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WO2000012732A2 - Sequences de ciblage d'organelle - Google Patents

Sequences de ciblage d'organelle Download PDF

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WO2000012732A2
WO2000012732A2 PCT/US1999/018955 US9918955W WO0012732A2 WO 2000012732 A2 WO2000012732 A2 WO 2000012732A2 US 9918955 W US9918955 W US 9918955W WO 0012732 A2 WO0012732 A2 WO 0012732A2
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sequence
plant
sequences
transit peptide
nos
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WO2000012732A3 (fr
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Robert J. Bensen
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Pioneer Hi Bred International Inc
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Pioneer Hi Bred International Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention is drawn to the genetic modification of plants, particularly to the targeting of proteins to cellular organelles.
  • Plastids are a class of plant organelles derived from proplastids and include chloroplasts, leucoplasts, amyloplasts, and chromoplasts.
  • the plastids are major sites of biosynthesis in plants. In addition to photosynthesis in the chloroplast, plastids are also sites of lipid biosynthesis, nitrate reduction to ammonium, and starch storage. And while plastids contain their own circular genome, most of the proteins localized to the plastids are encoded by the nuclear genome and are imported into the organelle from the cytoplasm.
  • the mechanism of protein import into the plastids has been most extensively studied in the chloroplast.
  • the chloroplast is a complex cellular organelle composed of three membranes: the inner envelope membrane, the outer envelope membrane, and the thylakoid membrane.
  • the membranes together enclose three aqueous compartments termed the intermediate space, the stroma, and the thylakoid lumem.
  • Proteins imported from the cytosol generally contain, at their amino terminus, short sequences referred to as "transit peptides" that are responsible for post-translational targeting of the protein to the chloroplast.
  • the import process is initiated by binding of precursor proteins to the chloroplast surface, followed by the subsequent translocation of the precursor protein across the chloroplast envelope membranes.
  • the transit peptide is typically an expendable part of the protein, and upon translocation into the chloroplast the amino acid sequence is cleaved from the precursor protein. Further sub-organellar sorting of the modified precursor takes place as appropriate.
  • N-terminus include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco),de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:169- 780; Schnell, D.J. et al. (1991) J. Biol. Chem. 26(5 ⁇ :3335-3342; 5- (enolpyruvyl)shikimate-3-phosphate synthase (EPSPS), Archer et al. (1990) J. Bioenerg. and Biomemb. 22 (6): 789-810; tryptophan synthase. Zhao, j. et al. (1995) J. Biol. Chem.
  • the peptides typically contain an exceptionally high content of basic and hydroxylated amino acids, such as serine and threonine.
  • the amino-terminal region is devoid of charged amino acids and lacks turn promoting amino acids such as glycine and proline.
  • the carboxy terminal domain is high in arginine and has a capacity for forming an amphipathic beta sheet secondary structure.
  • the length of the transit peptide is variable, commonly between 50 and 120 amino acids.
  • transit peptide sequences prove useful in recombinant DNA technology.
  • transit peptide sequences may be inserted into an expression cassette and serve to guide the expressed protein to the chl ⁇ ropast.
  • transit peptide signals have been useful in the localization of proteins responsible for herbicide or antibacterial resistance to the chloroplast.
  • the present invention provides methods and compositions for the subcellular localization of proteins. Specifically, the invention provides a means to direct the localization of a protein to a plant cell organelle, more particularly to a plant plastid.
  • Compositions of the present invention include the nucleotide and amino acid sequences of novel plastid targeting sequences, hence referred to as transit peptides. Such sequences find utility in the enhanced or modified localization of proteins to a plastid or compartment thereof.
  • compositions of the invention include, expression cassettes and transformation vectors comprising the isolated nucleotide sequences of the transit peptides. Also provided are transgenic plants, plant cells, and plant tissue that express proteins that have been localized to a plastid using the transit peptides of the present invention.
