EP0666865A1 - Synthases d'acides gras vegetales - Google Patents

Synthases d'acides gras vegetales

Info

Publication number
EP0666865A1
EP0666865A1 EP94900499A EP94900499A EP0666865A1 EP 0666865 A1 EP0666865 A1 EP 0666865A1 EP 94900499 A EP94900499 A EP 94900499A EP 94900499 A EP94900499 A EP 94900499A EP 0666865 A1 EP0666865 A1 EP 0666865A1
Authority
EP
European Patent Office
Prior art keywords
plant
synthase
sequence
plant cell
seed
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94900499A
Other languages
German (de)
English (en)
Other versions
EP0666865A4 (fr
Inventor
Vic. C. Knauf
Gregory A. Thompson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monsanto Co
Original Assignee
Calgene LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Calgene LLC filed Critical Calgene LLC
Publication of EP0666865A1 publication Critical patent/EP0666865A1/fr
Publication of EP0666865A4 publication Critical patent/EP0666865A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition

Definitions

  • 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.
  • 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.
  • 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.
  • having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content.
  • edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardio-vascular health reasons.
  • temperate substitutes for high saturate tropical oils such as palm and coconut, would also find uses in a variety of industrial and food applications.
  • 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) .
  • acetyl-ACP acyl carrier protein
  • 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 ⁇ -ketoacyl-ACP ( ⁇ -ketoayl-ACP synthase) , reduction of the keto-function to an alcohol ( ⁇ - ketoacyl-ACP reductase) , dehydration to form an enoyl-ACP ( ⁇ -hydroxyacyl-ACP dehydrase) , and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl- ACP reductase) .
  • ⁇ -ketoacyl-ACP synthase I catalyzes elongation up to palmitoyl-ACP (C16:0)
  • ⁇ -ketoacyl- ACP synthase II catalyzes the final elongation to stearoyl- ACP (C18:0) .
  • Common plant unsaturated fatty acids such as oleic, linoleic and ⁇ -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 ⁇ -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 ⁇ -12 desaturase and ⁇ -15 desaturase.
  • 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.
  • 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.
  • enzyme target (s) are identified and qualified, quantities of protein and purification protocols are needed for sequencing.
  • useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and plants containing such constructs are needed.
  • 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.
  • compositions and methods of use related to ⁇ -ketoacyl-ACP synthase hereinafter also referred to as “synthase".
  • methods and compositions of amino acid and nucleic acid sequences related to biologically active plant synthase(s) are described in WO 92/03564 which is hereby incorporated by reference in its entirety.
  • 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.
  • 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.
  • 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.
  • synthase protein may be coordinated expression of synthase factor A and synthase factor B may be desirable to provide optimal synthase-II type activity in plant cells.
  • synthase III gene with plant synthase proteins may also be desired.
  • other enzymes related to fatty acid synthesis include plant thioesterases, especially medium- chain thioesterases, desaturases, especially ⁇ -9 desaturases, and the like.
  • non-plant synthase protein sequences in plant cells are considered herein. Such sequences may be used alone or in conjunction with plant synthase proteins.
  • 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.
  • 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 C 2 to Ci 6 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 .
  • a plant synthase will be derived in whole or in part from a natural plant source.
  • 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.
  • the E. coli synthase protein encoded by the faJB gene is shown herein to have homology to plant synthase proteins.
  • synthase I enzymatic activity is provided by a homodimer of the fabB gene product.
  • faJH E. coli synthase III
  • 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
  • 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.
  • heterologous is meant any sequence which is not naturally found joined to the synthase sequence.
  • 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.
  • the synthase 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.
  • nucleic acids may encode biologically active synthases or sequences complementary to the sequence encoding a synthase to inhibit the production of endogenous plant synthase.
  • the amount of synthase available to the plant FAS complex is increased.
  • the amount of the synthase available to the plant FAS is decreased.
  • 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.
  • rate influencing synthase activity may reside in synthase I-type, synthase II-type, synthase III-type or in a combination of these enzymes.
  • 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" .
  • the regulatory regions will vary, including regions from structural genes from viruses, plasmid or chromosomal genes, or the like.
  • a wide variety of constitutive or regulatable promoters may be employed.
  • 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 ⁇ -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.
  • a transcription and translation initiation control regulatory region also known as a "promoter”
  • 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.
  • 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.
  • different promoters may be desired.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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) .
  • synthases are part of the FAS pathway of plastid organelles, such as the chloropla ⁇ t, 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.
  • 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.
  • 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.
  • 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.
  • sequences which are homologously related to or derivations from either R . coz ⁇ munis synthase I or II are considered obtainable from the present invention.
  • homologously 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.
  • probes are typically labeled in a detectable manner (for example with 32 P-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 gracilli ⁇ .
  • 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.
  • flanking regions may be subjected to resection, mutagenesis, etc.
  • transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence.
  • 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.
  • 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.
  • a transcription initiation region or transcription/translation initiation region may be used.
  • a transcription/translation initiation regulatory region is needed.
  • 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.
