. After a slide prehybridization step at 65 ℃ for 30 seconds, the hybridization mixture was injected and incubated with agitation for 17 hours at 65 ℃. The slides were then rinsed with AgilentGEWash #1 for 1 minute at 37 ℃, followed by a second rinse back with AgilentGEWash #2 for 1 minute at 30 ℃, and a final drying step with nitrogen gas for 2 minutes 30 seconds at 30 ℃. The slide was immediately scanned to minimize the effect of environmental oxides on signal intensity.

The array was scanned using an Agilent G2565CA microarray scanner (Agilent technologies, Santa Clara, Calif.) with SureScan high resolution technology. The operational flow for scanning each array defines the dye channel, the scan area and resolution, the TIFF file dynamic range, the PMT gain parameters, and the final image result settings. Once the array has been scanned, a Feature Extraction (FE) operation is then performed, using the parameters defined for placement and fit of the optimized grid, finding fixed point, weak outliers, calculating background bias, errors and proportions, and calculating quality control indicators. After the scanning and feature extraction procedures are completed, a TIFF file containing Cy3 images is generated along with quality control pointer reports and a final file (TXT) containing all raw data. The image file (TIFF) is used to check the general quality of the slides, incorporate the location (four corners) and intensity of the contrast present in the pair, and also to confirm the success of the hybridization, washing, scanning, and feature extraction processes. The FE Quality Control (QC) report provides coefficient of variation values that allow for the incorporation of designed positive and negative (prokaryotic versus artificial sequences) spiked controls as provided by agilent technologies (santa clara, CA) as the basis for measuring data diversity. The report also provides information about data distribution, uniformity, background, reproducibility, sensitivity, and general quality of the data. The TXT file containing all raw data was uploaded to GeneSpring (Agilent, santa clara, CA) for further analysis.

Data normalization and statistical analysis

After the scanning and feature extraction operation flow, the raw data file is uploaded to GeneSpringGX version 10.0.2(Agilent technologies, Santa Clara, Calif.) and a project is created, each array data file is defined as a sample and appropriate parameter values are specified. Samples with the same parameter values are processed with duplicates. How well defined samples are grouped with experimental conditions and used for interpretation of imaging and analysis data. Quality control of the samples was performed based on previously defined incorporation controls, parameters and interpretations to ensure data quality prior to starting the analysis and reported by GeneSpring presentation quality control pointers.

Data were normalized using the ensemble percentile shift normalization method to minimize systematic non-biological differences and normalize the arrays for cross-alignment. The algorithm converts the signal strength to a base 2 logarithm and arranges in increasing order, calculates the ranking 75^thPercentiles and subtracting this value from each logarithmic transformed signal intensity, yielding normalized intensity values. Data is filtered by selecting entities labeled Present (Present) and excluding entities labeled Marginal (Marginal) or missing (Absent) per single sample in the study. The filtered and normalized entity list is used as input for statistical analysis using TwoWayANOVA Method for correcting p-value critical value p<0.05, define DAP and genotype as parameters. Determines the expression of each newly introduced gene.

Results

The total RNA concentration and also the values obtained by labeling and amplifying the cRNA are optimized. Furthermore, the values of post-amplification concentration, efficiency of labeling with Cy3, and specific activity required for consistent and reliable results were excellent. Quality Control (QC) reports provided by the feature extraction protocol following each individual array scan provide coefficient of variation values for measuring data diversity on a positive and negative spiked control basis. All values obtained from the report show the best quality of data distribution, homogeneity, background, reproducibility and sensitivity. The GeneSpring (Agilent technologies, Santa Clara, Calif.) sample quality control index report used during this study provided important statistics to assist in evaluating reproducibility and reliability of the data obtained. The reported values for the technical duplicate array (3 per sample) groups are all within range and indicate that the data obtained is reliable (data not shown).

The original values defined for the reported homozygote (table 17) ("DAP" for post-pollination days) and blank (table 18) lines at each of the six time points during seed development represent the signal intensity values remaining after all Feature Extraction (FE) processing steps, including background subtraction and multiplicative negation, if necessary, have been completed. On the other hand, normalized values for homozygous (table 19) and null (table 20) lines have been processed using the global percentile shift normalization method responsible for technical variation to minimize systematic abiotic differences and normalize the arrays for cross-alignment.

The original (fig. 12) and normalized (fig. 13) numerical plots of the blank lines obtained at each time point during seed development confirmed that the genes were not present in the ω -9Nexera710 untransformed lines and thus no significant expression was detected. In the event 5197[14] -032.002 line shown in FIG. 14 (original) and FIG. 15 (normalized), a general trend was observed in which the accumulation of all gene transcripts progressively increased as seed development progressed. The first significant increase in transcript accumulation occurred during the 15 and 30DAP periods and reached maximum levels at DAP 42. The raw curve shown in FIG. 14 provides a visual representation of the relative hybridization intensity values obtained for each gene studied, while the normalized curve summarized in FIG. 15 represents-to minimize the general trend of the gene expression profile obtained from the systematic non-biological variation versus normalized cross-array alignment.

Example 12

Expression of algal PUFA synthase gene kits using alternative promoters

The use of additional transcriptional regulatory units to express the gene(s) encoding the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI proteins may in turn increase the DHA content of the canola. Identification and use of transcriptional regulatory units that are expressed early in development and for longer periods of time increases the level of DHA in canola seeds by promoting transcription of heterologous genes at early stages of seed development (e.g., 15-25 DAP) and thus prolonging DHA production. Examples of such transcriptional regulatory regions include, but are not limited to, the LfKCS3 promoter (U.S. Pat. No. 7,253,337), the FAE1 promoter (U.S. Pat. No. 6,784,342), and the ACP promoter (WO 1992/18634). The promoters are used alone or in combination to drive the expression of the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI expression cassettes previously described in the following plasmids; pDAB7361, pDAB7362, and pDAB 7363. Methods for replacing transcriptional regulatory regions within plasmids are well known in the art. To this end, the polynucleotide fragment comprising PvDlec2 promoter v2 was removed from pDAB7361, pDAB7362, or pDAB7363 (or the aforementioned plasmids used to construct pDAB7361, pDAB7362, or pDAB 7363) and replaced with the LfKCS3 or FAE1 promoter region. The newly created plasmid line was used to stably transform canola plants. Transgenic canola plants were isolated and assayed for molecular characteristics. The resulting LC-PUFA accumulation was determined and canola plants producing 0.01% to 15% DHA or 0.01% to 10% EPA were identified.

Construction of pDAB9166

The pDAB9166 plasmid (FIG. 26; SEQ ID NO:46) was constructed using a multiple site gateway L-R recombination reaction. pDAB9166 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains the LfKCS3 promoter v1, szpufaforfavav 3, and atu orf233' UTRv 1. The second PUFA synthase PTU contains the LfKCS3 promoter v1, SzPUFAOrfBv3 and AtuOrf233' UTRv 1. The third PUFA synthase PTU contains LfKCS3 promoter v1, hSzThPUFAOrfCv3 and AtuORF233' UTRV 1. The phosphopantetheinyl transferase PTU contains LfKCS3 promoter v1, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB9161, pDAB9162, pDAB9163, pDAB101484 and pDAB7333 were recombined to form pDAB 9166. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9167

The pDAB9167 plasmid (FIG. 27; SEQ ID NO:47) was constructed using a multiple site gateway L-R recombination reaction. pDAB9167 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains the LfKCS3 promoter v1, szpufaforfavav 3, and atu orf233' UTRv 1. The second PUFA synthase PTU contains the BoACP promoter v1, BoACP5'UTRv1, szpufaforfbv 3 and AtuOrf233' UTRv 1. The third PUFA synthase PTU contains LfKCS3 promoter v1, hSzThPUFAOrfCv3 and AtuORF233' UTRV 1. The phosphopantetheinyl transferase PTU contains a BoACP promoter v1, BoACP5'UTRV1, NoHetIV3 and AtuORF233' UTRV 1.

