WO1994010189A1 - Plant fatty acid synthases - Google Patents

Abstract

By this invention, compositions and methods of use related to β-ketoacyl-ACP synthase, hereinafter also referred to as 'synthase', are provided. Also of interest are methods and compositions of amino acid and nucleic acid sequences related to biologically active plant synthase(s) factors. Amino acid and nucleic acid for synthase protein factors are provided, as well as methods to utilize such sequences in constructs for production of genetically engineered plants having altered fatty acid compositions. In addition, uses of non-plant synthase proteins in plant genetic engineering methods are also considered.

Description

PLANT FATTY ACID SYNTHASES

This application is a continuation-in-part of USSN 07/971,182 filed on November 2, 1992.

Field of Invention

The present invention is directed to synthase enzymes relevant to fatty acid synthesis, amino acid and nucleic acid sequences related thereto, and methods of using such compositions in plants.

Introduction

Background Plant oils are used in a variety of industrial and edible uses. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources, are needed. Depending upon the intended oil use, various different fatty acid compositions are desired.

For example, in some instances having an oilseed with a higher ratio of oil to seed meal would be useful to obtain a desired oil at lower cost. This would be typical of a high value oil product. In some instances, having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content. In other uses, edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardio-vascular health reasons. And alternatively, temperate substitutes for high saturate tropical oils such as palm and coconut, would also find uses in a variety of industrial and food applications.

One means postulated to obtain such oils and/or modified fatty acid compositions is through the genetic engineering of plants. However, in order to genetically engineer plants one must have in place the means to transfer genetic material to the plant in a stable and heritable manner. Additionally, one must have nucleic acid sequences capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of such sequences, and the like. Moreover, it should be appreciated that in order to produce a desired phenotype requires that the Fatty Acid Synthetase (FAS) pathway of the plant is modified to the extent that the ratios of reactants are modulated or changed.

Higher plants appear to synthesize fatty acids via a common metabolic pathway. In developing seeds, where fatty acids attached to triglycerides are stored as a source of energy for further germination, the FAS pathway is located in the proplastids. The first step is the formation of acetyl-ACP (acyl carrier protein) from acety-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACP transacylase (ATA) . Elongation of acetyl-ACP to 16- and 18- carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a β-ketoacyl-ACP (β-ketoayl-ACP synthase) , reduction of the keto-function to an alcohol (β- ketoacyl-ACP reductase) , dehydration to form an enoyl-ACP (β-hydroxyacyl-ACP dehydrase) , and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl- ACP reductase) . β-ketoacyl-ACP synthase I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas β-ketoacyl- ACP synthase II catalyzes the final elongation to stearoyl- ACP (C18:0) . Common plant unsaturated fatty acids, such as oleic, linoleic and α-linolenic acids found in storage triglycerides, originate from the desaturation of stearoyl- ACP to form oleoyl-ACP (C18:l) in a reaction catalyzed by a soluble plastid Δ-9 desaturase (also often referred to as "stearoyl-ACP desaturase"). Molecular oxygen is required for desaturation in which reduced ferredoxin serves as an electron co-donor. Additional desaturation is effected sequentially by the actions of membrane bound Δ-12 desaturase and Δ-15 desaturase. These "desaturases" thus create mono- or polyunsaturated fatty acids respectively. A third β-ketoacyl-ACP synthase has been reported in S. oleracea leaves having activity specific toward very short acyl-ACPs. This acetoacyl-ACP synthase or "β- ketoacyl-ACP" synthase III has a preference to acetyl-CoA over acetyl-ACP, Jaworski, J.G. , et al . , Plant Phys . (1989) 50:41-44. It has been postulated that this enzyme may be an alternate pathway to begin FAS, instead of ATA.

Obtaining nucleic acid sequences capable of producing a phenotypic result in FAS, desaturation and/or incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of a protein source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants.

Thus, the identification of enzyme targets and useful plant sources for nucleic acid sequences of such enzyme targets capable of modifying fatty acid compositions are needed. Ideally an enzyme target will be amenable to one or more applications alone or in combination with other nucleic acid sequences, relating to increased/decreased oil production, the ratio of saturated to unsaturated fatty acids in the fatty acid pool, and/or to novel oils compositions as a result of the modifications to the fatty acid pool. Once enzyme target (s) are identified and qualified, quantities of protein and purification protocols are needed for sequencing. Ultimately, useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and plants containing such constructs are needed.

Brief Description of the Figures

Figure 1 provides cDNA and translated amino acid sequences of a 50kD R. communis synthase factor B gene. Preliminary cDNA sequence and the corresponding translational peptide sequence derived from the cDNA clone, pCGN2765 (2-8) , which encodes the 50kD synthase protein is shown. The cDNA includes both the postulated transit peptide sequence (amino acids 1-42) and the sequence encoding the mature protein.

Figure 2 provides the R . communis synthase factor B 2-8 sequence with additional 3' untranslated sequence. Figure 3 provides cDNA and translated amino acid sequences of a J?. communis 46kD synthase factor A gene.

Figures 4 and 5 provide cDNA and translated amino acid sequences of Brassica synthase factor B genes.

Figure 4 provides sequences of the cDNA insert of pCGN3248.

Figure 5 provides sequences of clone 4A. Figure 6 provides cDNA sequence of a Brassica synthase factor A gene. Comparison of the translated amino acid sequence to the R. communis factor A sequence reveals a possible frame shift mutation in the region near nucleotide 1120.

Figure 7 provides translated amino acid sequence of nucleotides 79-1119 of the Brassica synthase A gene sequence shown in Fig. 6. Figure 8 provides translated amino acid sequence of nucleotides 1127-1606 of the Brassica synthase A gene sequence shown in Fig. 6.

Figure 9 provides approximately 2 kb of genomic sequence of Bce4. Figure 10 provides a cDNA sequence and the corresponding translational peptide sequence derived from C. tinctorius desaturase. The cDNA includes both the plastid transit peptide sequence (amino acids 1-33) and the sequence encoding the mature protein.

Summary of the Invention By this invention, compositions and methods of use related to β-ketoacyl-ACP synthase, hereinafter also referred to as "synthase", are provided. Also of interest are methods and compositions of amino acid and nucleic acid sequences related to biologically active plant synthase(s) . Various plant synthase factor A and B proteins are described in WO 92/03564 which is hereby incorporated by reference in its entirety. As is described herein, synthase III constructs for expression in plant cells may also be used, either alone, or in conjunction with other plant synthase or fatty acid biosynthesis gene sequences to provide enhanced oil yields and/or altered compositions of plant seed oil.

Nucleic acid sequences encoding a synthase protein required for synthase activity in a host cell may be employed in nucleic acid constructs to modulate the amount of synthase activity present in the host cell. A synthase may be produced in host cells for harvest or as a means of effecting a contact between the synthase and its substrate. Host cells include prokaryotes and/or eukaryotes. Plant host cells containing recombinant constructs encoding a synthase protein, as well as plants and cells containing modified levels of synthase protein(s) are also provided. Additional nucleic acid sequences, such as those encoding transit peptides, may also be used, particularly where a full length close for a particular synthase protein is not available or where a non-plant synthase sequence is used. In addition, nucleic acid constructs may be designed to decrease expression of endogenous synthase in a plant cell as well. One example is the use of an anti-sense synthase sequence under the control of a promoter capable of expression in at least those plant cells which normally produce the enzyme.

Additionally, one may wish to coordinate expression of a synthase protein with the expression of other synthase proteins or other introduced sequences encoding enzymes related to fatty acid synthesis. For example, coordinated expression of synthase factor A and synthase factor B may be desirable to provide optimal synthase-II type activity in plant cells. Furthermore, coordinated expression of the synthase III gene with plant synthase proteins may also be desired. Examples of other enzymes related to fatty acid synthesis which may find use in conjunction with synthase proteins include plant thioesterases, especially medium- chain thioesterases, desaturases, especially Δ-9 desaturases, and the like. When nucleic acid constructs encoding such factors are prepared for introduction into a plant cell, the transcriptional initiation regions may be different from each other.

Furthermore, uses of non-plant synthase protein sequences in plant cells are considered herein. Such sequences may be used alone or in conjunction with plant synthase proteins. For example, constructs for expression of an E. coli synthase III protein in plant cells are described. Such constructs may be modified to provide optimal codons for expression in plant cells, as well as to provide transit peptide sequences to target the synthase protein to plastids for effect on the plant fatty acid synthesis reactions.