  • Figure 1 schematically illustrates a plasmid vector comprising a ubiquitin promoter operably linked to a transit peptide sequence of the invention operably linked to a gene of interest.
  • compositions and methods are provided for modulating the subcellular localization of proteins.
  • the compositions of the invention include maize transit peptide sequences that find use in modulating the cellular localization of a protein of interest.
  • the transit peptides of the invention finds use in the localization of proteins to plant organelles, particularly to plastids and compartments thereof.
  • plastid is intended a class of plant cell organelles comprising proplastids, leucoplasts, amyloplast, chromoplasts, and chloroplast.
  • plastid or compartment thereof is intended any plastid structure, membrane or compartment of a plastid.
  • a compartment thereof encompasses the intermediate envelope space, the stroma, the lumen, the outer envelope, the inner envelope and the thylakoid membrane.
  • a signal or targeting sequence is a structural peptide domain required for targeting of a given polypeptide to a subcellular organelle. subcellular compartment or secretion from the cell.
  • the transport of a protein of interest to a subcellular compartment is accomplished by operably linking the nucleotide sequence encoding a signal sequence to the 5' and/or 3' region of the gene encoding the protein of interest.
  • the targeting sequence influence where the protein of interest is ultimately compartmentalized.
  • transit peptide is intended a polypeptide that directs the transport of a nuclear encoded protein to a plastid or a compartment thereof.
  • the transit peptide sequence is located at the amino-terminus of a polypeptide.
  • the transit peptide may also be located at either the c-terminus or internally in the polypeptide.
  • the maize sequences provided by the present invention includes a maize transit peptide having homology to the maize light harvesting chlorophyll a/b binding protein (SEQ ID NOS: 1 and 2).
  • the present invention also provides a transit peptide having a homology to the maize ribulose bisphosphate carboxylase/oxygenase protein (SEQ ID NOS: 3 and 4).
  • Also provided are maize transit peptide sequences that share homology to transit peptide sequences of various non-maize gene products including, EPSP synthase (SEQ ID NOS: 5 and 6), tryptophan synthase component (SEQ ID NOS: 7 and 8), ribosomal protein L35 (SEQ ID NOS: 9 and 10), plastid ribosomal protein CL9 (SEQ ID NOS: 1 1 and 12), plastocyanin (SEQ ID NOS: 13 and 14), 3-dehydroquinate synthase (SEQ ID NOS: 15 and 16), plastid ribosomal protein CL15 (SEQ ID NOS: 17 and 18), chorismate synthase (SEQ ID NOS: 19 and 20), and choporphyringogen oxidase (SEQ ID NOS: 21 and 22).
  • EPSP synthase SEQ ID NOS: 5 and 6
  • tryptophan synthase component SEQ ID NOS: 7 and 8
  • the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 and fragments and variants thereof.
  • the invention encompasses isolated or substantially purified nucleic acid or polypeptide compositions.
  • An "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb. 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptides having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating polypeptides.
  • culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • Fragments and variants of the disclosed nucleotide sequences and the polypeptides encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence polypeptide encoded thereby.
  • Fragments of a nucleotide sequence may encode polypeptide fragments that retain the biological activity of the native polypeptide and hence facilitates the transport of a nuclear encoded protein to a plastid or a compartment thereof.
  • fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment polypeptides retaining biological activity.
  • fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.
  • a fragment of a transit peptide nucleotide sequence that encodes a biologically active portion of a transit peptide of the invention will encode at least 15, 25, 30, 40, 50, 60, 70, 80, 90, 100 contiguous amino acids, or up to the total number of amino acids present in a full-length transit peptide of the invention (for example, 135, 117, 144, 152, 150, 152, 145, 132, 134, 166, and 107 amino acids for SEQ ID NOS; 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, respectfully).
  • Fragments of a transit peptide nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a transit peptide.