Plasmids pDAB9161, pDAB9165, pDAB9163, pDAB101485 and pDAB7333 were recombined to form pDAB 9167. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7379

pDAB7379 is a binary plasmid constructed to contain a reconstituted, codon optimized version of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, andNoHetI. The SzACS-2 gene sequence was not included in this construct. The pDAB7379 plasmid (FIG. 28; SEQ ID NO:48) was constructed using the multiple site gateway L-R recombination reaction.

pDAB7379 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5'UTR, szpufaforfava 3, and atu orf233' UTRv 1. The second PUFA synthase PTU contains PvPhas promoter v3, PvPhas5'UTR, SzPUFAOrfBv3 and AtuORF233' UTRV 1. The third PUFA synthase PTU contains PvPhas promoter v3, PvPhas5'UTR, hSztHPUFAorfCv3 and AtuORF233' UTRV 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v3, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB7371, pDAB7372, pDAB7373, pDAB7374 were recombined with pDAB7333 to form pDAB 7379. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7380

pDAB7380 is a binary plasmid constructed to contain reconstructed, codon-optimized versions of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, and NoHetI. The SzACS-2 gene sequence is not included in this construct. The version of the phaseolin promoter used in this construct is modified essentially as described in Butoseal, 1989(the plant cell, Vol.1; 839-853), in which the 5 'part of the promoter is truncated and the 5' untranslated region of phaseolin is intact. The pDAB7380 plasmid (FIG. 29; SEQ ID NO:49) was constructed using a multiple site gateway L-R recombination reaction.

pDAB7380 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, szpufaforfava 3, and atu orf233' UTRv 1. The second PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, SzPUFAOrfBv3 and AtuORF233' UTRV 1. The third PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, hSztHPUFAorfCv3 and AtuORF233' UTRV 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v5, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB7375, pDAB7376, pDAB7377, pDAB7378 were recombined with pDAB7333 to form pDAB 7380. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9323

pDAB9323 is a binary plasmid constructed to contain the native, non-codon optimized version of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, SzACS-2, and NoHetI. The pDAB9323 plasmid (FIG. 30; SEQ ID NO:50) was constructed using a multiple site gateway L-R recombination reaction.

pDAB9323 contains three PUFA synthase PTUs, an acyl-CoA synthase PTU, a phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 2, PvPhas3' UTRv1 and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzPUFAOrfBv2, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). The third PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzPUFAOrfCv2, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). The acyl-CoA synthetase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzACS-2v2 gene, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). Phosphopantetheinyl transferase PTU contains PvPhas promoter v3, PvPhas5' UTR, NoHetIv2, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map).

Plasmids pDAB9307, pDAB9311, pDAB9315, pDAB9322 were recombined with pDAB7333 to form pDAB 9323. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv2, SzPUFAOrfBv2, SzPUFAOrfCv2, NoHetIv 2. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing six PTUs were then isolated and tested for incorporation of the six PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9330

pDAB9330 is a binary plasmid constructed to contain a re-constructed, codon-optimized version of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, SzACS-2 and NoHetI. The pDAB9330 plasmid (FIG. 31; SEQ ID NO:51) was constructed using a multiple site gateway L-R recombination reaction. pDAB9330 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 3, PvPhas3' UTRv1 and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzPUFAOrfBv3, PvPhas3' UTR and PvPhas3' MARv2 (not shown in the plasmid map). The third PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, hSzThPUFAOrfCv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). The acyl-CoA synthetase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzACS-2v3 gene, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). Phosphopantetheinyl transferase PTU contains PvPhas promoter v3, PvPhas5' UTR, NoHetIv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map).

Plasmids pDAB9324, pDAB9325, pDAB9326, pDAB9329 were recombined with pDAB7333 to form pDAB 9330. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, SzACS-2v3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing six PTUs were then isolated and tested for incorporation of the six PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9337

pDAB9337 is a binary plasmid constructed to contain reconstructed, codon-optimized versions of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, and NoHetI, the expression of which is driven by the phaseolin promoter. The pDAB9337 plasmid (FIG. 32; SEQ ID NO:52) was constructed using a multiple site gateway L-R recombination reaction.

pDAB9337 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 3, PvPhas3' UTRv1 and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzPUFAOrfBv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). The third PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, hSzThPUFAOrfCv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). Phosphopantetheinyl transferase PTU contains PvPhas promoter v3, PvPhas5' UTR, NoHetIv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map).

Plasmids pDAB9324, pDAB9325, pDAB9326, pDAB9328 and pDAB7333 were recombined to form pDAB 9337. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9338

pDAB9338 is a binary plasmid constructed to contain a reconstructed, codon-optimized version of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, and NoHetI. The phaseolin promoter was used to drive the expression of SzPUFAOrfA, while the PvDlec2 promoter was used to drive other transgenes. The pDAB9338 plasmid (FIG. 33; SEQ ID NO:53) was constructed using a multiple site gateway L-R recombination reaction.

pDAB9338 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 3, PvPhas3' UTRv1 and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB9324, pDAB7335, pDAB7336, pDAB7338 were recombined with pDAB7333 to form pDAB 9338. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9344

pDAB9344 is a binary plasmid constructed to contain reconstructed, codon-optimized versions of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, and NoHetI, all of which contain ribulose bisphosphate carboxylase small strand 1A (labeled SSU-TPv1) ligated to the amino terminus of the coding sequence. The phaseolin promoter is used to drive the expression of SzPUFAOrfA, and the PvDlec2 promoter is used to drive other transgenes.

The pDAB9344 plasmid (FIG. 34; SEQ ID NO:54) was constructed using a multiple site gateway L-R recombination reaction. pDAB9344 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 4, PvPhas3' UTRv1 and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzPUFAOrfBv4, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). The third PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, hSzThPUFAOrfCv4, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). Phosphopantetheinyl transferase PTU contains PvPhas promoter v3, PvPhas5' UTR, NoHetIv4, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map).

Plasmids pDAB9343, pDAB9342, pDAB9340, pDAB9331 were recombined with pDAB7333 to form pDAB 9344. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv4, SzPUFAOrfBv4, hsztthpufaorfcv 4, NoHetIv 4. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing six PTUs were then isolated and tested for incorporation of five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9396

pDAB9396 is a binary plasmid constructed to contain reconstructed, codon-optimized versions of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, SzACS-2, and NoHetI. The phaseolin promoter is used for driving the expression of SzPUFAOrfA and SzPUFAOrfB. The PvDlec2 promoter was used to drive other transgenes; hSzThPUFAOrfC, SzACS-2, and NoHetI.