Detailed Description of the Invention A plant synthase of this invention includes any sequence of amino acids, polypeptide, peptide fragment or other protein preparation, whether derived in whole or in part from natural or synthetic sources which demonstrates the ability to catalyze a condensation reaction between an acyl-ACP or acyl-CoA having a chain length of C₂ to Ci₆ and malonyl-ACP in a plant host cell. A plant synthase will be capable of catalyzing a synthase reaction in a plant host cell, i.e., in vivo, or in a plant cell-like environment, i.e., in vi tro . Typically, a plant synthase will be derived in whole or in part from a natural plant source. In addition, synthase from other sources such as bacteria or lower plants, may also be useful in plants and thus be considered a plant synthase in this invention. For example, the E. coli synthase protein encoded by the faJB gene is shown herein to have homology to plant synthase proteins. In E. coli , synthase I enzymatic activity is provided by a homodimer of the fabB gene product. Of particular interest is a gene for E. coli synthase III (faJH) . Constructs for expression of the bacterial gene in plant cells will include fusion constructs to incorporate chloroplast transit peptide sequences, such that the E. coli synthase III gene product is directed to the site of fatty acid synthesis. In this manner, the overall lipid yield may be increased by enhancing the first step in the

FAS pathway.

Synthase I demonstrates preferential activity towards acyl-ACPs having shorter carbon chains, C₂~Ci_4; synthase II demonstrates preferential activity towards acyl-ACPs having longer carbon chains, Ci₄~Ci₆- Synthase III demonstrates preferential activity towards acyl-CoAs having very short carbon chains, C₂ to C₆- Other plant synthases may also find applicability by this invention, including synthase III type activities.

Synthases include modified amino acid sequences, such as sequences which have been mutated, truncated, increased and the like, as well as such sequences which are partially or wholly artificially synthesized. Synthases and nucleic acid sequences encoding synthases may be obtained by partial or homogenous purification of plant extracts, protein modeling, nucleic acid probes, antibody preparations, or sequence comparisons, for example. Once purified synthase is obtained, it may be used to obtain other plant synthases by contacting an antibody specific to R . communis synthase with a plant synthase under conditions conducive to the formation of an antigen:antibody immunocomplex and the recovery of plant synthase which reacts thereto. Once the nucleic acid sequence encoding a synthase is obtained, it may be employed in probes for further screening or used in_ genetic engineering constructs for transcription or transcription and translation in host cells, especially plant host cells.

Recombinant constructs containing a nucleic acid sequence encoding a synthase and a heterologous nucleic acid sequence of interest may be prepared. By heterologous is meant any sequence which is not naturally found joined to the synthase sequence. Hence, by definition, a sequence joined to any modified synthase is not a wild-type sequence. Other examples include a synthase from one plant source which is integrated into the genome of a different plant host.

Constructs may be designed to produce synthase in either prokaryotic or eukaryotic cells. The increased expression of a synthase in a plant cell or decreased amount of endogenous synthase observed in a plant cell are of special interest. Moreover, in a nucleic acid construct for integration into a plant host genome, the synthase may be found in a "sense" or "anti-sense" orientation in relation to the direction of transcription. Thus, nucleic acids may encode biologically active synthases or sequences complementary to the sequence encoding a synthase to inhibit the production of endogenous plant synthase. By transcribing and translating a sense sequence in a plant host cell, the amount of synthase available to the plant FAS complex is increased. By transcribing or transcribing and translating an anti-sense sequence in a plant host cell, the amount of the synthase available to the plant FAS is decreased. Ideally, the anti-sense sequence is very highly homologous to the endogenous sequence. Other manners of decreasing the amount of synthase available to FAS may be employed, such as ribozymes or the screening of plant cells transformed with constructs containing sense sequences which in fact act to decrease synthase expression, within the scope of this invention. Other analogous methods may be applied by those of ordinary skill in the art.

Synthases may be used, alone or in combination, to catalyze the elongating condensation reactions of fatty acid synthesis depending upon the desired result. For example, rate influencing synthase activity may reside in synthase I-type, synthase II-type, synthase III-type or in a combination of these enzymes. Furthermore, synthase activities may rely on a combination of the various synthase factors as described in WO 92/03564.

Constructs which contain elements to provide the transcription and translation of a nucleic acid sequence of interest in a host cell are "expression cassettes" . Depending upon the host, the regulatory regions will vary, including regions from structural genes from viruses, plasmid or chromosomal genes, or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters may be employed. Among transcriptional initiation regions which have been described are regions from bacterial and yeast hosts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, including genes such as β-galactosidase, T7 poly erase, trp-lac (tac) , trp E and the like.

An expression cassette for expression of synthase in a plant cell will include, in the 5' to 3 ' direction of transcription, a transcription and translation initiation control regulatory region (also known as a "promoter") functional in a plant cell, a nucleic acid sequence encoding a synthase, and a transcription termination region. Numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable, e.g., inducible, transcription of the desaturase structural gene. Among transcriptional initiation regions used for plants are such regions associated with cauliflower mosaic viruses (35S, 19S) , and structural genes such as for nopaline synthase or mannopine synthase or napin and ACP promoters, etc. The transcription/translation initiation regions corresponding to such structural genes are found immediately 5 ' upstream to the respective start codons. Thus, depending upon the intended use, different promoters may be desired. Of special interest in this invention are the use of promoters which are capable of preferentially expressing the synthase in seed tissue, in particular, at early stages of seed oil formation. Selective modification of seed fatty acid/oils composition will reduce potential adverse effects to other plant tissues. Examples of such seed- specific promoters include the region immediately 5 ' upstream of a napin or seed ACP genes such as described in EP 0 255 378 (published 2/3/88) , desaturase genes such as described in Thompson et al ( Proc. Nat . Acad. Sci . (1991) 88 :2578-2582) , WO 92/03564 and Fig. 10 herein, or Bce-4 gene such as described in co-pending USSN 494,722, and Fig. 9 herein. Alternatively, the use of the 5' regulatory region associated with the plant synthase structural gene, i.e., the region immediately 5' upstream to a plant synthase structural gene and/or the transcription termination regions found immediately 3 ' downstream to the plant synthase structural gene, may often be desired. In general, promoters will be selected based upon their expression profile which may change given the particular application.

Sequences found in an anti-sense orientation may be found in cassettes which at least provide for transcription of the sequence encoding the synthase. By anti-sense is meant a DNA sequence in the 5' to 3' direction of transcription which encodes a sequence complementary to the sequence of interest. It is preferred that an "anti-sense synthase" be complementary to a plant synthase gene indigenous to the plant host. Any promoter capable of expression in a plant host which causes initiation of high levels of transcription in all storage tissues during seed development is sufficient. Seed specific promoters may be desired.

A DNA sequence of this invention may include genomic or cDNA sequence. A cDNA sequence may or may not contain pre-processing sequences, such as transit peptide sequences. Transit peptide sequences facilitate the delivery of the protein to a given organelle and are cleaved from the amino acid moiety upon entry into the organelle, releasing the "mature" protein (or enzyme) . As synthases are part of the FAS pathway of plastid organelles, such as the chloroplaεt, proplastid, etc., transit peptides may be required to direct the protein(s) to substrate. A transit peptide sequence from any plastid- translocating sources may be employed, such as from ACP, especially seed ACP, small subunit of ribulose bisphosphate carboxylase (RuBC) , plant desaturase or from the native sequence naturally associated with the respective synthase. The complete genomic sequence of a plant synthase may be obtained by the screening of a genomic library with a probe and isolating those sequences which hybridize thereto as described more fully below. Regulatory sequences immediately 5 ' , transcriptional and translational initiation regions, and 3', transcriptional and translational termination regions, to the synthase may be obtained and used with or without the synthase structural gene.

Other synthases and/or synthase nucleic sequences are obtainable from amino acid and DNA sequences provided herein. "Obtainable" refers to those plant synthases which have sufficiently similar sequence to that of the native sequence(s) of this invention to provide a biologically active synthase. One skilled in the art will readily recognize that antibody preparations, nucleic acid probes (DNA and RNA) and the like may be prepared and used to screen and recover synthases and/or synthase nucleic acid sequences from other sources. Thus, sequences which are homologously related to or derivations from either R . cozπmunis synthase I or II are considered obtainable from the present invention.

"Homologeously related" includes those nucleic acid sequences which are identical or conservatively substituted as compared to the native sequence. Typically, a homologously related nucleic acid sequence will show at least about 60% homology, and more preferably at least about 70% homology, between the R. communis synthase and the given plant synthase of interest, excluding any deletions which may be present. Homology is determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions.

Probes can be considerably shorter than the entire sequence, but should be at least about 10, preferably at least about 15, more preferably at least 20 or so nucleotides in length. Longer oligonucleotides are also useful, up to the full length of the gene encoding the polypeptide of interest. Both DNA and RNA can be used.

