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
  • 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
• • 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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0083—Miscellaneous (1.14.99)

Definitions

• Some embodiments generally relate to certain delta-9 desaturase enzymes, nucleic acids encoding these enzymes, and methods of expressing the same in a plant cell. Some embodiments relate to utilizing the activity of certain delta-9 desaturase enzymes to decrease the percent composition of saturated fatty acids in plant materials (e.g., seed) and increasing the percent composition of ⁇ -7 fatty acids. Further embodiments relate to utilizing seed- specific promoters to preferentially express delta-9 desaturase enzymes in seeds. Also disclosed herein are plants and plant materials produced by methods in particular embodiments, and oil produced by those plants which contains less than 3.5% or less than 2.7% saturated fatty acids. BACKGROUND
• Vegetable-derived oils have gradually replaced animal-derived oils and fats as the major source of dietary fat intake.
• saturated fat intake in most industrialized nations has remained at about 15% to 20% of total caloric consumption.
• USDA United States Department of Agriculture
• saturated fats make up less than 10% of daily caloric intake.
• current labeling guidelines issued by the USDA now require total saturated fatty acid levels be less than 1.0 g per 14 g serving to receive the "low-sat" label and less than 0.5 g per 14 g serving to receive the "no-sat” label.
• saturated and trans fats are not. Saturated fat and trans fat raise undesirable LDL cholesterol levels in the blood. Dietary cholesterol also raises LDL cholesterol and may contribute to heart disease even without raising LDL. Therefore, it is advisable to choose foods low in saturated fat, trans fat, and cholesterol as part of a healthful diet.
• oils whether of plant or animal origin, are determined predominately by the number of carbon and hydrogen atoms in the oil molecule, as well as the number and position of double bonds comprised in the fatty acid chain.
• Most oils derived from plants are composed of varying amounts of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) fatty acids.
• palmitic and stearic acids are designated as "saturated,” because their carbon chains are saturated with hydrogen atoms, and hence have no double bonds; they contain the maximal number of hydrogen atoms possible.
• oleic, linoleic, and linolenic acids are 18-carbon fatty acid chains having one, two, and three double bonds, respectively, therein.
• Oleic acid is typically considered a monounsaturated fatty acid
• linoleic and linolenic are considered to be polyunsaturated fatty acids.
• the U.S.D.A. definition of "no sat" oil products meaning those having less than 3.5% saturated fatty acid content, is calculated as the combined saturated fatty acid content by weight (as compared to the total amount of fatty acids).
• Canola oil has the lowest level of saturated fatty acids of all vegetable oils.
• “Canola” refers to rapeseed (Brassica) which has an erucic acid (C22:l ) content of at most 2% by weight, based on the total fatty acid content of a seed (preferably at most 0.5% by weight, and most preferably essentially 0% by weight), and which produces, after crushing, an air-dried meal containing less than 30 ⁇ /g of glucosinolates in defatted (oil-free) meal.
• rapeseed are distinguished by their edibility in comparison to more traditional varieties of the species.
• fatty acid synthesis occurs primarily in the plastid.
• the major product of fatty acid synthesis is palmitate (16:0), which appears to be efficiently elongated to stearate (18:0).
• the saturated fatty acids may then be desaturated by an enzyme known as acyl-ACP delta-9 desaturase, to introduce one or more carbon-carbon double bonds.
• acyl-ACP delta-9 desaturase an enzyme known as acyl-ACP delta-9 desaturase
• stearate may be rapidly desaturated by a plastidial delta-9 desaturase enzyme to yield oleate (18: 1).
• palmitate may also be desaturated to palmitoleate ( 16: 1 ) by the plastidial delta-9 desaturase, but this fatty acid appears in only trace quantities (0-0.2%) in most vegetable oils.
• the major products of fatty acid synthesis in the plastid are palmitate, stearate, and oleate.
• oleate is the major fatty acid synthesized, as the saturated fatty acids are present in much lower proportions.
• Newly-synthesized fatty acids are exported from the plastid to the cytoplasm. Subsequent desaturation of plant fatty acids in the cytoplasm appears to be limited to oleate, which may be desaturated to linoleate (18:2) and linolenate (18:3) by microsomal desaturases acting on oleoyl or lineoleoyl substrates esterified to phosphatidyl choline (PC). In addition, depending on the plant, oleate may be further modified by elongation (to 20: 1, 22: 1 , and/or 24: 1), or by the addition of functional groups. These fatty acids, along with the saturated fatty acids, palmitate and stearate, are then assembled into triglycerides in endoreticular membranes.