The pDAB9396 plasmid (FIG. 35; SEQ ID NO:55) was constructed using a multiple site gateway L-R recombination reaction. pDAB9396 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 3, PvPhas3' UTRv1 and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The acyl-CoA synthetase PTU contains PvPhas promoter v3, PvPhas5' UTR, SzACS-2v3 gene, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB9324, pDAB7335, pDAB7336, pDAB7339 were recombined with pDAB7333 to form pDAB 9338. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, SzACS-2v3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of six PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB101412

pDAB101412 is a binary plasmid constructed to contain a reconstituted, codon-optimized version of SzPUFAOrfA, SzPUFAOrfB, hSzThPUFAOrfC, SzACS-2, and NoHetI. The version of the phaseolin promoter used in this construct is modified essentially as described in Butoseal, 1989(the plant cell, Vol.1; 839-853), in which the 5 'part of the promoter is truncated and the 5' untranslated region of phaseolin is intact. The truncated phaseolin promoter sequences are identified in the present application as version 4(v4), version 5(v5), and version 6(v 6). The pDAB101412 plasmid (FIG. 36; SEQ ID NO:56) was constructed using a multiple site gateway L-R recombination reaction.

pDAB101412 contains three PUFA synthase PTUs, an acyl-CoA synthase PTU, a phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, szpufaforfava 3, and atu orf233' UTRv 1. The second PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, SzPUFAOrfBv3 and AtuORF233' UTRV 1. The third PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, hSztHPUFAorfCv3 and AtuORF233' UTRV 1. The acyl-CoA synthetase PTU contains PvPhas promoter v4, PvPhas5' UTR,2S5' UTR, SzACS-2v3 gene and AtuORF235' UTRV 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v5, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB7375, pDAB7376, pDAB7377, pDAB7398 were recombined with pDAB7333 to form pDAB 101412. Specifically, the five PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hSzThPUFAOrfCv3, SzACS-2v3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of six PTUs by restriction enzyme digestion and DNA sequencing.

Arabidopsis transformation with promoters expressed early in seed development

These plasmids were used to stably transform canola plants using the above procedure. Transgenic canola plants were isolated and assayed for molecular characteristics. Alternative constructs were used to generate canola plants containing greater amounts of DHA and LC-PUFAs. The resulting LC-PUFA accumulation was determined and canola plants producing 0.01% to 15% DHA or 0.01% to 15% LC-PUFA were identified.

Example 13

Co-expression of DGAT2 or ACCase and algal PUFA synthase gene sets in Arabidopsis

The oil content in canola plants is further modified by transformation with a chimeric DNA molecule encoding and expressing an acetyl CoA carboxylase (ACCase) or a type 2 diacylglycerol acyltransferase (DGAT 2). Co-expression of these genes with the algal PUFA synthase genes described above by breeding of Arabidopsis plants containing the ACCase or DGAT2 expression cassette and Arabidopsis plants containing the PUFA synthase genes; or transforming a canola plant with a gene stack (stack) containing ACCase or DGAT2 and a PUFA synthase gene. The regulatory units necessary for expression of the ACCase or DGAT2 coding sequences may include those described above. Sequences may also be expressed using additional regulatory units known in the art. The ACCase and DGAT2 expression cassettes were transformed into Arabidopsis using the transformation protocol described above. Transformation can occur with a molecular stack of ACCase or DGAT2 expression cassettes in combination with PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI expression cassettes; or by independent ACCase or DGAT2 expression cassettes linked to a selectable marker and subsequently mating with a canola plant containing expression cassettes for PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI. Positive transformants were isolated and molecular characterization was determined. Identifying a canola plant comprising a plant, plant seed, or plant oil concentrate having an increased accumulation of LC-PUFAs as compared to an untransformed control canola plant. Such increases may range from 1.2 to 20 fold.

Overexpression of ACCase in the cytoplasm can produce higher levels of malonyl-CoA. Canola plants or seeds containing increased levels of cytoplasmic malonyl-CoA can subsequently produce higher levels of long chain polyunsaturated fatty acids (LC-PUFAs) when algal PUFA synthase genes are present and expressed. The DGAT2 gene expressed in canola plants is capable of preferentially aligning and amplifying docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) into triglycerides. DGAT2 genes with a substrate preference for LC-PUFAs (see e.g. WO2009/085169) increase the integration of the fatty acids into Triglycerides (TAG). Such DGAT genes are useful for directing the integration of LC-PUFAs, especially DHA, into TAG and for increasing TAG production in plants and other organisms.

Example 14

Expression of higher levels of acyl-CoA synthetase in plants using native acyl-CoA synthetase gene sequences

An alternative version of an acyl-CoA synthetase gene from Schizochytrium is created by modifying the native gene sequence by removing the excess open reading frame region. This version is labeled "SzACS-2v4" and is juxtaposed to SEQ ID NO: 34. The sequence was synthesized from service provider DNA2.0 (Menlopack, CA). The coding sequences are incorporated into plant expression cassettes containing a promoter and 3' untranslated region, which are described in the examples. The resulting expression cassette was used to replace the acyl-CoA synthetase expression cassette-described above as "SzACS-2v3", seq id no:9, which was combined with PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC and 4' phosphopantetheinyl transferase HetI expression cassettes to construct pDAB7361, pDAB7362 and pDAB 7363. The novel plasmid containing the "SzACS-2v4" expression cassette was assigned a unique identification tag. The newly created plasmid line was used to stably transform canola plants. Transgenic canola plants were isolated and assayed for molecular characteristics. An alternative version of the gene "SzACS-2v4" produces canola plants containing greater amounts of DHA and LC-PUFAs. The resulting LC-PUFA accumulation was determined and canola plants producing 0.01% to 15% DHA or 0.01% to 10% EPA were identified.

Example 15

PUFA synthetase Activity of mature transgenic canola

PUFA synthase activity was detected as an extract from mature T1 transgenic canola seeds from plants produced using Agrobacterium vector pDAB7361 (event 5197[14] -032). The seeds were immersed in water for 3-4 hours and ground on dry ice in extraction buffer (200mM phosphate pH7.0, 1mM EDTA, 1mM DTT, 50mM NaCl, 5% glycerol, 1% PVPP, 0.52. mu.g/mL antinocidin, 0.58. mu.g/mL leupeptin, 0.83. mu.g/mL pepstatin A, 12. mu.g/mL LCK, 12. mu.g/mL mLTPCK, and 6. mu.g/mL soybean trypsin inhibitor) and microcentrifuged at 4 ℃ for 10min before removing the seed coat. The fat pad was removed and the resulting pellets were shake incubated with the higher ionic strength buffer prior to re-centrifugation. The fat pad and lipid layers were removed and the sample and aqueous supernatant passed through a Zeba desalting column pre-equilibrated with 50mM phosphate ph7.2, 1mM dtt, 10% glycerol, and 1mM edta. Seeds of untransformed Nexera710 were treated in parallel as a negative control group. Samples of both sets of seeds were tested using the HIP extraction and TLC method described in Metzetal, plantaphysiol. biochem.47:6(2009) (FIG. 16). The assay conditions were modified to include 2mM ADH, NADH regeneration system (glucose + glucose dehydrogenase), shaking was continued and the final malonyl-CoA concentration was 100. mu.M (0.064. mu. Ci/100. mu.L per assay). The resulting supernatant was tested normalized by volume and indicated that FFA formation could be measured after 60 min. This was not observed in the Nexera710 control, indicating that FFA formation is from DHA formation by PUFA synthases.

Example 16

OrfA pantetheination in canola by co-expressed HetI

OrfA contains nine acyl-carrying protein domains, each of which requires derivatization with a phosphopantetheinyl group by phosphopantetheinyl transferase (PPTase) to function. The degree of OrfA pantetheination by PPTaHetI in transgenic canola was assessed by assessing tryptic peptide fragments containing pantetheination sites from various OrfA samples using a nano-liquid chromatography mass spectrometer (nanolC-MS).