A genomic library prepared from the plant source of interest may be probed with conserved sequences from a R . communis synthase cDNA to identify homologously related sequences. Use of an entire R . communis synthase cDNA may be employed if shorter probe sequences are not identified. Positive clones are then analyzed by restriction enzyme digestion and/or sequencing. In this general manner, one or more sequences may be identified providing both the coding region, as well as the transcriptional regulatory elements of the synthase gene from such plant source. cDNA libraries prepared from other plant sources of interest may be screened as well, providing the coding region of synthase genes from such plant sources.

In use, probes are typically labeled in a detectable manner (for example with ³²P-labeled or biotinylated nucleotides) and are incubated with single-stranded DNA or RNA from the plant source in which the gene is sought, although unlabeled oligonucleotides are also useful. Hybridization is detected, typically using nitrocellulose paper or nylon membranes by means of the label after single-stranded and double-stranded (hybridized) DNA or DNA/RNA have been separated. Hybridization techniques suitable for use with oligonucleotides are well known to those skilled in the art. Thus, plant synthase genes may be isolated by various techniques from any convenient plant. Plant genes for synthases from developing seed obtained from other oilseed plants, such as C. tinctorius seed, rapeseed, cotton, corn, soybean cotyledons, jojoba nuts, coconut, peanuts, oil palm and the like are desired as well as from non-traditional oil sources, such as S. oleracea chloroplast, avocado mesocarp, Cuphea, California Bay and Euglena gracilliε. Synthases, especially synthase I, obtained from Cuphea may show specialized activities towards medium chain fatty acids. Such synthase may be of special interest for use in conjunction with a plant medium-chain thioesterase. Once the desired plant synthase sequence is obtained, it may be manipulated in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence. In addition, all or part of the sequence may be synthesized, where one or more codons may be modified to provide for a modified amino acid sequence, or one or more codon mutations may be introduced to provide for a convenient restriction'site or other purpose involved with construction or expression. The structural gene may be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like. For expression, the open reading frame coding for the plant synthase or functional fragment thereof will be joined at its 5' end to a transcriptional initiation regulatory control region. In some instances, such as modulation of plant synthase via a nucleic acid sequence encoding synthase in an anti-sense orientation, a transcription initiation region or transcription/translation initiation region may be used. In embodiments wherein the expression of the synthase protein is desired in a plant host, a transcription/translation initiation regulatory region, is needed. Additionally, modified promoters, i.e., having transcription initiation regions derived from one gene source and translation initiation regions derived from a different gene source or enhanced promoters, such as double 35S CaMV promoters, may be employed for some applications. As described above, of particular interest are those 5' upstream non-coding regions which are obtained rom genes regulated during seed maturation, particularly those preferentially expressed in plant embryo tissue, such as ACP-and napin-derived transcription initiation control regions. Such regulatory regions are active during lipid accumulation and therefore offer potential for greater control and/or effectiveness to modify the production of plant desaturase and/or modification of the fatty acid composition. Especially of interest are transcription initiation regions which are preferentially expressed in seed tissue, i.e., which are undetectable in other plant parts. For this purpose, the transcript initiation region of acyl carrier protein isolated from B. campestris seed and designated as "Beg 4-4" and a gene having an unknown function isolated from B. campestris seed and designated as "Bce-4" are also of substantial interest.

Briefly, Bce4 is found in immature embryo tissue at least as early as 11 days after anthesis (flowering) , peaking about 6 to 8 days later or 17-19 days post- anthesis, and becoming undetectable by 35 days post- anthesis . The timing of expression of the Bce4 gene closely follows that of lipid accumulation in seed tissue. Bce4 is primarily detected in seed embryo tissue and to a lesser extent found in the seed coat. Bce4 has not been detected in other plant tissues tested, root, stem and leaves.

Bce4 transcript initiation regions will contain at least 1 kb and more preferably about 5 to about 7.5 kb of sequence immediately 5' to the Bce4 structural gene.

The Beg 4-4 ACP message presents a similar expression profile to that of Bce4 and, therefore, also corresponds to lipid accumulation in the seed tissue. Beg 4-4 is not found in the seed coat and may show some differences in expression level, as compared to Bce4, when the Beg 4-4 5' non-coding sequence is used to regulate transcription or transcription and translation of a plant Δ-9 desaturase of this invention. The napin 1-2 message is found in early seed development and thus, also offers regulatory regions which can offer preferential transcriptional regulation of a desired DNA sequence of interest such as the plant desaturase DNA sequence of this invention during lipid accumulation. Napins are one of the two classes of storage proteins synthesized in developing Brassica embryos (Bhatty, et al . , Can J. Biochem. (1968) 46:1191-1197) and have been used to direct tissue-specific expression when reintroduced into the Brassica genome (Radke, et al . , Theor. Appl . Genet . (1988) 75:685-694).

As to regulatory transcript termination regions, these may be provided by the DNA sequence encoding the plant synthase or a convenient transcription termination region derived from a different gene source, especially the transcript termination region which is naturally associated with the transcript initiation region. Typically, the transcript termination region will contain at least about 1 kb, preferably about 3 kb of sequence 3 ' to the structural gene from which the termination region is derived. In developing the DNA construct, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector which is capable of replication in a bacterial host, e.g., E. coli . Numerous vectors exist that have been described in the literature. After each cloning, the plasmid may be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, insertion, resection, etc., so as to tailor the components of the desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance, with the manner of transformation of the host cell.

Normally, included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformed cells. The gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc. , complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the number of different host species into which the expression construct or components thereof are introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts. The manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. Various methods for plant cell transformation include the use of Ti- or Ri-plasmids, microinjection, electroporation, liposome fusion, DNA bombardment or the like. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation.

Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrojacterium host. The Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation) , either being permissible, so long as the vir genes are present in the transformed Agrobacterium host. The armed plasmid can give a mixture of normal plant cell and gall . A preferred method for the use of Agrojbacterium as the vehicle for transformation of plant cells employs a vector having a broad host range replication system, at least one T-DNA boundary and the DNA sequence or sequences of interest. Commonly used vectors include pRK2 or derivatives thereof. See, for example, Ditta et al . , PNAS USA, (1980) 77:7347-7351 and EPA 0 120 515, which are incorporated herein by reference. Normally, the vector will be free of genes coding for opines, oncogenes and vir- genes. Included with the expression construct and the T- DNA will be one or more markers, which allow for selection of transformed Agrojbacterium and transformed plant cells. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like. The particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.

The vector is used for introducing the DNA of interest into a plant cell by transformation into an Agrobacterium having vir-genes functional for transferring T-DNA into a plant cell. The Agrobacterium containing the broad host range vector construct is then used to infect plant cells under appropriate conditions for transfer of the desired DNA into the plant host cell under conditions where replication and normal expression will occur. This will also usually include transfer of the marker, so that cells containing the desired DNA may be readily selected.

The expression constructs may be employed with a wide variety of plant life, particularly plant life involved in the production of vegetable oils. These plants include, but are not limited to rapeseed, peanut, sunflower, C. tinctorius, cotton, Cuphea, soybean, and corn or palm.

For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed

Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils. The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.

EXAMPLES

Example 1. Analysis of Synthase Proteins

In purification of synthase proteins as discussed in WO 92/03564, synthase II activity was observed only when both the 46 and 50kD peptides were present in R . communis protein preparations, while synthase I activity was detected in preparations containing only the 50kD peptide. In addition, E. coli expression data demonstrated that both the 46kD and 50kD synthase factors (factors A and B, respectively) were required for synthase II type activity, and that synthase factor A contributes the longer chain fatty acyl substrate specificity to synthase II activity.

To determine whether synthase II activity requires two discrete proteins or a single heterodimer, covalent intramolecular bonds are introduced into the purified protein preparation and the products of this reaction identified by SDS-PAGE and Western analysis. A similar analysis is conducted with a synthase I preparation to determine if synthase I activity is provided by a single peptide, or a homodimer of the observed 50kD peptide. Twenty μg (400pmol of 46kD and 50kD peptides) of a purified synthase II preparation in "20 buffer", is combined with 40nmol of EGS (ethylene glycol is(succinimidyl succinate) ) in 10% Me2S0, to a final volume of 0.4ml. The reaction is stopped by addition of 0.045ml of 2M Tris-HCl, pH 7.0, and the protein is prepared immediately for gel electrophoresis by the addition of SDS-PAGE sample buffer containing β- mercaptoethanol. A purified preparation of synthase I is similarly treated, except that 600ng (12pmol of 50kD peptide) is combined with 8.3nmol of EGS in a volume of 85 μl. This reaction is stopped by addition of 9μl of 2M

Tris-HCl, pH 7.0. The crosslinked proteins are analyzed by SDS-PAGE, Western transfer and antibody blotting. Each of the two active sites on a molecule of EGS can form a covalent bond with any available amino group of the studied proteins, resulting in linkage of the two amino groups across an EGS bridge. With synthase II, at room temperature incubations of 10 or 30 minutes in from 0.ImM to lOmM EGS, only two major and two minor species of crosslinked proteins are formed. These proteins are observed to migrate on SDS-PAGE at about 124 and 107kD, suggesting that the proteins are dimerε. By Western analysis, all crosslinked products react positively with antibodies raised against both the 46 and 50kD peptides, indicating that both peptides are present in all products of crosslinking. The appearance of more than one dimer could reflect different conformations of the dimers depending on the number and the locations of intramolecular bond formations. Only after prolonged incubation, or with higher concentrations of EGS, are multimeric protein species formed. These results provide additional evidence that the synthase II protein is a heterodimer of the 46 and 50kD subunits. When synthase I protein is subjected to the same reactions, one major and two minor products are formed, each of which has a mobility of about 116kD. All three of these products react with antibody raised against the 50kD peptide, but not with antibodies raised against the 46kD peptide. These results suggest that the protein having synthase I activity is a homodimer of the 50kD peptide.