  • a fragment of a transit peptide nucleotide sequence may encode a biologically active portion of a transit peptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of a transit peptide can be prepared by isolating a portion of one of the transit peptide nucleotide sequences of the invention, expressing the encoded portion of the transit peptide(e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the transit peptide.
  • Nucleic acid molecules that are fragments of a transit peptide nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400 nucleotides, or up to the number of nucleotides present in a full-length transit peptide nucleotide sequence disclosed herein (for example, 407, 426, 455, 459, 560, 461, 437, 463, 448, 528, and 427 nucleotides for SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, respectively).
  • variants are intended substantially similar sequences.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the transit peptide of the invention.
  • Naturally occurring variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a transit peptide or protein of the invention.
  • nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, 70%, generally, 80%, preferably 85%, 90%, up to 95%, 98% sequence identity to its respective native nucleotide sequence.
  • variant polypeptide is intended as a polypeptide derived from the native polypeptide by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native polypeptide; deletion or addition of one or more amino acids at one or more sites in the native polypeptide; or substitution of one or more amino acids at one or more sites in the native protein.
  • Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • the transit polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the transit peptide can be prepared by mutations in the DNA.
  • Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 52:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:361-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al.
  • nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • polypeptides of the invention encompass both naturally occurring polypeptides as well as variations and modified forms thereof. Such variants will continue to possess the desired ability to facility the transport of a nuclear encoded protein to a plastid or compartment thereof. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
  • deletions, insertions, and substitutions of the polypeptides sequences encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by the ability of the isolated sequences to target and deliver a reporter protein to a plastid or compartment thereof . See, for example, de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 796-180, herein incorporated by reference.
  • Variant nucleotide sequences and polypeptides also encompass sequences and polypeptides derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different transit peptide sequences can be manipulated to create a new transit peptide sequence possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • sequence motifs encoding a domain of interest may be shuffled between the transit peptide sequences of the invention and other known signal sequences or transit peptide sequences to obtain a new nucleotide sequence coding for a transit peptide with an improved property of interest, such as an increased K m or an increased efficiency and/or specificity of plastid targeting.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10141-10151; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 75:436-438; Moore et al. (1997) J. Mol.
  • nucleotide sequences of the invention can be used to isolate coresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire transit peptide sequences set forth herein or to fragments thereof are encompassed by the present invention.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest.
  • Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds.
  • PCR PCR Strategies
  • cDNA fragments i.e., genomic or cDNA libraries
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the transit peptide sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • the entire nucleotide sequence encoding a transit peptide disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding transit peptide sequences and messenger RNAs.
  • probes include sequences that are unique among transit peptide sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • Such probes may be used to amplify corresponding transit peptide sequences from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • T m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem.
  • T m 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50%) of a complementary target sequence hybridizes to a perfectly matched probe.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • sequences that encode a transit peptide and hybridize to the nucleic acid sequences encoding a transit peptide disclosed herein will be at least 40%) to 50%) homologous, about 60% to 70% homologous, and even about 80%, 85%, 90%), 95%> to 98%) homologous or more with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%), about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.
  • sequence relationships between two or more nucleic acids or polynucleotides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30. 40, 50, 100, or longer.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; by the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci.
  • preferred computer alignment methods also include the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 275:403-410). Alignment is also often performed by inspection and manual alignment. Sequence alignments are performed using the default parameters of the alignment programs.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%>, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 80%, more preferably at least 90%>, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%>.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringent conditions encompass temperatures in the range of about 1 °C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 45:443.
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • Peptides that are "substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.
  • nucleotide sequences of the present invention may be operably linked to the nucleotide sequences encoding a protein of interest and thereby modulate the cellular localization of the protein.
  • modulate is intended any increase in the concentration of said protein in a plastid or compartment thereof beyond that which occurs in the absence of the transit peptide. Additionally, “modulate” refers to an increased rate of importation of said protein into the plastid as compared to the rate of importation in the absence of the transit peptide sequence.