• oleate may be desaturated to linoleate (18:2) and linolenate (18:3) by microsomal desaturases acting on oleoyl or lineo
• the plant acyl-ACP delta-9 desaturase enzyme is soluble. It is located in the plastid stroma, and uses newly-synthesized fatty acids esterified to ACP, predominantly stearyl- ACP, as substrates. This is in contrast to the other delta-9 desaturase enzymes, which are located in the endoplasmic reticular membrane (ER, or microsomal), use fatty acids esterified to Co-A as substrates, and desaturate both the saturated fatty acids, palmitate and stearate.
• U.S. Patents 5,723,595 and 6,706,950 relate to a plant desaturase.
• the yeast delta-9 desaturase gene has been isolated from Saccharomyces cerevisiae, cloned, and sequenced. Stukey et al. (1989) J. Biol. Chem. 264: 16537-44; Stukey et al. (1990) J. Biol. Chem. 265:20144-9. This yeast gene has been introduced into tobacco leaf tissue (Polashcok et al. (1991 ) FASEB J. 5:A1157; Polashok et al. (1992) Plant Physiol. 100:894-901), and was apparently expressed in this tissue. Further, this yeast gene was expressed in tomato. See Wang et al. (1996) J. Agric. Food Chem.
• a different fungal acyl-CoA delta-9 desaturase from Aspergillus nidulans has been introduced into canola, thereby achieving reduced saturated fatty acid levels in seed oil.
• U.S. Patent Application Publication US 2008/0260933 Al The A. nidulans acyl-CoA delta-9 desaturase provided greater depletion of stearate (61 -90%) than the more abundant palmitate fatty acids (36-49%) in the seed oil.
• novel fungal delta-9 desaturase enzymes comprising at least one nucleotide sequence encoding such a desaturase; and plants, plant materials (e.g., seed), plant parts, and plant commodity products comprising either of the foregoing.
• fungal delta-9 desaturase enzymes isolated from Magnaporthe grisea, Leptosphaeria nodorum, and Helicoverpa zea.
• Some examples include native and synthetic delta-9 desaturases that have a substrate preference for palmitic acid or stearic acid.
• Some embodiments comprise an isolated nucleic acid molecule encoding a delta-9 desaturase enzyme comprising an amino acid sequence being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.
• the nucleic acid molecule comprises a sequence being at least 60% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
• These and further embodiments may include an isolated delta-9 desaturase polypeptide comprising an amino acid sequence being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:l , SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.
• Particular embodiments take advantage of a delta-9 desaturase enzyme's activity, such that the percent composition of saturated fatty acids may be decreased in a plant, plant material (e.g., seed), and/or plant part comprising the plant cell, and/or a plant commodity product produced from any of the foregoing.
• ⁇ -7 fatty acids may concomitantly be increased in the plant, plant material, plant part, and/or plant commodity product.
• Further embodiments take advantage of seed- specific expression to further lower the level of saturated fatty acids in seed oil.
• Some embodiments include a method for decreasing the amount of saturated fatty acids in a plant, plant material, plant part, and/or plant commodity product, the method comprising transforming a plant cell with a nucleic acid molecule encoding a delta-9 desaturase polypeptide of the invention, such that the amount of saturated fatty acids in the cell is decreased. Some embodiments include a method for creating a genetically engineered plant that comprises decreased amounts of saturated fatty acids in the plant compared to a wild-type plant of the same species.
• FIG. 1 shows a plasmid map of pDAB7305.
• FIG. 3 shows the distribution of TSFA in T2 seed population from three selected transgenic events as compared to the negative control NEXERA 710TM canola plants. Dark dots represent seed progeny with a TSFA lower than 3.5 % (dark line). As indicated in the graph, the plants with a TSFA lower than 3.5% produced varying amounts of yield and possessed from 2 to 10 copies numbers of the pat transgene that is contained on the same T- strand integrant as the AnD9DS transgene.
• FIG. 4 illustrates the distribution of TSFA and saturated fatty acid percentage in canola single seed (wild type control plants are excluded so that the graphs depict the TSFA values of transgenic canola events).
• nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. ⁇ 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
• SEQ ID NO:l shows the amino acid sequence of Aspergillus nidulans acyl-CoA delta- 9 desaturase protein (referred to in some places as AnD9DS).