Example 2. Synthase Gene Sequence

The preparation of a cDNA libraries, using the methods as described in Alexander, et al . (Methods in Enzymology (1987) 154:41-64), and.the screening of the cDNA libraries for synthase cDNA clones are described in WO 92/03564. Sequences of plant synthase factor proteins A and B which have been shown to be required for synthase II activitiy are provided herein in Figures 1-8. It is noted that synthase factor B protein is also required for plant synthase I type activity (WO 92/03564) Sequence of an E. coli synthase III gene is found in Tsay et. al . ( J. Biol . Chem. (1992) 257:6807-6814)

Example 3. Expression Cassettes

In this example, expression cassettes suitable for insertion of synthase genes are described.

Expression cassettes utilizing 5 ' -upstream sequences and 3 ' -downstream sequences of genes preferentially expressed during seed development can be constructed from isolated DNA sequences of genes with an appropriate expression pattern. Examples of genes which are expressed during seed development in Brassica are a napin gene, 1-2, and an ACP gene, Bcg4-4, both described in European Patent Publication EP 0 255 378, and a Bce4 gene, as described below. The napin gene encodes a seed storage protein that is preferentially expressed in immature embryos which are actively producing storage proteins. The ACP gene encodes a protein which is an integral factor in the synthesis of fatty acids in the developing embryo and is preferentially expressed during fatty acid synthesis. Bce4 is a gene that produces a protein of unknown function that is preferentially expressed early in embryo development, at about 15-19 days post-anthesis, and is also detectable as early as 11 days post-anthesis. The sequence of Bce4 is shown in Figure 9. DNA sequences that control the expression of these genes can be isolated and sufficient portions of the 5 ' and

3 ' regulatory regions combined such that a synthase gene inserted between these sequences will be preferentially expressed early in seed development. This expression pattern will allow the synthase gene to affect fatty acid synthesis, which also occurs early in seed development.

For example, a 1.45 kb Xhol fragment containing 5' sequence and a 1.5 kb Sstl/Bglll fragment containing 3' sequence of the Bcg4-4 ACP gene can be combined in an ACP expression cassette using a variety of available DNA manipulation techniques. Similarly, a napin expression cassette can be prepared that contains approximately 1.725 kb of 5' sequence from an EcoRV site to immediately before the ATG start codon and approximately 1.25 kb of 3' sequence from an Xhol site approximately 18 bases past the TAG stop codon to a 3 ' Hindlll site of a 1-2 napin gene. A Bce4 expression cassette can be made by combining approximately 7.4 kb of 5' DNA sequence from an upstream PstI site to immediately before the ATG start codon with approximately 1.9 kb of 3' sequences from immediately after the TAA stop codon to a 3 ' PstI site.

Variations can be made in these expression cassettes such as increasing or decreasing the amounts of 5 ' and 3 ' sequences, combining the 5' sequences of one gene with the 3' sequences of a different gene (for example using the 1.3 kb 5' sequences of napin 1-2 with the 1.5 kb 3' sequences of ACP Bcg4-4 in an expression cassette) , or using other available 3' regulatory sequences, as long as these variations result in expression cassettes that allow for expression of the inserted synthase gene at an appropriate time during seed development.

A. Napin Seed Specific Expression Cassettes

1. Napin 1-2 pCGN1808 Expression Cassette An expression cassette utilizing 5' upstream sequences and 3 ' downstream sequences obtainable from B . campestris napin gene can be constructed as follows.

A 2.7 kb Xhol fragment of napin 1-2 (See, Figure 2 of EP 0 255 378, published February 3, 1988) containing 5' upstream sequences is subcloned into pCGN789 (a pUC based vector with the normal polylinker replaced by the synthetic linker which encodes the restriction digestion sites ScoRI,

Sail , Bglll , PstI , Xhol , BamHI , Hindlll ) and results in pCGN940. The majority of the napin coding region of pCGN940 was deleted by digestion with Sail and religation to form pCGN1800. Single-stranded DNA from pCGN1800 was used in an in vi tro mutagenesis reaction (Adelman et al. ,

DNA (1983) 2:183-193) using a synthetic oligonucleotide which inserted .EcoRV and Ncol restriction site at the junction of the promoter region and the ATG start codon of the napin gene. An appropriate mutant was identified by hybridization to the oligonucleotide used for the mutagenesis and sequence analysis and named pCGN1801. A 1.7 kb promoter fragment was subcloned from pCGN1801 by partial digestion with EcoRV and ligation to pCGN786 (a pCGN566 (polylinker in opposite orientation as pCGN565 described in WO 92/03564) chloramphenicol based vector with the synthetic linker described above in place of the normal polylinker) cut with BcoRI and blunted by filling in with DNA Polymerase I Klenow fragment to create pCGN1802.

A 2.1 kb Sail fragment of napin 1-2 containing 3' downstream sequences is subcloned into pCGN7δ9 (described above) and results in pCGN941. pCGN941 is digested with Xhol and Hindlll and the resulting approximately 1.6 kb of napin -3 ' sequences are inserted into Xhol-HindiII digested pCGNlδ02 to result in pCGNlδ03. In order to remove a 326 nucleotide Hindlll fragment inserted opposite to its natural orientation, as a result of the fact that there are 2 Hindlll sites in pCGNlδ03, the pCGN1803 is digested with Hindlll and religated. Following religation, a clone is selected which now contains only 1.25 kb of the original 1.6 napin 3' sequence. This clone, pCGN1808 is the napin 1-2 expression cassette and contains 1.725 kb of napin promoter sequences and 1.265 kb of napin 3' sequence with the unique cloning sites Sail, Bgll, PstI and Xhol in between. 2. Napin 1-2 pCGN3223 Expression Cassette Alternatively, pCGN1808 may be modified to contain flanking restriction sites to allow movement of only the expression sequences and not the antibiotic resistance marker to binary vectors such as pCGN1557 (McBride and Summerfelt, supra) . Synthetic oligonucleotides containing Jpnl, NotI and Hindlll restriction sites are annealed and ligated at the unique Hindlll site of pCGΝ1808, such that only one Hindlll site is recovered. The resulting plasmid, pCGN3200 contains unique Hindlll, NotI and Kpnl restriction sites at the 3 '-end of the napin 3 ' -regulatory sequences as confirmed by sequence analysis.

The majority of the napin expression cassette is subcloned from pCGΝ3200 by digestion with Hindlll and Sad and ligation to Hindlll and Sad digested pIC19R (Marsh, et al . (1984) Gene 32:481-465) to make pCGN3212. The extreme 5 ' -sequences of the napin promoter region are reconstructed by PCR using pCGN3200 as a template and two primers flanking the Sad site and the junction of the napin 5'- promoter and the pUC backbone of pCGN3200 from the pCGNlδOδ construct. The forward primer contains C al, Hindlll, NotI , and Kpnl restriction sites as well as nucleotides 408-423 of the napin 5 '-sequence (from the EcoRV site) and the reverse primer contains the complement to napin sequences 718-739 which include the unique Sad site in the 5' -promoter. The PCR was performed using a Perkin

Elmer/Cetus thermocycler according to manufacturer's specifications. The PCR fragment is subcloned as a blunt- ended fragment into pUC8 (Vieira and Messing (1982) Gene 15:259-268) and digested with Hindi to give pCGΝ3217. Sequence of pCGN3217 across the napin insert verifies that no improper nucleotides were introduced by PCR. The napin 5-sequences in pCGN3217 are ligated to the remainder of the napin expression cassette by digestion with Clal and Sad and ligation to pCGN3212 digested with Clal and SacI. The resulting expression cassette pCGN3221, is digested with

Hindlll and the napin expression sequences are gel purified away and ligated to pIC20H (Marsh, supra) digested with Hindlll. The final expression cassette is pCGN3223, which contains in an ampicillin resistant background, essentially identical 1T725 napin 5' and 1.265 3' regulatory sequences as found in pCGN1808. The regulatory regions are flanked with Hindlll, NotI and Jpnl restriction sites and unique

Sail, Bglll, PstI, and Xhol cloning sites are located between the 5 ' and 3 ' noncoding regions.