  • the nucleotide sequences of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will comprise a trancriptional initiation and translational termination sequence functional in plants operably linked to a nucleic acid sequence encoding a transit peptide of the invention, operably linked to a nucleotide encoding a protein of interest.
  • the cassette may contain at least one additional sequence to be cotransformed into the organism. Alternatively, the additional sequences can be provided on another expression cassette.
  • “Operably linked” refers to a functional linkage between a promoter and a second sequence, wherein the promoter inititates and mediates transcription of DNA sequences corresponding to the second sequence. "Operably linked” also refers to a functional linkage between 2 or more distinct nucleotide sequences such that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. Operably linking the transit peptide-coding sequences with the nucleotide sequences encoding a protein of interest may require the manipulation of one or more of the DNA sequences.
  • a convenient restriction site or a linker sequences that acts as a non-specific spacer that may permit better recognition of the amino-terminal transit sequence may be introduced.
  • Expression of the coding sequences of a protein of interest operably linked to sequences of the transit peptide produces a hybrid polypeptide, or so-called fusion protein.
  • hybrid polypeptide By "hybrid" polypeptide is intended the coding sequences for the transit peptide is foreign to the coding sequences for the protein of interest, and hence, the two coding sequences are not natively expressed as a polypeptide in the plant cell.
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be a natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. While it may be preferable to express the sequences using heterologous promoters, the native promoter sequences of either the gene of interest or the transit peptide sequence may be used.
  • Such constructs would change expression levels of the gene of interest in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered. It is recognized that a variety of promoters will be useful in the invention, the choice of which will depend in part upon the desired level of expression of the protein of interest. It is recognized that the levels of expression can be controlled to modulate the levels of expression in the plant cell. Constitutive and tissue specific promoters are of particular interest. Such constitutive promoters include, for example, the core promoter of the Rsyn7 (copending U.S. Application Serial No. 08/661,601); the core CaMV 35S promoter (Odell et al. (1985) Nature 573:810-812); rice actin (McElroy et al.
  • Tissue-specific promoters can be utilized to target enhanced expression within a particular plant tissue.
  • Tissue-specific promoters include Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):192-803; Hansen et al. (1997) Mol. Gen Genet. 254 (3):331 -343; Russell et al. (1997) Transgenic Res. 6(2):151-168; Rinehart et al. (1996) Plant Physiol.
  • Leaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12 (2) :255-265; Kwon et al. (1994) Plant Physiol. 105:351- 67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):113-118; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:611-614; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell. 2:1261-1272; Munroe et al.
  • the expression cassette contains a plurality of restriction sites to insert both the gene of interest and the transit peptide sequence 3' of the designated promoter.
  • the transit peptide sequences of the invention may be operably linked to the gene of interest at the 3' terminus, 5' terminus or internally. Preferably, the sequences of the invention will be placed at the 5' end.
  • the nucleic acids included in the expression cassette may be optimized for expression in a plastid or compartment thereof to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acid sequences may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by reference.
  • the gene of interest encoding a protein to be localized the a plastid or compartment thereof is linked to the transit peptide nucleic acid sequence in such a way that upon translation and import into a plastid or compartment thereof the transit peptide is cleaved from the protein of interest.
  • Methods for preparing transit peptide chimeras are known in the art and are described in the following publications and issued patents. See de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:169-180; Pilon et al. (1995) J. Biol. Chem. 270f5):3882-3893; U.S. Patent No. 5,633,444; U.S. Patent No. 5,498,544.
  • the gene of interest may be native or analogous or foreign or heterologous to the plant host. By foreign is intended that the gene of interest is not found in the native plant into which it is introduced.
  • the gene of interest may also be nuclear encoded or plastid encoded. Generally, the proteins selected for targeting to the plastids are heterologous to the transformed cell and nuclear encoded.
  • Genes of interest include, for example, any protein whose localization in the plastid will modify agronomically important traits such as oil, starch, and protein content. Other modified traits include herbicide, disease, and insect resistance.