• SEQ ID NO:2 shows the nucleic acid sequence of the v3 of the Aspergillus nidulas acyl-CoA delta-9 desaturase gene (referred to in some places as AnD9DS).
• SEQ ID NO:5 shows the nucleic acid sequence of the third PTU of pDAB7305.
• SEQ ID NO: 13 is an exemplary intronless MgD9DS clone
• SEQ ID NO: 14 shows an exemplary nucleic acid sequence encoding a first Leptosphaeria nodorum acyl-CoA delta-9 desaturase, referred to in some places as LnD9DS-l
• SEQ ID NO: 16 shows a coding region from an exemplary native delta-9 desaturase gene from M. grisea (labeled as MgD9DS vl).
• SEQ ID NO: 17 shows a coding region from an exemplary native delta-9 desaturase gene from Helicoverpa zea (labeled as HzD9DS
• SEQ ID NO: 18 shows a coding region from an exemplary native delta-9 desaturase
• SEQ ID NO:21 shows the sequence of an exemplary canola-optimized delta-9 desaturase gene from L. nodorum (LnD9DS-2 v2).
• SEQ ID NO:22 shows the sequence of a further exemplary canola-optimized delta-9 desaturase gene from L. nodorum (LnD9DS-2 v3).
• SEQ ID NO:23 shows the sequence of a further exemplary canola-optimized delta-9 desaturase gene from H. zea (HzD9DS v3).
• SEQ ID NO:24 shows an exemplary nucleic acid sequence encoding an Aspergillus nidulans delta-9 desaturase, referred to in some places as AnD9DS v2.
• SEQ ID NO:25 shows a second exemplary nucleic acid sequence encoding an A. nidulans delta-9 desaturase, referred to in some places as AnD9DS v3.
• SEQ ID NO:26 shows the amino acid sequence of an exemplary native delta-9 desaturase from M. grisea (MgD9DS).
• SEQ ID NO:27 shows the amino acid sequence of an exemplary native delta-9 desaturase from H. zea (HzD9DS).
• SEQ ID NO:28 shows the amino acid sequence of an exemplary native delta-9 desaturase from L. nodorum (LnD9DS-2).
• SEQ ID NO:29 shows the amino acid sequence encoded by nucleic acids as exemplified by SEQ ID NOs:24-25 (AnD9DS).
• SEQ ID NO:30 shows the amino acid sequence of another exemplary AnD9DS desahirase.
• SEQ ID NO:31 shows the amino acid sequence of an exemplary native delta-9 desaturase (ScOLEl) from Saccharomyces cerevisiae.
• SEQ ID NO:32 shows the N-terminal 68 residues (1-68) of an exemplary AnD9DS desaturase.
• SEQ ID NO:33 shows the C-terminal 175 residues (281-455) of an exemplary AnD9DS desaturase.
• nucleic acid molecules encoding a delta-9 desaturase polypeptide comprising a nucleotide sequence being at least 60% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
• the nucleic acid molecule may further comprise a gene regulatory element operably linked to the delta-9 desaturase polypeptide-encoding sequence.
• a gene regulatory element may be a phaseolin promoter, a phaseolin 5' untranslated region, a phaseolin 3' untranslated region ("UTR"), an Agrobacterium tumefaciens ORF1 3' untranslated region, a Cassava vein Mosaic Virus promoter, a Nicotiana tabacum RB7 Matrix Attachment Region, a T-strand border sequence, a LfKCS3 promoter, and FAE 1 promoter.
• the gene regulatory elements may be phaseolin promoter and phaseolin 5' UTR, and Lesquerella fenderi LfKCS3 promoter, such that two copies of the AND9DS is present, one copy controlled by the phaseolin promoter and 5'UTR, and a second copy controlled by the LfKCS3 promoter.