B. Bce4 Expression Cassette

An expression cassette for seed specific expression can also be constructed from Bce4 gene sequences, such as those represented in Figure 9. Genomic clones having regulatory sequences of the Bce4 gene may be isolated from a Brassica campestris genomic library using Bce4 sequences as probe. For example, an approximately 20 kb BamHI fragment is isolated and designated as clone P1C1. The approximately 20 kb insert of clone P1C1 is released by BamHI digestion and inserted into the BamHI site of the binary vector pCGΝ1547 (see below), producing pCGN1853. The PstI fragment of pCGN1853, containing the Bce4 gene, is inserted into the PstI site of pUClδ (Norrander, et al . (1983) Gene 25:101-106), producing pCGN1857. The plasmid pCGNlδ57 was deposited with the ATCC, Rockville, MD on

March 9, 1990, accession number 6δ251. The Clal fragment of pCGN1657, containing the Bce4 gene is ligated into Clal digested Bluescript KS+ (Stratagene; La Jolla, CA) , producing pCGNlδ64. Single stranded DNA is made from pCGN1864 and altered by in vi tro mutagenesis as described by Adelman et al . (DNA (1983) 2:183-193) using oligonucleotides having homology to Bce4 sequences 5 ' and 3 ' of the translated start and stop codons and also coding for restriction digest sites. The resulting plasmid, pCGN1866, contains Xhol and BamHI sites (from BCE45P) immediately 5 ' to the Bce4 start codon and BamHI and S al sites (from BCE43P) immediately 3' to the Bce4 stop codon. The Clal fragment of pCGNlδ66, containing the mutagenized sequences, is inserted into the Clal site of pCGN2016 (described below), producing pCGNlδ66C. The Clal fragment of pCGN1866C is used to replace the corresponding wild-type Clal fragment of pCGN1867 (described below) to produce pCGN166δ. Bce4 coding sequences are removed by digestion of pCGNlδδδ with BamHI and recircularization of the plasmid to produce pCGN1870. The Bce4 expression cassette, pCGN1870, contains 7.4 kb of 5' regulatory sequence and 1.9 kb of 3 ' regulatory sequence derived from the Bce4 genomic clone separated by the cloning sites, Xhol , BamHI , and Smal.

PCGN1867

The BamHI and Smal sites of pUC18 (Norrander et al . , (1983) supra) are removed by BamHI-Smal digestion and recircularization of the plasmid, without repair of the ends, to produce pCGN1862. The PstI fragment of pCGNlδ57, containing the Bce4 gene, is inserted into the PstI site of pCGN1862 to produce pCGN1867. PCGN2016 The multiple cloning sites of pUC12-Cm (Buckley, K. , Ph.D. Thesis, UCSD, CA (1985)) are replaced by those of pUC18 to produce pCGN565. The Hhal fragment of pCGN565, containing the chloramphenicol resistance gene is excised, blunted by use of mung bean nuclease, and inserted into the EcoRV site of Bluescript KS- (Stratagene; La Jolla, CA) to create pCGN2008. The chloramphenicol resistance gene of pCGN200δ is removed by BcoRI-Hindlll digestion. After treatment with Klenow enzyme to blunt the ends, the fragment carrying the chloramphenicol resistance gene is inserted into the Dral site of Bluescript KS-, replacing the ampicillin resistance gene of Bluescript KS-, to produce pCGN2016. C. ACP Expression Cassette

An expression cassette utilizing 5 ' -upstream sequences and 3 ' -downstream sequences obtainable from B. campestris ACP gene can be constructed as follows. A 1.45kb Xhol fragment of Beg 4-4 containing 5 ' -upstream sequences is subcloned into the cloning/sequencing vector Bluescript÷ (Stratagene Cloning Systems, San Diego, CA) . The resulting construct, pCGNl941, is digested with Xhol and ligated to a chloramphenicol resistant Bluescript M13+ vector, pCGN2015 digested with Xhol. pCGN2015 is described in WO 92/05364. This alters the antibiotic resistance of the plasmid from penicillin resistance to chloramphenicol resistance. The chloramphenicol resistant plasmid is pCGN1953. 3 ' -sequences of Beg 4-4 are contained on an Sstl/Bglll fragment cloned in the Sstl/BamHI sites of M13 Bluescript÷ vector. This plasmid is named pCGN1940. pCGN1940 is modified by in vitro site-directed mutagenesis (Adelman et al . , DNA (1963) 2:163-193) using the synthetic oligonucleotide (SEQ ID NO:51) 5'-

CTTAAGAAGTAACCCGGGCTGCAGTTTTAGTATTAAGAG-3 ' to insert Smal and PstI restriction sites immediately following the stop codon of the reading frame for the ACP gene 18 nucleotides from the SstI site. The 3 ' -noncoding sequences from this modified plasmid, pCGNl950, are moved as a PsI-Smal fragment into pCGN1953 cut with PstI and Smal. The resulting plasmid pCGN1977 comprises the ACP expression cassette with the unique restriction sites EcoRV, EcoRI and PstI available between the 1.45kb 5' and 1.5 kb of 3'- noncoding sequences for the cloning of genes to be expressed under regulation of these ACP gene regions.

Example 4. Synthase Constructs A. Preparation of Plant Transformation Vectors

Synthase cDNA sequences can be inserted in expression cassettes containing plant regulatory regions using a variety of DNA manipulation techniques. In this manner, synthase constructs in either the sense or anti-sense orientation are prepared. If convenient restriction sites are present in the synthase clones, they may be inserted into the expression cassette by digesting with the restriction endonucleases and ligation into the cassette that has been digested at one or more of the available cloning sites. If convenient restriction sites are not available in the clones, the DNA of either the cassette or the synthase gene(s), can be modified in a variety of ways to facilitate cloning of the synthase gene(s) into the cassette. Examples of methods to modify the DNA include by PCR, synthetic linker or adaptor ligation, in vitro site- directed mutagenesis (Adelman et al . , supra), filling in or cutting back of overhanging 5' or 3 ' ends, and the like. These and other methods of manipulating DNA are well known to those of ordinary skill in the art. The fragment containing the synthase gene in the expression cassette, 5' sequences/synthase/3 ' sequences, is then cloned into a binary vector, such as described by McBride and Summerfelt (Pi .Mol . Biol . (1990) 14:269-276), for Agrobacterium transformation. Other binary vectors are known in the art and may also be used for synthase cassettes. The binary vector containing the expression cassette and the synthase gene is transformed into Agrojbacterium tumefaciens, such as strain EHA101 (Hood, et al . , J. Bacteriol . (1986) 158:1291-1301) as per the method of Holsters, et al . , (Mol . Gen. Genet . (1978) 153:181-187), and used to generate transformed plants as described in Example 5. B. Synthase Factor A Constructs 1. Sense orientation

Constructs containing sense synthase sequences under the control of plant regulatory regions for expression in plant cells may be prepared as follows. The R . communis synthase factor A cDNA, 1-1A, is altered by in vitro mutagenesis to insert a BamHI restriction site at the 5' end of the cDNA insert and Xhol and Smal sites immediately 3' of the translation stop codon. The resulting construct, pCGN2781, is digested with BamHI and Xhol and ligated into Bglll and Xhol digested pCGN3223, the above described napin expression cassette, resulting in pCGN2765. The napin/factor A/napin region of pCGN27δ5 is obtained by digestion with Asp71δ and ligated into Asp718 digested pCGN1557 (McBride et al. ; supra) , resulting in pCGN2767. 2. Antisense Orientation. For antisense synthase A, a BamHI/EcoRV fragment of the B . campestris synthase factor A cDNA clone, pCGN4300 (nucleotides 218-1535) , is treated to create blunt ends, and subcloned into the EcoRV site of the ACP expression cassette, pCGN1977, to create pCGN4304. A KpnI/Xbal fragment containing the ACP 5 ' /antisense Brassica factor A/ACP 3' fragment is inserted into KpnI /Xbal digested binary vector pCGN1557 (McBride and Summerfelt, supra) resulting in plant transformation construct pCGN4306. To insert the Brassica synthase factor A cDNA clone into a napin cassette, the BamHI/EcoRV fragment of pCGN4300

(nucleotides 218-1535) is blunted and ligated into the

Bglll-digested napin cassette, pCGN3223, which has been similarly treated to provide a blunt ended DNA molecule.

The resulting plasmid is pCGN4305. An Asp718 fragment containing the napin 5 '/antisense Brassica A/napin 3' fragment is subcloned into binary vector pCGN1557 to produce a construct for plant transformation, pCGN4324. 3. Transit Fusion Constructs.