  • transcription initiation regions which are preferentially expressed in seed tissue, i.e., which are undetectable in other plant parts.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • further manipulation such as restriction, insertion of new fragments, ligation, deletion, insertion, resection, etc., so as to tailor the components of the desired sequence.
  • 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.
  • a cytotoxic agent e.g. antibiotic, heavy metal, toxin, etc.
  • complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g (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.
  • EGS ethylene glycol is(succinimidyl succinate)
  • 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 ⁇ - 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 ⁇ l. This reaction is stopped by addition of 9 ⁇ l of 2M
  • 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.
  • 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 dimer ⁇ .
  • 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.
  • 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
  • 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.
  • 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.
  • 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 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.
  • pCGN941 is digested with Xhol and Hindlll and the resulting approximately 1.6 kb of napin -3 ' sequences are inserted into Xhol-HindiII digested pCGNl ⁇ 02 to result in pCGNl ⁇ 03.
  • the pCGN1803 is digested with Hindlll and religated.
  • 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 pCG ⁇ 1808, 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 pCG ⁇ 3200 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 pCGNl ⁇ O ⁇ 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.
  • 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.
  • 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 pCG ⁇ 1547 (see below), producing pCGN1853.
  • the PstI fragment of pCGN1853, containing the Bce4 gene is inserted into the PstI site of pUCl ⁇ (Norrander, et al . (1983) Gene 25:101-106), producing pCGN1857.
  • the plasmid pCGNl ⁇ 57 was deposited with the ATCC, Rockville, MD on
  • 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 pCGNl ⁇ 66 containing the mutagenized sequences, is inserted into the Clal site of pCGN2016 (described below), producing pCGNl ⁇ 66C.
  • the Clal fragment of pCGN1866C is used to replace the corresponding wild-type Clal fragment of pCGN1867 (described below) to produce pCGN166 ⁇ .
  • Bce4 coding sequences are removed by digestion of pCGNl ⁇ 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.
  • the BamHI and Smal sites of pUC18 are removed by BamHI-Smal digestion and recircularization of the plasmid, without repair of the ends, to produce pCGN1862.
  • the PstI fragment of pCGNl ⁇ 57, 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 pCGN200 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 pCGN27 ⁇ 5 is obtained by digestion with Asp71 ⁇ and ligated into Asp718 digested pCGN1557 (McBride et al. ; supra) , resulting in pCGN2767.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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 PCR product is digested with PstI and ligated to pUC8 (Vieira and Messing (1982) Gene 15:2359-268) digested with PstI to produce pCGN3220.
  • 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 PCG ⁇ 3229. 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.
  • 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.
  • 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 ⁇ -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.
  • ACP/synthase III fusion fragment is inserted into an appropriate cassette containing plant regulatory regions.
  • the ACP/synthase III fragment is obtained by digestion with Bglll and Xhol and ligated into pCGN3223.
  • 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.
  • 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.
  • rapa ACP clone contains a Ddel site within the codons for amino acids 11-12 (Ser-Lys) of the mature protein region.
  • 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.
  • additional constructs for transcriptional control of the synthase III fusion under various other plant regulatory elements may be similarly prepared.
  • 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.
  • 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.
  • 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 ⁇ g/1) , nicotinic acid (50 ⁇ g/1) , glycine (200 ⁇ g/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 ⁇ Einsteins per square meter per second ( ⁇ Em- 2 S _1 ) .
  • Hypocotyl ⁇ are excised from 7 day old seedling ⁇ , 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 ino ⁇ itol, 1.3 mg/1 thiamine-HCl, 200 mg KH2PO4 with 3% ⁇ ucrose, 2,4-D (1.0 mg/1), 0.6% Phytagar, and pH adjusted to 5.8 prior to autoclaving (MSO/1/0 medium) .
  • MS salt base Carolina Biological
  • a sterile filter paper disc (Whatman 3 mm) i ⁇ placed on top of the feeder layer prior to u ⁇ e. Tobacco suspension cultures are subcultured weekly by tran ⁇ fer 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) .
  • hypocotyl explants are preincubated on feeder plates for 24 h. at 22°C in continuous light of intensity 30 ⁇ Ern "
  • hypocotyl explant ⁇ 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.
  • Transgenic Arabidop ⁇ is thaliana plants may also be obtained by AgroJbacterium-mediated transformation using ⁇ imilar technique ⁇ .
  • AgroJbacterium-mediated transformation using ⁇ imilar technique ⁇ .
  • a u ⁇ eful method ha ⁇ been described by Valverkens et al . , ( Proc . Nat . Acad. Sci . (1988) 85:5536-5540) .
  • DNA sequences of interest may be introduced as expres ⁇ ion cassette ⁇ , compri ⁇ ing at lea ⁇ t 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.
  • tungsten or gold particles of a size ranging from 0.5 ⁇ _M-3 ⁇ M 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.
  • Ti ⁇ sue used as the target for bombardment may be from cotyledonary explants, shoot meristems, immature leaflet ⁇ , or anther ⁇ .
  • the particle ⁇ are placed in the barrel at variable distances ranging from lcm-14cm from the barrel mouth.
  • the tis ⁇ ue to be bombarded is placed beneath the stopping plate; te ⁇ ting i ⁇ performed on the tissue at distance ⁇ up to 20 cm.
  • the tissue is protected by a nylon net or a combination of nylon nets with mesh ranging from 10 ⁇ M to 300 ⁇ M.
  • plants may be regenerated following the method of Atreya, et al . , ( Plant Science Letters (1984) 34:379-383) .
  • embryo axis tis ⁇ ue or cotyledon ⁇ egments are placed on MS medium (Murashige and Skoog, Physio . Plant . (1962) 15:473) (MS plu ⁇ 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/m 2 ) .
  • the plantlets are tran ⁇ ferred to pot ⁇ containing ⁇ terile ⁇ oil, are kept in the shade for 3-5 days are and finally moved to greenhouse.
  • the putative transgenic shoots are rooted.
  • Seed ⁇ from 15 Arabidop ⁇ is plants transformed with pCGN2797 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 syntha ⁇ e protein by We ⁇ tern analysis.
  • a u ⁇ eful GC temperature program for these analyses is as follows: 200'C for zero minutes, followed by a 5 degree ⁇ 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 acid ⁇ for seeds from the non-expressing transformants. The total saturated fatty acid level in transformant #6 is 10.64%.