• the several copies of the nucleic acid encoding a delta-9 desaturase polypeptide may be under the control of other regulatory elements, including the Saccharomyces cerevisiae delta-9 desaturase promoter, the delta-9 desaturase 3'UTR/terminator, the olel gene promoter, the Phaseolus vulgaris phaseolin 3' untranslated region, the Phaseolus vulgaris phaseolin matrix attachment region, the Agrobacterium tumefaciens Mannopine Synthase promoter, the Agrobacterium tumefaciens ORF23 3' untranslated region, the Cassava vein Mosaic Virus Promoter, the Agrobacterium tumefaciens ORFl 3' untranslated region, the Nicotiana tabacum RB7 Matrix Attachment Region, Overdrive, T-stand border sequences, the LfKCS3 promoter, FAE 1 promoter,
• delta-9 desaturase polypeptides comprising an amino acid sequence being at least 80% identical to a sequence selected from the group consisting of SEQ ID NOT as well as nucleic acid molecules encoding such delta-9 desaturase polypeptides, such as SEQ
• nucleic acid molecules and delta-9 desaturase polypeptides may be expressed in a plant material, cell, tissue, or whole plant, to decrease the amount of saturated fatty acids in the plant material, cells, tissues, or whole plants, relative to the amount observed in a wild-type plant of the same species.
• Alternative embodiments of the invention include methods for decreasing the amount of saturated fatty acids in the plant material, cell, tissue, or whole plant. Such methods may comprise transforming a plant material, cell, tissue, or whole plant with at least one of the aforementioned nucleic acid molecules, such that the amount of saturated fatty acids in the plant material, cell, tissue, or whole plant is decreased.
• Particular embodiments include methods for preferentially decreasing palmitic and/or stearic fatty acids in a plant material, cell, tissue, or whole plant.
• Methods disclosed herein may be performed, for example, on plants, or plant materials derived from plants (e.g., plants of the genus Arabidopsis, or canola).
• a particular embodiment is drawn to methods for creating or regenerating a genetically engineered plant comprising decreased amounts of saturated fatty acids in the plant compared to a wild-type plant of the same species, the method comprising transforming a plant cell or material with at least one of the aforementioned nucleic acid molecules; and culturing the transformed plant material to obtain a plant.
• Plants, plant materials, plant cells, and seeds obtained by any of the aforementioned methods are also disclosed.
• Metabolic pathway refers to a series of chemical reactions occurring within a cell, catalyzed by enzymes, to achieve either the formation of a metabolic product, or the initiation of another metabolic pathway.
• a metabolic pathway may involve several or many steps, and may compete with a different metabolic pathway for specific reaction substrates.
• the product of one metabolic pathway may be a substrate for yet another metabolic pathway.
• Metabolic engineering refers to the rational design of strategies to alter one or more metabolic pathways in a cell, such that the step-by-step modification of an initial substance into a product having the exact chemical structure desired is achieved within the overall scheme of the total metabolic pathways operative in the cell.
• Desaturase refers to a polypeptide that can desaturate (i.e., introduce a double bond) in one or more fatty acids to produce a fatty acid or precursor of interest.
• a plant-soluble fatty acid desaturase enzyme may introduce a double bond regiospecifically into a saturated acyl-ACP substrate.
• Acyl-CoA desaturases introduce a double bond regiospecifically into a saturated fatty acyl-CoA substrate. The reaction involves activation of molecular oxygen by a two-electron reduced diiron center coordinated by a four- helix bundle that forms the core of the desaturase architecture.
• acyl-CoA delta-9 desaturases are particularly interest in some embodiments.
• delta-9-18:0'-ACP desaturase is required by all plants for the maintenance of membrane fluidity. While this enzyme primarily desaturates stearoyl-ACP, it is also active to a minor extent with palmitoyl-ACP.
• Progeny plant refers to any plant, or plant material obtained therefrom, that may be obtained by plant breeding methods. Plant breeding methods are well-known in the art, and include natural breeding, artificial breeding, selective breeding involving DNA molecular marker analysis, transgenics, and commercial breeding.
• Plant material refers to any cell or tissue obtained from a plant.
• Nucleic acid molecule A polymeric form of nucleotides, which can include both sense and anti-sense strands of R A, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
• a nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide.
• a "nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double- stranded forms of DNA.
• a nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non- naturally occurring nucleotide linkages.
• Nucleic acid molecules can be modified chemically or biochemically, or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of ordinary skill in the art. Such modification include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendent moieties (for example, peptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.).
• the term "nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexe
• a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence.
• a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
• operably linked nucleic acid sequences are generally contiguous and, where necessary to join two protein-coding regions, in the same reading frame. However, nucleic acids need not be contiguous to be operably linked.