Due to the possibility that the R . communis factor A cDNA clone does not encode the entire transit peptide, DNA constructs are prepared to fuse the transit peptide encoding region from the Brassica factor A cDNA clone (including the V-A-A-C-M-S conserved region) to the mature peptide encoding region from the R. communis factor A clone. The constructs are designed such that the encoding region for the transit peptide and first 24 amino acids of the mature R. communis protein (lysine residue at nucleotides 365-367 of sequence shown in Figure 3 is the presumed N-terminus) is replaced with the corresponding region from the Brassica clone.

Two different 5' regions of the Brassica factor A cDNA are obtained by PCR. The "short" version includes nucleotides 79-423 of the sequence provided in Figure 6. This region encodes from the first methionine residue of the Brassica factor A cDNA to the histidine residue at position 115. The "long" version includes nucleotides 7- 423 of the sequence shown in Figure 6, and thus includes a portion of the factor A 5' untranslated region. Each of these Brassica synthase factor A fragments also contains Sail and EcoRV restriction sites at their 5' and 3' ends, respectively, which were provided in the oligonucleotide primers used in the PCR. The insertion of the 3' EcoRV site alters the codon for the 116 Asp from "GAC" to "GAT". An J?. communis synthase factor A DNA fragment, containing the encoding region for amino acids 145-540 of the sequence shown in Figure 3, and a stop codon, is also obtained by PCR. The coding sequence "GATATC" is chosen for the Asp'and J e amino acids at positions 145-146 in order to provide an EcoRV site at the 5 ' end of this fragment. The PCR primers are designed such that an Xhol site is inserted at the 3 ' end of this fragment immediately following the "TGA" stop codon.

The Brassica and R . communis synthase factor A fragments are fused by ligation of their respective 3 ' and 5' EcoRV sites. The fusion fragment is obtained by digestion with Sail and Xhol and ligated into the napin expression cassette, pCGN3223. The construct containing the "short" Brassica factor A is designated pCGN4313, and the construct containing the "long" Brassica factor A is designated pCGN4314. The Kpnl fragments containing the napin 5 '/synthase A fusion/napin 3' fragments are subcloned into binary vector pCGN1557 to produce vectors for plant transformation. The "short" Brassica factor A construct is designated pCGN4319, and the "long" Brassica factor A construct is designated pCGN4320.

A third fusion construct with the mature R . communis synthase factor A is prepared which incorporates the transit peptide encoding sequence of the C. tinctorius desaturase shown in Figure 10. A DNA fragment encoding amino acids 121-540 of Figure 3 (the mature R . communis synthase factor A and the asparagine residue immediately N- terminal to the mature peptide) is obtained by PCR. In addition, the sequence, CCATGGCC is included in the forward oligonucleotide primer and added at the 5' terminus of this fragment, such that an Ncol restriction site precedes the synthase sequence, and methionine and alanine residues are added to the synthase peptide encoding region. An Xhol restriction site is provided immediately following the stop codon at the 3 ' terminus.

The C. tinctorius desaturase cDNA clone shown in Figure 10, pCGN2754, is modified by PCR to insert PstI, Smal and Xhol sites to flank the coding region. The PCR product is digested with PstI and ligated to pUC8 (Vieira and Messing (1982) Gene 15:2359-268) digested with PstI to produce pCGN3220. The large NcoI/SacI fragment of pCGN3220 containing the pUC8 vector and the C. tinctorius desaturase cDNA sequence 5 ' to the Ncol site and 3 ' to the Sad site is gel purified and ligated to the gel-purified NcoI/SacI internal fragment of pCGN2754 resulting in pCGN3222. The coding region of the C. tinctorius desaturase from pCGN3222 is cloned into the pCGN3223 napin cassette by digestion with Xhol and ligation to pCGN3223 digested with Xhol and Sail, resulting in pCGN3229. pCGN3229 is digested with Ncol and Xhol to remove the mature desaturase encoding region. The R . communis synthase factor A fragment described above is digested with Ncol and Xhol and ligated to the Ncol /Xhol digested PCGΝ3229. This results in pCGN4308, the napin 5 ' /desaturase:synthase A/napin 3' fusion construct. pCGN4308 is digested with Asp718 and subcloned into binary vector pCGN1557 to produce plant transformation construct pCGN4318. C. • S nthase III Constructs

Fusion constructs of the bacterial synthase III encoding sequence and various plant transit peptide encoding sequences may be prepared. These constructs are used for generation of transgenic plants, wherein the bacterial synthase is incorporated into the chloroplasts for interaction with the plant fatty acid synthesis enzymes. A fusion of the Brassica ACP transit peptide encoding sequence from a B. rapa (formerly campestris) seed ACP cDNA (Rose et al. (1987) Nuc . Acids Res . 15:7197) and the β- ketoacyl-acyl carrier protein synthase III gene (fa H) from E. coli K-12 (Tsay et al. (1992) J. Biol Chem. 257:6807- 6814) , is prepared as follows. The B. rapa ACP transit peptide encoding region plus the 5 ' untranslated sequence is obtained by PCR, wherein the oligonucleotide primers are designed such that an BamHI site is added immediately 5' to the Xhol site at the 5' end of the B. rapa cDNA clone, and an Nhel site is inserted immediately 3' to the cysteine codon at the 3' end of the transit peptide encoding region. The faJbH encoding region is obtained by PCR from E. coli DNA, with oligonucleotide primers designed such that an Nhel site is inserted immediately 5 ' to the Ν-terminal methionine codon, and Xhol and Smal sites are inserted immediately 3' to the TAG stop codon. The Nhel site adds an alanine and serine encoding region immediately 5 ' to the faJbH N-terminal methionine. The ACP and synthase III fragments are obtained by ligation at the inserted Nhel restriction sites. The

ACP/synthase III fusion fragment is inserted into an appropriate cassette containing plant regulatory regions.

For example, for regulation under the napin regulatory regions, the ACP/synthase III fragment is obtained by digestion with Bglll and Xhol and ligated into pCGN3223. Similarly, constructs wherein the fusion ACP/synthase III fragment is positioned for control under various other plant regulatory regions may be obtained. Other regulatory regions of interest include the Bce4 and ACP regions for seed expression, as well as 35S, double 35S, or T-DNA promoter regions (such as mas and nos) , to provide for constitutive expression in various plant tissues. Constitutive expression may be desirable to test for uptake into chloroplasts of the synthase protein produced by such constructs, for example by electroporation into plant protoplasts and Western analysis. Constitutive expression is also useful for analysis of effects of the expression of synthase in various plant tissues, such as leaves, roots and stems.

Additional ACP/synthase III fusion constructs may be prepared which include various portions of the ACP mature protein encoding region in addition to the ACP transit peptide encoding region. For example, a fusion containing the B. rapa ACP transit encoding sequence plus coding sequence for an additional 12 amino acids of the mature ACP protein is prepared. The faJbH encoding region is obtained by PCR from E. coli DNA, with oligonucleotide primers designed such that an Ddel site, "CTAAG" is inserted immediately 5' to the N-terminal methionine codon, and Xhol and Smal sites are inserted immediately 3 ' to the TAG stop codon. The B. rapa ACP clone contains a Ddel site within the codons for amino acids 11-12 (Ser-Lys) of the mature protein region. Thus, the B. rapa ACP transit plus 12 fragment is obtained by digestion with Ddel and an appropriate site 5' to the ATG start codon. This fragment is ligated to the synthase III fragment at the Ddel site to form the ACP transit + 12/synthase III fusion. This fusion is. inserted into a napin expression cassette by digestion with Xhol and ligation to Xhol digested pCGN3223. As discussed above, additional constructs for transcriptional control of the synthase III fusion under various other plant regulatory elements may be similarly prepared. In addition to the ACP transit peptide discussed above, various other plant transit peptides are known in the art, and may be used in a similar manner. For example, the Brassica synthase A transit peptide used above in R . communis synthase fusion constructs, may also find use in conjunction with the bacterial synthase III. Similarly, other known transit peptides, such as those for SSU, stearoyl ACP desaturase and other nuclear encoded chloroplast proteins may be substituted for the ACP transit peptide. D. Constructs Containing More than One Synthase Gene

If more than one synthase gene is required to obtain an optimum effect in plants, the genes may be expressed under regulation of the same promoter, or alternatively under regulation of two different promoters that are preferentially expressed in developing seeds, such as the napin, ACP, and Bce4 sequences described above. The constructs may then be introduced into plants in the same binary vector, or introduced simultaneously in different binary vectors. For example, for expression of both synthase factors A and B in plant cells, a construct is prepared where R . communis synthase factor A and R . communis synthase factor B genes are each under the control of napin regulatory regions in the same binary vector. The napin/factor A/napin region of pCGN2785 is obtained by digestion with Asp718 and ligated into Asp718 digested pCGN1557 (McBride et al . ; supra) , resulting in PCGN2787. pCGN2787 is digested at the unique PstI site and treated with T4 polymerase to fill in the 3' overhang, and digested with calf intestinal alkaline phosphatase to dephosphorylate the 5 ' termini to prevent self-ligation of pCGN2787. The napin/factor B/napin region of pCGN2786 is obtained by digestion with Hindlll and the Klenow fragment of DNA polymerase to provide a blunt-ended DNA fragment, which is then ligated to the T4 polymerase blunt-ended pCGN2787 DNA. The resulting construct, pCGN2797, contains the R. communis synthase factors A and B, each positioned for expression from a napin promoter region.