Recombinant holo and apoOrfA polypeptide standard lines were made in E.coli by co-expression with or without HetI. Expression of OrfA in the absence of HetI results in a nonfunctional protein, since the endogenous PPTAses of E.coli are unable to add pantetheinyl groups (Hauvermalee)tal, Lipids41: 739-; 2006). Conversely, expression in conjunction with HetI results in a highly pantetheinated OrfA protein. To extract e.coli expressed OrfA, 0.5L of frozen cells of recombinant cell culture medium were resuspended in 20mL of extraction buffer: 20mM Tris pH7.0, 1mg/mL lysozyme, 1mM EDTA, 1mM PMSF, 1mM DTT, 0.52. mu.g/mL antinocidin, 0.58. mu.g/mL leupeptin, 0.83. mu.g/mL pepstatin A, 12. mu.g/mL LCK, 12. mu.g/mL PCK, 6. mu.g/mL soybean trypsin inhibitor. After cleavage with Dnase and 4mMMgCl ₂The extracts were processed, clarified by centrifugation and the supernatant frozen at-80 ℃.

Extraction from event 5197[14 ] Using the aforementioned extraction methods]-032.002 OrfA produced by a rehydrated mature canola plant isolate for use in an in vitro test for PUFA synthase produced by canola. OrfA proteins from both E.coli standards and Arabidopsis thaliana were enzymatically digested and analyzed by nanolC-MS using an Agilent ChipCube nano-chromatographic inlet together with MS analysis by an Agilent QTOF mass spectrometer (model 6530). Automation of QTOF programming MS²Analysis to generate peptide sequence data during chromatography. The method is mainly characterized in that the mass spectrometer is programmed to perform full-scan MS scanning and then perform automatic MS scanning on three ions with the most abundant content²To generate MS²Sequence spectrum. Ions were then removed from the MS after 2 passes occurred²Removal, exclusion time 30 sec. The internal reference is continuously implanted during the nanospray to generate reference ions (at m/z299.29446 and 1221.99064) for QTOF internal calibration. Ions commonly found from calibration inventory residues are defined as exclusion ions to avoid spurious MS of the plasma²And (6) scanning. MS scanning is performed and ranges from m/z 295-2400. MS (Mass Spectrometry) ²The scanning is performed and is in the range of m/z 59-3000. Performing automated MS²The following sequence of charge states is preferred: +2>+3>(>+3)>Is unknown>+1。

The tandem germplasm line was extracted with MascotDistiller (matrix science, London UK; version 2.3.2). Charge state deconvolution and deisotopic are not performed. All MS/MS spectra were analyzed using Mascot (matrix science, London, UK; version 2.2.06) and X! Tandem (www.thegpm.org; version 2007.01.01.1). Mascot and X! Tandem are both set to search a protein sequence database containing the full-length sequence of the OrfA protein exhibiting trypsin digestion specificity. Mascot and X! Tandem are searched with a fragment ion mass tolerance of 0.30Da and a parent ion tolerance of 10.0 ppm. Oxidation of methionine and phosphopantetheine of serine were specified as variable modifications in Mascot and X! Tandem.

Scaffold (version Scaffold-2-05-02, proteome software, Portland, OR) was used to verify MS/MS-based peptide and protein recognition. Peptide recognition is accepted if it can be established with greater than 95.0% probability as indicated by the peptide Prophet algorithm (kelleret al, anal. chem.74:5383-92 (2002)). Protein recognition is accepted if it can be established with a probability greater than 99.0% and contains at least 2 recognized peptides. Protein probability is assigned by the protein Prophet algorithm (Nesvizhskii, AnalChem.75:4646-58 (2003)). Proteins containing similar peptides and which cannot be distinguished by MS/MS analysis alone were grouped to fit the principles of brevity. The database searched for the apo form of tryptic peptides corresponding to pantetheinated site 1(SEQ ID NO:78TGYETDMIEADMELETELGIDSIK) and pantetheinated site 29(SEQ ID NO:77 TGYETDMIESDMELETELGIDSIK). No direct evidence of pantetheinated peptides was observed.

To estimate the degree of pantetheinization of OrfA sites 2-9 isolated from canola, the amount of apo2-9 peptide compared to six different reference peptides in other regions of the OrfA molecule was measured (Table 21).

TABLE 21 peptides used to calculate the relative amount of apo2-9 peptide in the OrfA cleavage section. "Start" refers to the starting position of a specified peptide in a full-length protein. The apo2-9 start position refers to the first appearance of the peptide in the protein sequence. The abbreviation "z" refers to charge and the abbreviation m/z refers to mass divided by charge.

In E.coli derived proteins (without HetI), the internal ratio of apo2-9 peptide to the reference peptide was considered an estimate of no pantetheination, whereas in E.coli derived proteins expressed in synergy HetI, the internal ratio was considered an estimate of high pantetheination. These internal ratios assume equal molar abundance of the reference peptide, regardless of the origin of the OrfA protein (fig. 17). The ratio of apo2-9 peptide to each of the six reference peptides was calculated and averaged. (three ratios of six reference peptides were calculated.) furthermore, three ratios of six reference peptides relative to each other (ref1/ref2, ref3/ref4 and ref5/ref6) were calculated to confirm that the reference peptides did not differ significantly between the three OrfA samples (FIG. 17) and are suitable for calculating the relative amount of apo2-9 peptide present.

In contrast to the calculated ratios for the reference peptides, the ratios of apo2-9 to each of the reference peptides showed-compared to the OrfA standard without HetI-significantly lower levels of apo2-9 peptide in both the OrfA/HetI and mustard isoforms (fig. 18). A brief explanation of these results is that the pantetheinated sites on the apo2-9 peptide are substantially occupied by phosphopantetheinyl groups, thereby significantly reducing the molar abundance of the apo2-9 peptide. It is indicated that the Arabidopsis-expressed PPTase, HetI are functionally capable of activating OrfA of transgenic canola, and that the Arabidopsis-expressed OrfAACP unit is functionally sufficient.

Example 17

Additional constructs

Introduction of promoter diversity to reduce replication of regulatory units

Gene silencing is a phenomenon that has been observed in transgenic canola events when offspring are produced. Several review articles discuss Transcriptional Gene Silencing (TGS) and post-transcriptional gene silencing (PTGS), such as Waterhouseetal, 2001(Nature411:834-842), VauchherendFagard, 2001 (Trendsinunggenetics 17(1):29-35, and Okamoto and Hirochika,2001(Trends plant Sci.6 (11): 527-534). in plants, gene silencing can be triggered by duplication of a transgenic polynucleotide sequence (a tandem repeat transgene sequence, an inverted repeat transgene sequence, or multiple chromosomal inserts) or when a sequence homologous to the target gene sequence is carried by T-DNA infecting a plant virus or Agrobacterium tumefaciens.

Furthermore, the duplication of the transgenic polynucleotide sequence may act as a trigger for construct instability. Multiple transgene sequences sharing high levels of sequence similarity can be reverse-folded onto one another. Rearrangement may occur via homologous recombination in which intervening sequences of DNA are excised. As a result, DNA fragments located between the repeated transgenic polynucleotide sequences are excised.

One strategy in designing plasmid vectors is to introduce promoter diversity into the construct by incorporating multiple unique seed-specific promoters that maintain high levels of expression for each transgene. Introduction of promoter sequence diversity into plasmid vectors can reduce gene silencing and improve plasmid stability. The multiple seed-specific promoter includes PvDlec2, phaseolin, and Napin (u.s. patent No. 5,608,152). These promoters are more similar in promoter activity such as tissue specificity, expression level, duration of expression, and the like.