• regulatory element refers to a nucleic acid molecule having gene regulatory activity; i.e., one that has the ability to affect the transcription or translation of an operably-1 inked transcribable nucleic acid molecule. Regulatory elements such as promoters, leaders, introns, and transcription termination regions are non-coding nucleic acid molecules having gene regulatory activity which play an integral part in the overall expression of genes in living cells. Isolated regulatory elements that function in plants are therefore useful for modifying plant phenotypes through the techniques of molecular engineering. By “regulatory element,” it is intended a series of nucleotides that determines if, when, and at what level a particular gene is expressed. The regulatory DNA sequences specifically interact with regulatory proteins or other proteins.
• gene regulatory activity refers to a nucleic acid molecule capable of affecting transcription or translation of an operably linked nucleic acid molecule.
• An isolated nucleic acid molecule having gene regulatory activity may provide temporal or spatial expression or modulate levels and rates of expression of the operably linked nucleic acid molecule.
• An isolated nucleic acid molecule having gene regulatory activity may comprise a promoter, intron, leader, or 3' transcriptional termination region.
• Promoters refers to a nucleic acid molecule that is involved in recognition and binding of RNA polymerase II or other proteins such as transcription factors (trans-acting protein factors that regulate transcription) to initiate transcription of an operably linked gene. Promoters may themselves contain sub-elements such as cis-elements or enhancer domains that effect the transcription of operably linked genes.
• a "plant promoter” is a native or non-native promoter that is functional in plant cells. A plant promoter can be used as a 5' regulatory element for modulating expression of an operably linked gene or genes. Plant promoters may be defined by their temporal, spatial, or developmental expression pattern.
• the nucleic acid molecules described herein may comprise nucleic acid sequences comprising promoters.
• sequence identity refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
• sequence similarity When 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, hydrophobicity, or steric effects), and therefore do not change the functional properties of the molecule. Therefore, when sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution at the site of the non-identical residue. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or “similarity.” Techniques for making this adjustment are well known to those of ordinary skill in the art.
• such techniques involve scoring a conservative substitution as a partial, rather than a full, mismatch, thereby increasing the percentage sequence identity. For example, where an identical amino acid is given a score between 0 and 1, and a non-conservative substitution is given a score of 0, a conservative substitution is given a score between 0 and 1.
• the scoring of conservative substitutions may be calculated, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, CA).
• the term "percentage of sequence identity” may refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the 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 nucleotide 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 comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
• NCBI National Center for Biotechnology Information
• Blast.cgi Basic Local Alignment Search Tool
• the "Blast 2 sequences" function of the BLAST program (bl2seq) is employed using the default parameters. Specific parameters may be adjusted within the discretion of one of skill in the art, to for example, provide a penalty for a mismatch or reward for a match.
• transformed refers to a cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid molecule, such as a construct.
• the introduced nucleic acid molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced polynucleotide molecule is inherited by subsequent progeny.
• a "transgenic” or “transformed” cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a transgenic plant as a parent in, for example, a cross and exhibiting an altered phenotype resulting from the presence of a foreign nucleic acid molecule.
• nucleic acid molecules of the present invention comprise a gene regulatory element ⁇ e.g., a promoter).
• Promoters may be selected on the basis of the cell type into which the vector construct will be inserted. Promoters which function in bacteria, yeast, and plants are well-known in the art. The promoters may also be selected on the basis of their regulatory features. Examples of such features include enhancement of transcriptional activity, inducibility, tissue-specificity, and developmental stage-specificity. In plants, promoters that are inducible, of viral or synthetic origin, constitutively active, temporally regulated, and spatially regulated have been described. See, e.g., Poszkowski et al. (1989) EMBO J. 3:2719; Odell et al. (1985) Nature 313:810; and Chau et al. (1989) Science 244: 174-81).
• Useful inducible promoters include, for example, promoters induced by salicylic acid or polyacrylic acids induced by application of safeners (substituted benzenesulfonamide herbicides), heat-shock promoters, a nitrate-inducible promoter derived from the spinach nitrate reductase transcribable nucleic acid molecule sequence, hormone-inducible promoters, and light-inducible promoters associated with the small subunit of RuBP carboxylase and LHCP families.
• a heterologous gene(s) it may be preferred to reengineer the gene(s) so that it is more efficiently expressed in the expression host cell (e.g. , a plant cell, for example, canola, rice, tobacco, maize, cotton, and soybean). Therefore, an optional additional step in the design of a gene encoding a delta-9 desaturase for plant expression (i.e., in addition to the provision of one or more gene regulatory elements) is reengineering of a heterologous gene protein coding region for optimal expression.