Abstract

L'invention se rapporte à des compositions et des procédés d'utilisation relatifs à la synthase β-cétoacyl-ACP, également appelée ci-après 'synthase'. L'invention se rapporte également à des procédés et compositions d'intérêt telles que des compositions de séquences d'acide aminé et d'acide nucléique relatives aux facteurs de la (des) synthase(s) végétale(s) ayant une activité biologique, ainsi qu'à l'acide aminé et l'acide nucléique des facteurs de protéine de synthase, et à des procédés d'utilisation de ces séquences dans des produits de recombinaison afin d'obtenir des plantes produites par génie génétique possédant des compositions d'acides gras modifiées. De plus, des utilisations de protéines de synthase non végétales dans les procédés de génie génétique sont aussi prises en considération.
EP94900499A 1992-11-02 1993-11-02 Synthases d'acides gras vegetales. Withdrawn EP0666865A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US97118292A 1992-11-02 1992-11-02
US971182 1992-11-02
PCT/US1993/010526 WO1994010189A1 (fr) 1992-11-02 1993-11-02 Synthases d'acides gras vegetales

Publications (2)

Publication Number Publication Date
EP0666865A1 true EP0666865A1 (fr) 1995-08-16
EP0666865A4 EP0666865A4 (fr) 1996-08-21

Family

ID=25518029

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94900499A Withdrawn EP0666865A4 (fr) 1992-11-02 1993-11-02 Synthases d'acides gras vegetales.