  • genes of interest may include, but are not limited to, the small subunit of ribulose bisphosphate carboxylase (Rubisco), Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342; ferrodoxin, Pilon et al. (1995) J Biol. Chem. 270(8) -.3882-3893; light harvesting chlorophyll a/b binding protein, Lamppa et al. (1988) J. Biol. Chem. 265:14996-14999, Reiski FeS protein, Madueno et al. (1994) J. Biol. Chem. 269(26): 17458- 17463; plastocyanin , Lawrence et al.
  • genes encoding proteins involved in herbicide resistance are genes encoding proteins involved in herbicide resistance.
  • the herbicide resistance is imparted by 5- enolpyruvylshikimate-3-phosphate synthase.
  • fragments and variants of the various proteins of interest may also be used with the transit peptide sequences of the invention.
  • the 5-enolpyruvylshikimate-3-phosphate synthase may be altered such that the protein product is less sensitive to herbicide inhibition. See for example, U.S. Patent No. 5,188,642.
  • the gene of interest may be a reporter gene.
  • Reporter genes are generally known in the art.
  • the reporter gene used should not be expressed endogenously. Ideally the reporter gene will exhibit low background activity and should not interfere with plant biochemical and physiological activities.
  • the products expressed by the reporter gene should be stable and readily detectable. It is important that the reporter gene expression should be able to be assayed by a non-destructive, quantitative, sensitive, easy to perform and inexpensive method. Examples of suitable reporter genes known in the art can be found in, for example, Jefferson et al. ( 1991 ) in Plant Molecular Biology Manual (Gelvin et al. eds.) pp. 1-33, Kluwer Academic Publishers; DeWet et al. (1987) Mol. Cell. Biol.
  • the transit peptide sequences of the invention may be native or analogous or foreign or heterologous to either the host plant or to the gene of interest. By foreign is intended that the transit peptide sequence is not found in the native host plant or is not naturally encoded by the gene of interest.
  • the DNA sequence encoding the transit peptide may be chemically synthesized either wholly or in part from the known sequence of the transit peptide.
  • the gene(s) of interest and the transit peptide sequences of the invention may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol.
  • the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS USA 56:6126-6130); poty virus leaders, for example. TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511 ; Christopherson et al. ( 992) Proc. Natl. Acad.
  • the above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
  • the present invention also relates to the introduction of the transformation constructs into plant protoplasts, calli, tissues, or organ explants and the regeneration of transformed plants expressing the recombinant constructs of the invention.
  • the expression cassette sequences of the present invention may be used for transformation of any plant species, including, but not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa).
  • rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea).
  • Lactuca sativa Lactuca sativa
  • green beans Paneolus vulgaris
  • lima beans Phaselus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (M ⁇ crophyll ⁇ hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus ellioti ⁇ ), ponderosa pine (Pinus ponder osa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine
  • plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, wheat, tobacco, etc.), more preferably corn and soybean plants, yet more preferably com plants. Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot. targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.
  • the modified plant may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell. Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
  • a reporter gene such as ⁇ -glucuronidase (GUS), chloramphenicol acetyl transferase (CAT), or green fluorescent protein (GFP) is operably linked to the transit peptide sequence.
  • GUS ⁇ -glucuronidase
  • CAT chloramphenicol acetyl transferase
  • GFP green fluorescent protein
  • GFPm is derived from a back translation of the GFP protein sequence using maize preferred codons and is shown in SEQ ID NO:23. Sequence analysis is performed using the Wisconsin Sequence Analysis Package from Genetics Computer Group, Madison, WI. The nucleotide sequence is assembled from a series of synthetic oligonucleotides. Cloning sites within the GFPm include a 5' flanking BamH restriction site, an4/7III site at the start codon, a 3' flanking Hpal site or a 7.g7II site converting the stop codon to an isoleucine.
  • GFPm modified green fluorescent protein
  • the ubiquitin promoter is inserted upstream of the GFPm fusion protein. Also engineered into the expression cassette is an intron and a Pinll termination sequence.
  • Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing a transit peptide sequence cloned into a flanking restriction sites of GFPm to create an inframe fusion. These sequences are operably linked to a ubiquitin promoter ( Figure 1). Also contained on this plasmid is the selectable marker gene, PAT, (Wohlleben et al. (1988) Gene 70:25-31) that confers resistance to the herbicide Bialaphos. Transformation is performed as follows. All media recipes are in the Appendix. Preparation of Target Tissue
  • the ears are surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • the immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5- cm target zone in preparation for bombardment.
  • a plasmid vector comprising a transit peptide sequence sequences cloned into restriction sites resulting in an inframe fusion with GFPm, and operably linked to a ubiquitin promoter, and containing a PAT selectable marker is precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCl precipitation procedure as follows:
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer.
  • the final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes.
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 ⁇ l 100%) ethanol is added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
  • sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PS1, with a total often aliquots taken from each tube of prepared particles/DNA.
  • the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established.
  • Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1 -2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Screening for GFP expression is carried out at each transfer using a Xenon and/or Mercury light source with the appropriate filters for GFP visualization.
  • GFP expressing colonies are identified they are monitored regularly for new growth and expression using the Xenon light source.
  • Plant cells containing GFP are regenerated by transferring the callus to 288 medium containing MS salts, 1 mg/L IAA, 0.5 mg/L zeatin and 4% sucrose. The callus is placed in the light. As plantlets develop they are transferred to tubes containing 272K, hormone-free MS medium and 3% sucrose. The percentage of green fluorescent colonies that regenerated into whole plants can be determined.
  • the ability of the tansit peptide to target GFP to the plastid is determined in stable transgenic maize cells using epifluorescent microscopy and image enhancement software. Samples of calli from the transformed maize plants are fixed in FAA and are examined with UV filters to visualize GFP localization in the plastid.
  • Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.
  • Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.

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Abstract

L'invention traite de compositions et de procédes permettant de moduler le repérage infracellulaire de protéines dans une cellule. Les compositions renferment notamment des séquences nucléotidiques et d'acides aminés de séquences peptidiques de transit du maïs. De telles séquences sont utiles en matière de repérage renforcé ou modifié d'une protéine sur un plaste ou un compartiment de celui-ci.
PCT/US1999/018955 1998-08-28 1999-08-25 Sequences de ciblage d'organelle Ceased WO2000012732A2 (fr)

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WO2005123929A3 (fr) * 2004-06-09 2006-03-30 Pioneer Hi Bred Int Peptides de transit vers des plastes
EP1645633A2 (fr) 2004-10-05 2006-04-12 SunGene GmbH Cassettes d'expression constitutives pour la régulation de l'expression chez les plantes.
EP1655364A2 (fr) 2004-11-05 2006-05-10 BASF Plant Science GmbH Cassettes d'expression pour une expression préférentielle dans les graines
EP1662000A2 (fr) 2004-11-25 2006-05-31 SunGene GmbH Cassettes d'expression pour l'expression préférée dans des cellules stomatiques de plantes
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WO2006089950A2 (fr) 2005-02-26 2006-08-31 Basf Plant Science Gmbh Cassettes d'expression destinees a une expression preferentielle de semences chez des plantes
WO2006120197A2 (fr) 2005-05-10 2006-11-16 Basf Plant Science Gmbh Cassettes d'expression pour l'expression preferentielle de semence dans des plantes
EP2186903A2 (fr) 2005-02-09 2010-05-19 BASF Plant Science GmbH Cassettes d'expression pour la régulation de l'expression chez les plantes monocotylédones
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WO2003008585A3 (fr) * 2001-07-18 2004-02-12 Biogemma Fr Acides nucleiques codant pour un polypeptide isum2a et utilisation de ces acides nucleiques pour l'obtention de plantes transformees produisant des graines affectees dans le developpement du germe
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