Construction of pDAB7733

The pDAB7733 binary plasmid (FIG. 37; SEQ ID NO:57) was constructed using a multiple site gateway L-R recombination reaction. pDAB7733 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, szpufaforfava 3, and atu orf233' UTRv 1. The second PUFA synthase PTU contains Bnanapin promoter v1, Bnanapin C5'UTR, SzPUFAOrfBv3 and Bnanapin C3' UTRV 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v5, PvPhas5'UTR, NoHetIv3 and AtuOrf233' UTRV 1.

Plasmids pDAB7375, pDAB7731, pDAB7336, pDAB7378 were recombined with pDAB7333 to form pDAB 7733. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB7734

The pDAB7734 binary plasmid (FIG. 38; SEQ ID NO:58) was constructed using a multiple site gateway L-R recombination reaction. pDAB7734 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, SzPUFAOrfBv3 and AtuORF233' UTRV 1. The third PUFA synthase PTU contains Bnanapin promoter v1, Bnanapin C5'UTR, hSztHPUFAOrfCv3 and Bnanapin C3' UTRV 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB7334, pDAB7376, pDAB7732, pDAB7338 were recombined with pDAB7333 to form pDAB 7734. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB101493

The pDAB101493 binary plasmid (FIG. 39; SEQ ID NO:59) was constructed using a multiple site gateway L-R recombination reaction. pDAB101493 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvPhas promoter v4, PvPhas5'UTR, SzPUFAOrfBv3 and AtuORF233' UTRV 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v5, PvPhas5'UTR, NoHetIv3 and AtuOrf233' UTRV 1.

Plasmids pDAB7334, pDAB7376, pDAB7336, pDAB7378 were recombined with pDAB7333 to form pDAB 101493. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB109507

The pDAB109507 plasmid (FIG. 40; SEQ ID NO:60) was constructed using a multiple site gateway L-R recombination reaction. pDAB109507 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 3, PvPhas3' UTRv1, and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains Bnanapin promoter v1, Bnanapin C5'UTR, SzPUFAOrfBv3 and Bnanapin C3' UTRV 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The phosphopantetheinyl transferase PTU contains the BoACP promoter/5 'UTRV1, NoHetIv3 and AtuOrf233' UTRV 1.

Plasmids pDAB9324, pDAB7731, pDAB7336, pDAB101485 and pDAB7333 were recombined to form pDAB 109507. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB109508

The pDAB109508 plasmid (FIG. 41; SEQ ID NO:61) was constructed using a multiple site gateway L-R recombination reaction. pDAB109508 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvPhas promoter v3, PvPhas5' UTR, szpufaovav 3, PvPhas3' UTRv1, and PvPhas3' MARv2 (not shown on the plasmid map). The second PUFA synthase PTU contains Bnanapin promoter v1, Bnanapin C5'UTR, SzPUFAOrfBv3 and Bnanapin C3' UTRV 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB9324, pDAB7731, pDAB7336, pDAB7338 and pDAB7333 were recombined to form pDAB 109508. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB109509

The pDAB109509 plasmid (FIG. 42; SEQ ID NO:62) was constructed using a multiple site gateway L-R recombination reaction. pDAB109509 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The phosphopantetheinyl transferase PTU contains the BoACP promoter/5 'UTRV1, NoHetIv3 and AtuOrf233' UTRV 1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB101485 were recombined with pDAB7333 to form pDAB 109509. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Rearranging the order of binary constructs PTUs to reduce Long Gene sequence segmentation

Szpufaforfamptu was placed at the 3' end of the binary construct to test whether PTU cassette order could reduce fragmentation and rearrangement separating transformation events. SzPUFAOrfA is a large open reading area (8, 700 8,700b.p.) containing nine tandem acyl carrier protein repeats. In the first series of completed constructs, the szpufaforfavtu line was placed first integrated into the plant chromosome. The szpufaofaftu is then linked to the remaining PUFA synthesis-related genes PTUs, as these reduce the molecular size. Molecular analysis of the SzPUFAOrfA coding region indicated that some transformed canola and arabidopsis events contained a fragmentation insert. Alternative construct designs are described in which the order of the PUFA synthase PTUs has been changed to the following configuration; hSzThPUFAOrfCPTU, SzPUFAOrfBPTU, NoHetIPTU, SzPUFAOrfAPTU, and PATPTU. Altering the position of szpufaforfamptu on the binary construct was done to reduce fragmentation and rearrangement of the isolated transgenic event.

Construction of pDAB9151

The pDAB9151 plasmid (FIG. 43; SEQ ID NO:63) was constructed using a multiple site gateway L-R recombination reaction. pDAB9151 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfbv 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1. The final PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfva 3 and At2SSSP terminator v 1.

Plasmids pDAB9148, pDAB7335, pDAB9149, pDAB9150 were recombined with pDAB7333 to form pDAB 9151. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: hSzThPUFAOrfCv3, SzPUFAOrfBv3, NoHetIv3, SzPUFAOrfAv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF 13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Altering the direction of transcription of binary construct PTUs to introduce construct diversity

An alternative construct design involves altering the sequence of PUFA synthase PTUs and the direction of transcription of the gene expression cassettes. In the first series of completed constructs, each gene expression cassette was placed in the same orientation ("head to tail", where the promoter of one gene expression cassette was adjacent to the 3' UTR of the second gene expression cassette). The following constructs illustrate the strategy of placing gene expression cassette lines in different orientations and using alternative promoters. In these embodiments, one gene expression cassette is joined in reverse orientation to a second gene expression cassette such that the promoters of the two gene expression cassettes are engineered to be adjacent to each other. This configuration is described as a "head joint" configuration. Other configurations are described in the examples, in which one gene expression cassette is joined in reverse to a second gene expression cassette such that the 3' UTRs of the two gene expression cassettes are engineered to be adjacent to each other. This configuration is described as a "tail-to-tail" configuration. To alleviate read-through that may occur with this type of design, a bidirectional Orf23/24 terminator is placed between the two PTUs. These configurations are proposed to increase the expression of the transgene, thereby producing higher concentrations and contents of LC-PUFA and DHA fatty acids.

Construction of pDAB108207

The pDAB108207 plasmid (FIG. 44; SEQ ID NO:64) was constructed using a multiple site gateway L-R recombination reaction. pDAB108207 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v6, PvPhas5' UTR, NoHetIv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map). The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5'UTR, hsztthpufaorfcv 3, At2SSSP terminator v1 and atu orf233' UTRv 1. The third PUFA synthase PTU contains PvPhas promoter v6, PvPhas5'UTR, SzPUFAOrfBv3, PvPhas3' UTR, PvPhas3'MARv2 (not shown on the plasmid map) and AtuORF233' UTRV 1.

Plasmids pDAB7334, pDAB101489, pDAB108205, pDAB108206 and pDAB7333 were recombined to form pDAB 108207. Specifically, in the T-strand DNA border region of the plant transformation binary pDAB7333, szpufaforfava 3 and NoHetIv3 lines were placed in tail-to-tail orientation; NoHetIv3 and hSzThPUFAOrfCv3 are placed in the direction of the head joint; the hSzThPUFAOrfCv3 and SzPUFAOrfB are placed in the tail-to-tail direction. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv3, hszthwpufaorfcv 3, SzPUFAOrfBv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB108208

The pDAB108208 plasmid (FIG. 45; SEQ ID NO:65) was constructed using a multiple site gateway L-R recombination reaction. pDAB108208 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v4, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvPhas promoter v5, PvPhas5'UTR, SzPUFAOrfBv3, PvPhas3' UTR, PvPhas3'MARv2 (not shown on the plasmid map), and AtuORF233' UTRV 1.