Country Status (4)

Country Link
EP (1) EP0666865A4 (fr)
JP (1) JPH08502891A (fr)
CA (1) CA2148358A1 (fr)
WO (1) WO1994010189A1 (fr)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6483008B1 (en) * 1990-08-15 2002-11-19 Calgene Llc Methods for producing plants with elevated oleic acid content
US5959175A (en) * 1997-04-09 1999-09-28 Thomas; Terry L. Sunflower albumin 5' regulatory region for the modification of plant seed lipid composition
AR013633A1 (es) 1997-04-11 2001-01-10 Calgene Llc METODO PARA LA ALTERACIoN DE LA COMPOSICIoN DE ÁCIDOS GRASOS DE CADENA MEDIA EN SEMILLAS VEGETALES QUE EXPRESAN UNA TIOESTERASA QUE PREFIERE CADENA MEDIA VEGETAL HETERoLOGA.
EP1624066A3 (fr) * 1997-04-11 2007-03-28 Calgene LLC Synthetases d'acides gras vegetaux et utilisation pour des procedes ameliores de production d'acides gras a chaine moyenne
WO1998050569A2 (fr) 1997-05-05 1998-11-12 Dow Agrosciences Llc Sequences nucleotidiques des genes codant pour la thio-esterase de la proteine transporteuse d'acyles d'oleoyle du mais et pour la thio-esterase de la proteine transporteuse d'acyles de palmitoyle et leur utilisation pour modifier la teneur en acides gras d'une huile
US6046029A (en) * 1997-11-24 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acids encoding human fatty acid synthase-like protein
US6365802B2 (en) * 1998-08-14 2002-04-02 Calgene Llc Methods for increasing stearate content in soybean oil
DE19926456A1 (de) * 1999-06-10 2000-12-14 Norddeutsche Pflanzenzucht Han Verfahren zur Erhöhung des Fettsäuregehalts in Pflanzensamen
US7067722B2 (en) 1999-08-26 2006-06-27 Monsanto Technology Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
US7531718B2 (en) 1999-08-26 2009-05-12 Monsanto Technology, L.L.C. Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
DE60037735T2 (de) 1999-08-26 2009-01-02 Calgene Llc, Davis Pflanzen mit veränderten mehrfachungesättigten fettsäuren
AU2001274402A1 (en) * 2000-05-24 2001-12-03 The University Of British Columbia Gene regulatory region that promotes early seed-specific transcription
US6706950B2 (en) 2000-07-25 2004-03-16 Calgene Llc Nucleic acid sequences encoding β-ketoacyl-ACP synthase and uses thereof
US7166771B2 (en) 2002-06-21 2007-01-23 Monsanto Technology Llc Coordinated decrease and increase of gene expression of more than one gene using transgenic constructs
US7566813B2 (en) 2002-03-21 2009-07-28 Monsanto Technology, L.L.C. Nucleic acid constructs and methods for producing altered seed oil compositions
BR0308614A (pt) 2002-03-21 2005-02-09 Monsanto Technology Llc Construções de ácidos nucléicos e processos para a produção de composições oleosas de sementes alteradas
CN101421406B (zh) 2006-02-13 2016-08-31 孟山都技术有限公司 用于产生改变的种子油组成的核酸构建体和方法
CA2738474C (fr) 2008-09-29 2020-05-12 Monsanto Technology Llc Evenement transgenique de soja t mon87705 et procedes pour la detection de celui-ci