Example 5. Plant Transformation

In this example, an AgroJbacterium-mediated plant transformation is described and Brassica napus is exemplified. Also, a DNA-bombardment plant transformation is described and peanut transformation is exemplified. A. Agrobacterium Mediated Transformation

Transformation of Brassica species is described by Radke et al . ( Theor. Appl . Genet . ( 1988 ) 75:685-694; Plant Cell Reports (1992) 11:499-505) . Seeds of Brassica napus cv. Delta are soaked in 95% ethanol for 2 min, surface sterilized in a 1.0% solution of sodium hypochlorite containing a drop of Tween 20 for 45 min. , and rinsed three times in sterile, distilled water. Seeds are then plated in Magenta boxes with 1/lOth concentration of Murashige minimal organics medium (Gibco) supplemented with pyrodoxine (50 μg/1) , nicotinic acid (50 μg/1) , glycine (200 μg/1), and 0.6% Phytagar (Gibco) pH 5.8. Seeds are germinated in a culture room at 22°C in a 16 h photoperiod with cool fluorescent and red light of intensity approximately 65 μEinsteins per square meter per second (μEm-²S^_1) .

Hypocotylε are excised from 7 day old seedlingε, cut into pieces approximately 4 mm in length, and plated on feeder plates (Horsch et al . 1985) . Feeder plates are prepared one day before use by plating 1.0 ml of a tobacco suspension culture onto a petri plate (100x25 mm) containing about 30 ml MS salt base (Carolina Biological) 100 mg/1 inoεitol, 1.3 mg/1 thiamine-HCl, 200 mg KH2PO4 with 3% εucrose, 2,4-D (1.0 mg/1), 0.6% Phytagar, and pH adjusted to 5.8 prior to autoclaving (MSO/1/0 medium) . A sterile filter paper disc (Whatman 3 mm) iε placed on top of the feeder layer prior to uεe. Tobacco suspension cultures are subcultured weekly by tranεfer of 10 ml of culture into 100 ml fresh MS medium as described for the feeder plates with 2,4-D (0.2 mg/1), Kinetin (0.1 mg/1) .

All hypocotyl explants are preincubated on feeder plates for 24 h. at 22°C in continuous light of intensity 30 μErn^"

²S^_1 to 65 μEM^-2S^_1. Single colonies of A. tumefaciens strain EHA101 containing a binary plasmid are transferred to 5 ml MG/L broth and grown overnight at 30°C. Per liter, MG/L broth contains 5g mannitol, 1 g L-glutamic acid or 1.15 g sodium glutamate, 0.25 g kH2 04, 0.10 g NaCL, 0.10 g MGSO4-7H20, 1 mg biotin, 5 g tryptone, and 2.5 g yeast extract, and the broth is adjusted to pH 7.0. Hypocotyl explantε are immersed in 7-12 ml MG/L broth with bacteria diluted to 1x10^s bacteria/ml and after 10-20 min. are placed onto feeder plates._. After 48 h of co-incubation with Agrobacterium, the hypocotyl explants are transferred to B5 0/1/0 callus induction medium which contains filter sterilized carbenicillin (500 mg/1, added after autoclaving) and kanamycin sulfate (Boehringer Mannheim) at concentrations of 25 mg/1. After 3-7 days in culture at 65 μEm^"2S~¹ to 75 μEm^_2S^~ continuous light, callus tissue is visible on the cut surface and the hypocotyl explants are transferred to shoot induction medium, B5BZ (B5 salts and vitamins supplemented with 3 mg/1 benzylaminopurine, 1 mg/1 zeatin, 1% sucrose, 0.6% Phytagar and pH adjusted to 5.8) . This medium also contains carbenicillin (500 mg/1) and kanamycin sulfate (25 mg/1) . Hypocotyl explants are subcultured onto fresh shoot induction medium every two weeks.

Shoots regenerate from the hypocotyl calli after one to three months. Green shoots at least 1 cm tall are excised from the calli and placed on medium containing B5 salts and vitamins, 1% sucrose, carbenicillin (300 mg/1), kanamycin sulfate (50 mg/1) and 0.6% Phytagar) and placed in a culture room with conditions as described for seed germination. After 2-4 weeks shootε which remain green are cut at the baεe and transferred to Magenta boxes containing root induction medium (B5 salts and vitamins, 1% sucrose, 2 mg/1 indolebutyric acid, 50 mg/1 kanamycin sulfate and 0.6% Phytagar) . Green rooted shoots are tested for NPT II activity.

Transgenic Arabidopεis thaliana plants may also be obtained by AgroJbacterium-mediated transformation using εimilar techniqueε. For example, a uεeful method haε been described by Valverkens et al . , ( Proc . Nat . Acad. Sci . (1988) 85:5536-5540) . B. Transformation by Particle Bombardment

DNA sequences of interest may be introduced as expresεion cassetteε, compriεing at leaεt a promoter region, a gene of interest, and a termination region, into a plant genome via particle bombardment as described in European Patent Application 332 855 and in co-pending application USSN 07/225,332, filed July 27, 1988.

Briefly, tungsten or gold particles of a size ranging from 0.5μ_M-3μM are coated with DNA of an expression cassette. This DNA may be in the form of an^' aqueous mixture or a dry DNA/particle precipitate.

Tiεsue used as the target for bombardment may be from cotyledonary explants, shoot meristems, immature leafletε, or antherε.

The bombardment of the tissue with the DNA-coated particleε iε carried out uεing a Bioliεtics™ particle gun

(Dupont; Wilmington, DE) . The particleε are placed in the barrel at variable distances ranging from lcm-14cm from the barrel mouth. The tisεue to be bombarded is placed beneath the stopping plate; teεting iε performed on the tissue at distanceε up to 20 cm. At the moment of diεcharge, the tissue is protected by a nylon net or a combination of nylon nets with mesh ranging from 10μM to 300μM. Following bombardment, plants may be regenerated following the method of Atreya, et al . , ( Plant Science Letters (1984) 34:379-383) . Briefly, embryo axis tisεue or cotyledon εegments are placed on MS medium (Murashige and Skoog, Physio . Plant . (1962) 15:473) (MS pluε 2.0 mg.l 6- benzyladenine (BA) for the cotyledon segments) and incubated in the dark for 1 week at 25 ± 2°C and are subsequently transferred to continuous cool white fluorescent light (6.8 W/m²) . On the 10th day of culture, the plantlets are tranεferred to potε containing εterile εoil, are kept in the shade for 3-5 days are and finally moved to greenhouse.

The putative transgenic shoots are rooted.

Integration of exogenous DNA into the plant genome may be confirmed by various methods known to those skilled in the art.

Example 6. Analysis of Transformed Plants

Seedε from 15 Arabidopεis plants transformed with pCGN2797 (napin 5'/_R. communis synthase factor A/napin 3' and napin 5'/J?. communis synthase factor B/napin 3') were analyzed for the presence of R. communis synthase proteins. Five of these plants test positive, by Western analysis, for expression of the 50kD R. communis synthase factor B protein. Cross-reactivity of the R. communis synthase factor A polyclonal antibody with the corresponding Brassica synthase protein, prevents detection of this synthaεe protein by Weεtern analysis.

Two of the plants which tested positive for expression of the 50kD R. communis synthase protein, transformants #5 and #6 have been analysed to determine the fatty acid composition of their seed oil. Several non-expressing transformants and a non-transformed control were similarly analyzed. Seed fatty acid composition iε determined by the acid ethanolyεis method according eεsentially as described by Browεe et al . (Anal . Biochem. (1986) 152:141-145) . Briefly, 100 εeedε of each sample are treated with 1 ml of 5% H2SO4 in MeOH and heated in a 90°C water bath for two hours to convert the fatty acids to fatty acid methyl esters (FAMEs) . An internal standard (C17:0) is added to each sample (250ml of a lmg/ml solution in tolulene) prior to the heating step. The samples are allowed to cool, after which 1 ml 0.9% NaCl in H2O is added to aid in phaεe εeparation. Hexane (250ml to each vial) is added to extract the FAMEs, and the sampleε are then vortexed and centrifuged to separate the phases. The hexane layer is removed and transferred to a GC autosampler for injected on the GC. A uεeful GC temperature program for these analyses is as follows: 200'C for zero minutes, followed by a 5 degreeε per minute temperature ramp to a final temperature of 250"C, which is held for 6 minutes. Data is reported as % of total fatty acids in Table I below.