Plasmids pDAB108200, pDAB101490, pDAB108201, pDAB108202 and pDAB7333 were recombined to form pDAB 108208. Specifically, in the T-strand DNA border region of the plant transformation binary pDAB7333, szpufaforfava 3 and NoHetIv3 lines were placed in the head-to-head orientation; NoHetIv3 and hSzThPUFAOrfCv3 are placed in a tail-to-tail orientation; the hSzThPUFAOrfCv3 and SzPUFAOrfB are placed in the direction of the head joint. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv3, hszthwpufaorfcv 3, SzPUFAOrfBv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB108209

The pDAB108209 plasmid (FIG. 46; SEQ ID NO:66) was constructed using a multiple site gateway L-R recombination reaction. pDAB108209 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v4, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains the PvPhas promoter v5, PvPhas5' UTR, SzPUFAOrfBv3, PvPhas3' UTR and PvPhas3' MARv2 (not indicated on the plasmid map), and random DNA spacers.

Plasmids pDAB108200, pDAB108204, pDAB108201, pDAB108202 and pDAB7333 were recombined to form pDAB 108209. Specifically, in the T-strand DNA border region of the plant transformation binary pDAB7333, szpufaforfava 3 and NoHetIv3 lines were placed in the head-to-head orientation; NoHetIv3 and hSzThPUFAOrfCv3 are placed in a tail-to-tail orientation; the hSzThPUFAOrfCv3 and SzPUFAOrfB are placed in the direction of the head joint. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv3, hszthwpufaorfcv 3, SzPUFAOrfBv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Doubling 3' UTRs and including spacer DNA to reduce transcriptional interference

Transcriptional interference can occur when multiple genes are stacked in a row, thereby resulting in reduced expression of downstream genes. This phenomenon is due to transcriptional read-through of the 3' UTR and terminator to the next promoter-transcriptional unit. Alternative construct designs that consist of two strategies for reducing transcriptional interference and transcriptional interference are described. The first strategy uses two terminators/3' UTRs stacked between individual DHA gene expression cassettes to limit read-through to the next gene expression cassette. The second strategy is to insert approximately one thousand pairs of spacer DNA bases between gene expression cassettes, thereby reducing transcriptional interference.

Construction of pDAB108207

The pDAB108207 plasmid (FIG. 44; SEQ ID NO:64) was constructed using a multiple site gateway L-R recombination reaction. pDAB108207 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvPhas promoter v3, PvPhas5'UTR, SzPUFAOrfBv3, PvPhas3' UTR, PvPhas3'MARv2 (not shown on the plasmid map), and AtuORF233' UTRV 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5'UTR, hsztthpufaorfcv 3, At2SSSP terminator v1 and atu orf233' UTRv 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v6, PvPhas5' UTR, NoHetIv3, PvPhas3' UTRV1 and PvPhas3' MARV2 (not shown on the plasmid map).

Plasmids pDAB7334, pDAB101489, pDAB108205, pDAB108206 and pDAB7333 were recombined to form pDAB 108207. Specifically, in the T-strand DNA border region of the plant transformation binary pDAB7333, szpufafafav 3 and NoHetIv3 lines were placed in tail-to-tail orientation and atu orf233' UTR line was placed between two PTUs; NoHetIv3 and hSzThPUFAOrfCv3 are placed in the direction of the head joint; the hSzThPUFAOrfCv3 and SzPUFAOrfB lines are placed in head-to-tail orientation and the AtuORF233' UTR line is placed between two PTUs. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv3, hszthwpufaorfcv 3, SzPUFAOrfBv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB108208

The pDAB108208 plasmid (FIG. 45; SEQ ID NO:65) was constructed using a multiple site gateway L-R recombination reaction. pDAB108208 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains PvPhas promoter v5, PvPhas5'UTR, SzPUFAOrfBv3, PvPhas3' UTR, PvPhas3'MARv2 (not shown on the plasmid map) and AtuORF233' UTRV 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v4, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB108200, pDAB101490, pDAB108201, pDAB108202 and pDAB7333 were recombined to form pDAB 108208. Specifically, in the T-strand DNA border region of the plant transformation binary pDAB7333, szpufaforfava 3 and NoHetIv3 lines were placed in the head-to-head orientation; NoHetIv3 and hSzThPUFAOrfCv3 are placed in the tail-to-tail orientation and the AtuORF233' UTR is placed between two PTUs; the hSzThPUFAOrfCv3 and SzPUFAOrfB are placed in the direction of the head joint. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv3, hszthwpufaorfcv 3, SzPUFAOrfBv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB108209

The pDAB108209 plasmid (FIG. 46; SEQ ID NO:66) was constructed using a multiple site gateway L-R recombination reaction. pDAB108209 contains three PUFA synthase PTUs, one acyl-CoA synthase PTU, one phosphopantetheinyl transferase PTU and one phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains the PvPhas promoter v5, PvPhas5' UTR, SzPUFAOrfBv3, PvPhas3' UTR, PvPhas3' MARv2 (not shown on the plasmid map), and random DNA spacers. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v4, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB108200, pDAB108204, pDAB108201, pDAB108202 and pDAB7333 were recombined to form pDAB 108209. Specifically, in the T-strand DNA border region of the plant transformation binary pDAB7333, szpufaforfava 3 and NoHetIv3 lines were placed in the head-to-head orientation; NoHetIv3 and hSzThPUFAOrfCv3 are positioned in the tail-to-tail orientation and a one thousand base pair spacer is positioned between two PTUs; the hSzThPUFAOrfCv3 and SzPUFAOrfB are placed in the direction of the head joint. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv3, hszthwpufaorfcv 3, SzPUFAOrfBv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Use of an alternative 3' UTR-terminator to limit transcriptional read-through

Due to the limited number of proprietary 3 'UTR-terminators, Agrobacterium ORF233' UTR-terminator was mainly used to terminate transcription. Recently it was shown that in Arabidopsis thaliana, the ZmLipase3' UTR-terminator line is more efficient at terminating transcriptional readthrough. To this end, one version of the construct utilized ZmLipase3 'UTR-terminator in combination with PvDlec2 promoter to test whether this 3' UTR could reduce transcriptional read-through of upstream genes, thereby reducing transcriptional interference.

Construction of pDAB9159

The pDAB9159 plasmid (FIG. 47; SEQ ID NO:67) was constructed using a multiple site gateway L-R recombination reaction. pDAB9159 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5'UTR, szpufaforfava 3, and ZmLip3' UTRv 1. The second PUFA synthase PTU contains the PvPhas promoter v3, PvPhas5'UTR, SzPUFAOrfBv3, and ZmLip3' UTRv 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5'UTR, hsztthpufaorfcv 3 and ZmLip3' UTRv 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v3, PvPhas5'UTR, NoHetIv3 and ZmLip3' UTRV 1.

Plasmids pDAB9152, pDAB9153, pDAB9154, pDAB9155 and pDAB7333 were recombined to form pDAB 9159. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB9147

The pDAB9147 plasmid (FIG. 48; SEQ ID NO:68) was constructed using a multiple site gateway L-R recombination reaction. pDAB9147 contains three PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5'UTR, szpufaforfav 3, At2SSSP terminator v1, and ZmLip3' UTRv 1. The second PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, SzPUFAOrfBv3 and At2SSSP terminator v 1. The third PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, hsztthpufaorfcv 3 and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvDlec2 promoter v2, 2S5' UTR, NoHetIv3 and At2SSSP terminator v 1.