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013972A1 (fr) * 1990-03-16 1991-09-19 Calgene, Inc. Desaturases de plantes - compositions et emplois
WO1991016421A1 (fr) * 1990-04-26 1991-10-31 Calgene, Inc. Thioesterases vegetales
WO1992003564A1 (fr) * 1990-08-15 1992-03-05 Calgene, Inc. Synthases d'acides gras de plantes
WO1993010240A1 (fr) * 1991-11-15 1993-05-27 E.I. Du Pont De Nemours And Company GENES DE β-CETOACYL-ACP-SYNTHETASE II, OBTENUS A PARTIR DE PLANTES

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5110728A (en) * 1986-07-31 1992-05-05 Calgene, Inc. Acyl carrier protein - DNA sequence and synthesis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013972A1 (fr) * 1990-03-16 1991-09-19 Calgene, Inc. Desaturases de plantes - compositions et emplois
WO1991016421A1 (fr) * 1990-04-26 1991-10-31 Calgene, Inc. Thioesterases vegetales
WO1992003564A1 (fr) * 1990-08-15 1992-03-05 Calgene, Inc. Synthases d'acides gras de plantes
WO1993010240A1 (fr) * 1991-11-15 1993-05-27 E.I. Du Pont De Nemours And Company GENES DE β-CETOACYL-ACP-SYNTHETASE II, OBTENUS A PARTIR DE PLANTES

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCIENCE, vol. 252, 5 April 1991, pages 80-87, XP002014300 SOMERVILLE, C., ET AL.: "PLANT LIPIDS: METABOLISM, MUTANTS, AND MEMBRANES" *
See also references of WO9410189A1 *

Also Published As

Publication number Publication date
EP0666865A4 (fr) 1996-08-21
WO1994010189A1 (fr) 1994-05-11
JPH08502891A (ja) 1996-04-02
CA2148358A1 (fr) 1994-05-11

Similar Documents

Publication Publication Date Title
EP0472722B1 (fr) Adn codant pour des desaturases de plantes et leurs emplois
EP0495096B1 (fr) Synthases d'acides gras de plantes
US7053267B2 (en) Plant seed oils
US6426447B1 (en) Plant seed oils
US5723761A (en) Plant acyl-ACP thioesterase sequences
CA2368414C (fr) Procedes servant a produire des plantes possedant une teneur importante en acides oleiques
US6828475B1 (en) Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
US5679881A (en) Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
WO1994010189A1 (fr) Synthases d'acides gras vegetales
US20080260933A1 (en) Certain Plants with "No Saturate" or Reduced Saturate Levels of Fatty Acids in Seeds, and Oil Derived from the Seeds
JPH0614667A (ja) デサチュラーゼを使用しての植物油の改変
US20180030463A1 (en) Certain plants with "no saturate" or reduced saturate levels of fatty acids in seeds, and oil derived from the seeds
AU763296B2 (en) Limanthes oil genes
WO1993010241A1 (fr) ACYL-CoA GRAS: ALCOOL ACYLTRANSFERASE GRASSE
JP2017537633A (ja) 低飽和脂肪酸または無飽和脂肪酸トランスジェニックキャノーラの生成
US20090138992A1 (en) Limnanthes oil genes
JPH09501325A (ja) 形質転換植物における蝋エステル
CA2205657A1 (fr) Sequences de stearoyl-(proteine vectrice d'acyle)-thioesterase vegetales et procedes pour augmenter la teneur en stearate des huiles de graines vegetales
WO1997012047A9 (fr) Sequences de stearoyl-(proteine vectrice d'acyle)-thioesterase vegetales et procedes pour augmenter la teneur en stearate des huiles de graines vegetales
KR20130026487A (ko) 식물 종자 내에 오메가-７ 지방산의 축적
Zebarjadi et al. Transformation of rapeseed (Brassica napus L.) plants with sense and antisense constructs of the fatty acid elongase gene
EP1624066A2 (fr) Synthétases d'acides gras végétaux et utilisation pour des procédés ameliores de production d'acides gras à chaine moyenne

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19941007

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

RHK1 Main classification (correction)

Ipc: C12N 15/82

A4 Supplementary search report drawn up and despatched

Effective date: 19960708

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

DA4 Supplementary search report drawn up and despatched (deleted)
RA4 Supplementary search report drawn up and despatched (corrected)

Effective date: 19960708

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: CALGENE LLC

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030603