• Particular embodiments include redesigned genes that have been optimized to increase the expression level (i.e. produce more protein) in a transgenic canola plant cell or Arabidopsis plant cell than in a canola plant cell or Arabidopsis plant cell transformed with the naturally-occurring heterologous gene sequence.
• Some embodiments are directed to a method of producing a transformed cell that comprises one or more nucleic acid molecule(s) comprising a nucleic acid sequence at least 60% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
• glyphosate resistance may be conferred by a herbicide resistance gene.
• Other selection devices can also be implemented, including for example and without limitation, tolerance to phosphinothricin, bialaphos, and positive selection mechanisms (Joersbro et al. (1998) Mol. Breed. 4:11 1-7), and are considered within the scope of embodiments of the present invention.
• the regenerated transgenic plants may be self-pollinated to provide homozygous transgenic plants.
• pollen obtained from the regenerated transgenic plants may be crossed with non-transgenic plants, preferably inbred lines of agronomically important species.
• pollen from non-transgenic plants may be used to pollinate the regenerated transgenic plants.
• the transgenic plant may pass along the transformed nucleic acid sequence to its progeny.
• the transgenic plant is preferably homozygous for the transformed nucleic acid sequence and transmits that sequence to all of its offspring upon, and as a result of, sexual reproduction.
• Progeny may be grown from seeds produced by the transgenic plant. These additional plants may then be self-pollinated to generate a true breeding line of plants.
• a transgenic seed may comprise a delta-9 desaturase polypeptide comprising an amino acid sequence being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:l , SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, and SEQ ID NO:33.
• AnD9DS coding sequence (An delta 9 desaturase v3)
• Agrobacterium tumefaciens ORF 23 3' untranslated region (AtuORF23 3'UTR; U.S. Patent No. 5,428, 147).
• the third PTU (SEQ ID NO:5) was comprised of Agrobacterium tumefaciens Mannopine Synthase promoter (AtuMas promoter; Barker, R. F., Idler, K. B., Thompson, D. V., Kemp, J.
• Example 2 Agrobacterium-mediated transformation of Canola (Brassica napus) hypocotyls Agrobacterium Preparation
• the Agrobacterium strain containing the pDAB7305 binary plasmid was streaked out on YEP media (Bacto PeptoneTM 20.0 gm/L and Yeast Extract 10.0 gm/L) plates containing streptomycin (100 mg/ml) and spectinomycin (50 mg/mL) and incubated for 2 days at 28°C.
• the propagated Agrobacterium strain containing the pDAB7305 binary plasmid was scraped from the 2-day streak plate using a sterile inoculation loop.
• Callus induction on selection medium After 3 days of co-cultivation, the hypocotyl segments were individually transferred with forceps onto callus induction medium, MSK1D1H1 (MS, 1 mg/L kinetin, 1 mg/L 2,4-D, 0.5 gm/L MES, 5 mg/L AgN0 3 , 300 mg/L TimentinTM, 200 mg/L carbenicillin, 1 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar) with growth regime set at 22-26°C. The hypocotyl segments were anchored on the medium but were not deeply embedded into the medium.
• MSK1D1H1 MS, 1 mg/L kinetin, 1 mg/L 2,4-D, 0.5 gm/L MES, 5 mg/L AgN0 3 , 300 mg/L TimentinTM, 200 mg/L carbenicillin, 1 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar
• MSB3Z1 H3 MS, 3 mg/L BAP, 1 mg/L Zeatin, 0.5 gm/L MES, 5 mg/L AgN0 3 , 300 mg/1 TimentinTM, 200 mg/L carbenicillin, 3 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar
• growth regime set at 22- 26°C.
• Root induction After 14 days of culturing on the shoot elongation medium, the isolated shoots were transferred to MSMEST medium (MS, 0.5 g/L MES, 300 mg/L TimentinTM, 2% sucrose, 0.7% TC Agar) for root induction at 22-26 °C. Any shoots which did not produce roots after incubation in the first transfer to MSMEST medium were transferred for a second or third round of incubation on MSMEST medium until the shoots developed roots.