Plasmids pDAB9146, pDAB7335, pDAB7336, pDAB7338 were recombined with pDAB7333 to form pDAB 9147. Specifically, the four PTUs lines described above were placed in a head-to-tail orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, SzPUFAOrfBv3, hsztthpufaorfcv 3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of the five PTUs by restriction enzyme digestion and DNA sequencing.

Delivery of DHA genes with two separate T-DNAs

An alternative construct design involves constructing two separate binary vectors, the first containing a subset of PUFA synthase genes on one T-DNA, and the second containing the remaining PUFA synthase genes on a second T-DNA. These binary vectors are used individually to transform sexually crossed plants, thereby producing progeny containing all PUFA synthase gene expression constructs. An alternative method for making transgenic plants is to co-transform both binary vectors into canola tissue and select or screen for a single plant containing both T-chains.

Construction of pDAB108224

The pDAB108224 plasmid (FIG. 49; SEQ ID NO:69) was constructed using a multiple site gateway L-R recombination reaction. pDAB108224 contains a PUFA synthase PTU, a phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfav 3, and At2SSSP terminator v 1. Phosphopantetheinyl transferase PTU contains PvPhas promoter v4, PvPhas5'UTR, NoHetIv3 and AtuORF233' UTRV 1.

Plasmids pDAB108216, pDAB108221 and pDAB7333 were recombined to form pDAB 108224. Specifically, szpufafaovav 3 and NoHetIv3 line were placed in head-to-head orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfAv3, NoHetIv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of three PTUs by restriction enzyme digestion and DNA sequencing.

Construction of pDAB108225

The pDAB108225 plasmid (FIG. 50; SEQ ID NO:70) was constructed using a multiple site gateway L-R recombination reaction. pDAB108225 contains two PUFA synthase PTUs and an phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains PvDlec2 promoter v2, 2S5' UTR, szpufaforfbv 3, and At2SSSP terminator v 1. The second PUFA synthase PTU contains the PvPhas promoter v4, SzPUFAOrfBv3 and AtuORF233' UTRV 1.

Plasmids pDAB108217, pDAB108222 and pDAB7333 were recombined to form pDAB 108225. Specifically, the SzPUFAOrfBv3 and hSzThPUFAOrfCv3 lines were placed in the head-adapter orientation within the T-strand DNA border region of the plant transformation binary pDAB 7333. The gene sequence is as follows: SzPUFAOrfBv3, hszthpufaofcv 3. pDAB 7333-contains, among other regulatory units, e.g. overdrive and T-strand border sequences (T-DNA border a and T-DNA border B) -also phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PATv5, AtuORF 13' UTRV 4. Recombinant plasmids containing five PTUs were then isolated and tested for incorporation of three PTUs by restriction enzyme digestion and DNA sequencing.

Transformation of canola with constructs containing alternative designs

These plasmids were used to stably transform canola plants using the above procedure. Transgenic canola plants were isolated and assayed for molecular characteristics. Alternative constructs were used to generate canola plants containing greater amounts of DHA and LC-PUFAs. The resulting LC-PUFA accumulation was determined and canola plants producing 0.01% to 15% DHA or 0.01% to 15% LC-PUFA were identified.

Example 18

Alternative construct design for Arabidopsis thaliana transformation and subsequent LC-PUFA and DHA manufacture

Arabidopsis plants were transformed with Agrobacterium tumefaciens strains containing a pDAB101493, pDAB7362, pDAB7369, pDAB101412, or pDAB7380 binary vector. The floral dip transformation protocol described by CloughandDrent (1998) was used for transformation. Cloughandparent, "floralclip: asimplifiedmethodformingrobacterium-mediatetransmormationof Arabidopsis, "planta J.,16:735-743, 1998. Transformed arabidopsis plants were obtained and molecular confirmation of the presence of the transgene was accomplished. Generation of T from transgenic Arabidopsis events₁Plants were grown to maturity in the greenhouse. The plants are self-pollinated and the resulting T is collected at maturity₂And (5) seeds. Single seed analysis via FAMEsGC-FID to determine T₂LC-PUFA and DHA content in Arabidopsis seeds. Tissues were analyzed by the FAMEsGC-FID method described in the previous examples. Arabidopsis thaliana plant T₁Plant single T₂The seeds contain 0.00% to 0.95% DHA and 0.00% to 1.50% total LC-PUFA. Individual T₁Respective T of plants₂The LC-PUFA and DHA content of the seeds is shown in fig. 51.

Example 19

Transformation of a non-high oleic canola variety with a PUFA synthase genome (DH12075)

Rape variety DH12075 was transformed with agrobacterium tumefaciens comprising plasmid pDAB7362 by an embryonic axis transformation method essentially as described in example 4. Unlike the genetic background of Nexera710, DH12075 is not a "high oleic" variety. Recovering T having the appearance of pat Gene ₀DH12075 plants were analyzed for the presence of all five DHA genomes (PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthase and 4' phosphopantetheinyl transferase HetI) by the molecular analysis method described in example 5. Event 001-₀A plant. Growing it to maturity in a growth chamber and collecting T₁And (5) seeds. Single T of event 006 was analyzed as described in example 6₁Seeds showed that 31 of the 48 seeds analyzed contained between 0A level of DHA between 19% and 0.86% DHA. Make 113T₁Seeds were planted, grown in growth chambers and leaf tissue samples were analyzed as described in example 4 to determine the zygosity of individual plants. qPCR analysis determined 23 plants to be homozygous PAT genes and also showed a common separation of five DHA genes, indicating the presence of a single locus. T for event 006 using pat and OrfA probes₁Southern analysis of plant tissues indicated the presence of one additional copy of the OrfA gene. Homozygous plants were grown to maturity and seeds were harvested. Batch T from each of the plants₂FAME analysis of seed samples showed 23 homozygous T₂Lines 17 in the plants produced LC-PUFAs with a DHA content between 0.17 and 0.72%. Five T ₂The seed sample contained between 0.08% and 0.16% EPA, all LC-PUFA of the LC-PUFA production event (DHA + EPA + DPA [ n-6 ]]) Is between 0.33% and 1.35%. Table 22a shows two DHA-containing batches T of event 006₂Complete fatty acid profile of the sample. For eight homozygous T₁48 individual T's of the line₂Seeds were analyzed for single seeds and the average DHA content of the seeds is shown in table 23. The single T with the DHA content of 1.31 percent is detected₂And (5) seeds. Table 22b shows four DHA containing Ts₂Complete fatty acid configuration of the seeds. The data show that DHA can be produced in canola with a genetic background of less than 72% oleic acid content via transformation with a PUFA synthase genome.

TABLE 23 eight homozygous events 006T with DH12075 Gene background₁T of canola plant₂Average DHA content of seeds (48 seeds analyzed per plant).

The foregoing description of the invention has been presented for purposes of illustration and description. Again, this description is not intended to limit the invention to the form disclosed herein.