Seeds from transformant #5 contain 3.95% C16:0, and seeds from #6 have a 4.59% C16:0. Seeds from the non- expressing transformants and the non-transformed control had C16:0 percentages ranging from 5.85 to 6.63%. Total saturated fatty acids in seeds from #5 were 9.74%, compared to 12.47% total saturated fatty acids for seeds from the non-transformed control and a range of 11.57%-13.33% total saturated fatty acidε for seeds from the non-expressing transformants. The total saturated fatty acid level in transformant #6 is 10.64%.

TABLE I

18:1 18:2 18:3 20:0 20:1 20:2 22:0 22:1 22:2 24:0 SΛTS % % % % % % % %

18.10 25.36 18.56 2.44 21.16 1.81 0.34 2.21 0.10 0.23 12.44

18.62 25.21 18.75 2.26 21.21 1.70 0.31 2.09 0.12 0.21 12.06

17.68 27.99 18.82 2.30 20.58 2.13 0.35 2.69 0.16 0.21 9.74

20.95 25.30 17.71 2.28 21.33 1.75 0.32 2.04 0.09 0.22 10.64

19.24 25.98 17.46 2.23 21.43 1.80 0.33 2.14 0.14 0.19 11.57

16.48 27.66 17.07 2.71 20.59 2.16 0.38 2.24 0.14 0.24 13.33

19.43 24.93 17.86 2.36 21.47 1.81 0.32 2.07 0.09 0.21 12.10

19.98 24.28 18.84 2.23 21.16 1.59 0.33 2.02 0.09 0.18 11.83

20.85 24.05 18.72 2.30 20.83 1.59 0.30 1.75 0.06 0.16 11.94

18.15 25.77 19.37 2.35 19.85 2.00 0.35 2.00 0.11 0.21 12.47

The above results demonstrate the ability to use synthase DNA sequences in plant genetic engineering methods for production of transgenic plantε having modified seed oil compositions.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.

All publications and patent applications are herein incorporated by referenced to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claim.

Claims

What iε claimed iε:

1. A recombinant DNA construct comprising, in the 5' to 3 ' direction of transcription, a promoter functional in a plant cell, a β-ketoacyl-ACP synthaεe protein encoding εequence and a transcriptional termination regulatory region 3' to said synthase protein encoding sequence, wherein said β-ketoacyl-ACP synthase protein is from a non- plant source.

2. The construct of Claim 1, wherein said synthase protein encoding sequence is oriented for transcription of a sense sequence.

3. The construct of Claim 2, wherein said construct comprises, immediately 5' to said synthase protein encoding sequence, a plant sequence encoding a transit peptide.

4. The construct of Claim 3, wherein said synthase protein encoding sequence is the E. coli faJbH gene sequence.

5. The construct of Claim 3, wherein said transit peptide encoding sequence is from an acyl-carrier protein gene.

6. The construct of Claim 1 wherein said promoter iε from a gene that iε preferentially expreεsed in plant seed tisεue.

7. A plant cell comprising a construct according to Claim 1.

8. The plant cell of Claim 7 further comprising a second recombinant DNA construct providing for transcription in said plant cell of a substantial portion of a sequence encoding a protein associated with lipid synthesiε.

9. The cell of Claim 8 wherein said protein is a desaturase or a thioesteraεe.

10. The cell of Claim 7 or 8 wherein said plant cell is a Brassica plant cell.

11. A recombinant DNA construct comprising, in the 5' to 3 ' direction of transcription, a promoter functional in a plant cell, a plant transit peptide encoding sequence, a mature R . communis β-ketoacyl-ACP synthase factor A encoding sequence and a transcriptional termination regulatory region 3 ' to said synthase protein encoding sequence, wherein said transit peptide encoding sequence is not naturally associated with said R. communis β-ketoacyl- ACP synthase factor A encoding sequence.

12. The construct of Claim 11, wherein said plant transit peptide encoding sequence is from a gene encoding a

Brassica β-ketoacyl-ACP synthaεe protein.

13. The construct of Claim 12, wherein said Brassica synthase protein is synthase factor A.

14. The construct of Claim 11, wherein said plant transit peptide encoding sequence is from a gene encoding a stearoyl-ACP desaturase protein.

15. A plant cell comprising a construct according to

Claim 11.

16. The cell of Claim 15 wherein said plant cell is a Brassica plant cell.

17. A transgenic plant cell comprising a non-plant β-ketoacyl-ACP synthase protein expressed from a recombinant DNA sequence.

18. The cell of Claim 17 wherein said non-plant synthase protein is the β-ketoacyl-ACP synthase protein encoded by E. coli fabH.

19. A method of producing a non-plant β-ketoacyl-ACP synthase protein in a plant cell or progeny thereof comprising growing a plant cell or progeny thereof comprising a construct according to Claim 1, under conditions which will permit the production of said β- ketoacyl-ACP synthase protein.

20. A plant cell comprising a non-plant β-ketoacyl- ACP synthase protein produced according to Claim 19.

21. A plant cell of Claim 20 wherein said construct iε integrated into the genome of said plant cell.

22. A method of modifying the fatty acid composition in a plant cell comprising: growing a plant cell having integrated in its genome a DNA construct, said construct comprising in the 5' to 3 ' direction of transcription, a promoter functional in said plant cell, a non-plant β-ketoacyl-ACP synthase protein encoding sequence, and a transcriptional termination region functional in εaid plant cell, under conditions which will permit the expression of said synthase protein encoding sequence.

23. The method of Claim 22 wherein εaid εynthase protein encoding sequence is a sense sequence.

24. The method of Claim 23 wherein said synthase protein is the β-ketoacyl-ACP synthase protein encoded by

E. coli fabH .

25. A method of modifying the fatty acid composition in a plant cell comprising: growing a plant cell having integrated in its genome a DNA construct according to Claim 11, under conditions which will permit the transcription of said synthase protein encoding sequence.

26. The method of Claim 22 or 25 wherein said plant cell is an oilseed plant seed cell.

27. A plant cell having a modified free fatty acid composition produced according to the method of Claim 26.

28. A plant seed having a modified fatty acid composition as compared to a seed of said plant having a native fatty acid composition, produced according to a method comprising: growing a plant to seed, wherein said plant has integrated in its genome a recombinant DNA sequence comprising a non-plant β-ketoacyl-ACP synthase protein encoding sequence under the transcriptional control of regulatory elements functional in seed during lipid accumulation, under conditions which will promote the activity of said regulatory elements, and harvesting said seed.

29. The seed of Claim 28 wherein said synthase protein is the β-ketoacyl-ACP synthase protein encoded by E. coli fabH.

30. A plant seed having a modified fatty acid composition as compared to a seed of said plant having a native fatty acid composition, produced according to a method comprising: growing a plant to εeed, wherein εaid plant haε integrated in its genome a recombinant DNA construct according to Claim 11, under conditions which will promote the activity of said regulatory elements, and harvesting said seed.

31. The seed of Claim 28 or 30 wherein εaid plant iε an oilseed plant.

32. The seed of Claim 31 wherein said plant is Brassica .

33. A method of modifying the fatty acid composition of triglycerides produced from an oilseed crop plant comprising: growing a plant cell having integrated in its genome a DNA construct, said construct comprising in the 5' to 3 ' direction of tranεcription, a promoter functional in said plant cell and a non-plant β-ketoacyl-ACP synthase protein encoding sequence, under conditions which will permit the transcription of said synthaεe protein encoding sequence.

34. The method of Claim 33 wherein said synthase protein encoding sequence is a sense sequence.

35. The method of Claim 34 wherein said synthase protein is the β-ketoacyl-ACP synthase protein encoded by

E. coli fabH .

36. A method of modifying the fatty acid composition of triglycerideε produced from an oilseed crop plant comprising: growing a plant cell having integrated in its genome a DNA construct according to Claim 11, under conditionε which will permit the tranεcription of said synthase protein encoding sequence.

37. The method of Claim 33 or 36 wherein said plant cell iε a seed cell.

38. A plant cell having a modified fatty acid composition of triglycerides produced according to the method of Claim 37.

39. The method of Claim 37 wherein εaid oilseed crop plant is selected from the group consiεting of rapeεeed, sunflower, safflower, cotton, cuphea, soybean, peanut, coconut, oil palm and corn.

40. A plant seed oil separated from a seed produced according to Claim 31.

41. The oil of Claim 40, wherein said plant is

Brassica .