The various aspects, embodiments, and options described herein can be combined in any and all variations.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims (17)

1. A process for the manufacture of an oil comprising at least one PUFA, which process comprises recovering a PUFA from a crude oil

Comprises the following steps:

a first nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase system that produces at least one PUFA, wherein the PUFA synthase system comprises the amino acid sequences of SEQ ID NOs:1-3, wherein the first nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1;

a second nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) of seq id No. 5 that transfers a phosphopantetheinyl cofactor to an ACP domain of a PUFA synthase system, wherein the second nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1; and

a third nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) of SEQ ID NO. 4, which catalyzes the conversion of a long chain PUFA Free Fatty Acid (FFA) to acyl-CoA, wherein the third nucleic acid sequence is operably linked to a promoter selected from the group consisting of: genetically modifying the rape plants, progeny, cells, tissues, seeds or parts thereof to recover oil, such as PvDlec2, LfKCS3 and FAE1,

wherein the recovered oil comprises 0.19% to 1.6% DHA (docosahexaenoic acid (C22:6, n-3)), 0.09% to 0.34% DPA (C22:5, n-6 or n-3), and 0.05% to 0.27% EPA (eicosapentaenoic acid (C20:5, n-3)).

2. A process for the manufacture of an oil comprising at least one PUFA, which process comprises contacting

Comprises the following steps:

a first nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase system that produces at least one PUFA, wherein the PUFA synthase system comprises the amino acid sequences of SEQ ID NOs:1-3, wherein the first nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1;

a second nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) of seq id No. 5 that transfers a phosphopantetheinyl cofactor to an ACP domain of a PUFA synthase system, wherein the second nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1; and

a third nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) of SEQ ID NO. 4, which catalyzes the conversion of a long chain PUFA Free Fatty Acid (FFA) to acyl-CoA, wherein the third nucleic acid sequence is operably linked to a promoter selected from the group consisting of: genetically modified oilseed rape plants, progeny, cells, tissues, seeds or parts thereof, of PvDlec2, LfKCS3 and FAE1,

wherein the oil comprises 0.19% to 1.6% DHA (docosahexaenoic acid (C22:6, n-3)), 0.09% to 0.34% DPA (C22:5, n-6 or n-3) and 0.05% to 0.27% EPA (eicosapentaenoic acid (C20:5, n-3)).

3. A process for the manufacture of at least one PUFA in a seed oil, the process comprising recovering a PUFA from a feedstock

Comprises the following steps:

a first nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase system that produces at least one PUFA, wherein the PUFA synthase system comprises the amino acid sequences of SEQ ID NOs:1-3, wherein the first nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1;

a second nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) of seq id No. 5 that transfers a phosphopantetheinyl cofactor to an ACP domain of a PUFA synthase system, wherein the second nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1; and

a third nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) of SEQ ID NO. 4, which catalyzes the conversion of a long chain PUFA Free Fatty Acid (FFA) to acyl-CoA, wherein the third nucleic acid sequence is operably linked to a promoter selected from the group consisting of: genetically modifying seed recovered oil of a rape plant, progeny, cells, tissues or parts thereof with PvDlec2, LfKCS3 and FAE1,

wherein the recovered oil comprises 0.19% to 1.6% DHA (docosahexaenoic acid (C22:6, n-3)), 0.09% to 0.34% DPA (C22:5, n-6 or n-3), and 0.05% to 0.27% EPA (eicosapentaenoic acid (C20:5, n-3)).

4. A process for the manufacture of at least one PUFA in a seed oil, the process comprising contacting

Comprises the following steps:

a first nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase system that produces at least one PUFA, wherein the PUFA synthase system comprises the amino acid sequences of SEQ ID NOs:1-3, wherein the first nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1;

a second nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) of seq id No. 5 that transfers a phosphopantetheinyl cofactor to an ACP domain of a PUFA synthase system, wherein the second nucleic acid sequence is operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3, and FAE 1; and

a third nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) of SEQ ID NO. 4, which catalyzes the conversion of a long chain PUFA Free Fatty Acid (FFA) to acyl-CoA, wherein the third nucleic acid sequence is operably linked to a promoter selected from the group consisting of: genetically modified oilseed rape plants, progeny, cells, tissues, seeds or parts thereof, of PvDlec2, LfKCS3 and FAE1,

wherein the seed oil comprises 0.19% to 1.6% DHA (docosahexaenoic acid (C22:6, n-3)), 0.09% to 0.34% DPA (C22:5, n-6 or n-3) and 0.05% to 0.27% EPA (eicosapentaenoic acid (C20:5, n-3)).

5. A method of producing a genetically modified oilseed rape plant, progeny, cell, tissue, seed or part thereof which produces a genetically modified oilseed rape plant comprising 0.19% to 1.6% DHA (docosahexaenoic acid (C22:6, n-3)), 0.09% to 0.34% DPA (C22:5, n-6 or n-3) and 0.05% to 0.27% EPA (eicosapentaenoic acid (C20:5, n-3)) oil, the method comprising transforming the oilseed rape plant or plant cell with the following nucleic acid sequences: a nucleic acid sequence encoding an algal PUFA synthase that produces at least one polyunsaturated fatty acid (PUFA), wherein the PUFA synthase system comprises the amino acid sequences of SEQ ID NOs: 1-3; a nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) of seq id No. 5 that transfers a phosphopantetheinyl cofactor to an algal PUFA synthase ACP domain; and a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) according to SEQ ID NO. 4, which catalyzes the conversion of long chain PUFA Free Fatty Acids (FFA) to acyl-CoA.

6. An oil obtained from a genetically modified oilseed rape plant, progeny, cell, tissue, seed or part thereof by the method of any one of claims 1 to 4, wherein the oil comprises 0.19% to 1.6% DHA (docosahexaenoic acid (C22:6, n-3)), 0.09% to 0.34% DPA (C22:5, n-6 or n-3) and 0.05% to 0.27% EPA (eicosapentaenoic acid (C20:5, n-3)).

7. A seed oil obtained from the genetically modified canola plant, progeny, cell, tissue, seed, or part thereof produced by the method of claim 5.

8. A food product comprising the oil of claim 6 or 7.

9. A functional food comprising the oil of claim 6 or 7.

10. A pharmaceutical product comprising the oil of claim 6 or 7.

11. The method of claim 5, wherein the oilseed rape plant or plant cell is transformed with the nucleic acid sequence of SEQ ID NO 35 or SEQ ID NO 36.

12. The method of any one of claims 1-5, wherein the nucleic acid sequence encoding the PUFA synthase system comprises:

a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO. 6;

a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO. 7; and

a nucleic acid sequence which is at least 80% identical to the nucleic acid sequence of SEQ ID NO. 8.

13. The method of any one of claims 1-5 and 12, wherein the nucleic acid sequence encoding PPTase is at least 80% identical to the nucleic acid sequence of seq id No. 10.

14. The method of any one of claims 1-5, 12, and 13, wherein the nucleic acid sequence encoding ACoAS comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence of seq id No. 9.

15. The method of any one of claims 1-5, 12 and 13, wherein the nucleic acid sequence encoding ACoAS comprises the nucleic acid sequence of seq id No. 34.

16. The method of any one of claims 1-5, 12, 13 and 15, wherein the nucleic acid sequence encoding the PUFA synthase system, the nucleic acid sequence encoding the PPTase and the nucleic acid sequence encoding the ACoAS are comprised in a single recombinant expression vector.

17. The method of any one of claims 1-5, 12, 13, 15 and 16, wherein the genetically modified oilseed rape plant, progeny, cells, tissue, seed or part thereof comprises a nucleic acid sequence encoding an acetyl CoA carboxylase (ACCase) and/or a nucleic acid sequence encoding a type 2 diacylglycerol acyltransferase (DGAT 2).