• MSMEST medium MS, 0.5 g/L MES, 300 mg/L TimentinTM, 2% sucrose, 0.7% TC Agar
• PCR analysis Transformed canola hypocotyl segments which regenerated into shoots comprising roots were further analyzed via a PCR molecular confirmation assay. Leaf tissue was obtained from the green shoots and tested via the PCR for the presence of the pat selectable marker gene. Any chlorotic shoots were discarded and not subjected to the PCR analysis. Samples that were identified as positive for presence of the pat selectable marker gene were kept and cultured on the MSMEST medium to continue development and elongation of the shoots and roots. The samples that were identified as not containing the pat selectable marker gene negative according to the PCR analysis were discarded.
• the transformed canola plants comprising shoots and roots that were PCR-positive for the presence of the pat selectable marker gene were transplanted into soil in a greenhouse. After establishment of the canola plants within soil, the canola plants were further analyzed to quantitate the copy number of the pat gene expression cassette via an InvaderTM quantitative PCR assay and Southern blotting. Transgenic To canola plants which were confirmed to contain at least one copy of the pat gene expression cassette were advanced for further analysis of the seed fatty acid profile.
• T 0 canola plants i.e., T
• canola seeds were analyzed via a FAME analysis method to identify events which comprise a reduction in total saturated fatty acids (total saturated fatty acid content was determined by summing all of the saturated fatty acids, including short and long chain fatty acids) as compared to control plants.
• Example 3 FAME analysis of Tj canola seeds obtained from transgenic pDAB7305 canola plants
• Segregating T] canola seeds were analyzed via a FAME analysis method to identify To canola events which produced Ti canola seeds comprising a reduction in total saturated fatty acids (C14:0, C16:0, CI 8:0, C20:0, C22:0, C24:0) as compared to seeds obtained from control plants grown in the same conditions.
• the sum of all Total Saturated Fatty Acids (TSFA) were quantitated and compared to a negative control plant.
• the FAME analysis was completed using the protocol described below on a single Tj canola seed. A total of 24 single Ti canola seed from each individual canola To event were assayed and the TSFA results from each single were quantitated.
• the resulting FAMEs were analyzed by GC-FID using a capillary column BPX 70 from SGE (15 m x 0.25 mm x 0.25 ⁇ ). Each FAME was identified by retention time and quantified by the injection of a rapeseed oil reference mix from Matreya LLC (Pleasant Gap, PA) as a calibration standard with addition of appropriate long chain fatty acids (Nu-Chek Prep, Elysian MN).
• Event 2182[12]-138.001 Event 2182[12]- 125.001 , and Event 2182[ 12]- 1 56.001 ) were identified and selected for advancement to the T) generation based on the FAME results which indicated a significant reduction in TSFA as compared to control canola plants.
• MUFA Mono Unsaturated Fatty Acid
• PUFA Poly Unsaturated Fatty Acid
• Table 1 Summary composition of single Ti seed TSFA, MUFA and PUFA accumulations obtained from three transgenic canola events as compared to several NEXERA 710TM non-transformed control plants. N* indicates the number of individual T] seed analyzed for each plant progeny.
• TSFA of the transgenic canola events is reduced significantly as compared to the NEXERA 710TM non-transformed control plants. Concomitant to the reduction of TSFA an increase in MUFA content (CI 8: 1 and C16: l) was observed.
• the increase in MUFA content is the direct result from the over-expression of the AnD9DS introducing a double bond at the 9 th carbon ( ⁇ 9) from the carboxylic function of saturated fatty acid.
• the PUFA content did not increase with the accumulation of MUFA substrate of phosphoglycerolipid desaturase FAD2 synthesizing CI 8:2.
• the TSFA (%) of the transgenic plants of the subject disclosure were quantitated and compared to the TSFA (%) obtained from the positive control, 218-1 1.30HL transgenic canola plants and the negative control, NEXERA 710TM plants.
• a small number of transgenic plants of the subject disclosure were identified to contain higher levels of TSFA, at levels similar to the negative control NEXERA 710TM plants. These plants are sibling nulls which resulted from segregation of the transgenes during self-fertilization, and do not contain any actively expressing copies of the transgenes of the subject disclosure.
• T 2 canola lines were further analyzed to determine which canola lines contained low copy numbers of the pDAB7305 T-strand integrant and produced the high T 2 seed yield. (FIG. 3).
• T 2 canola plant lines were transferred to the greenhouse, grown to maturity and self-fertilized. The T 2 canola plant lines were further analyzed molecularly and the T3 seed was harvested for fatty acid profile analysis via FAME assay.
• T 2 canola lines which were selected based on highest yield (seed weight), lowest PAT copy number (Tl plants) and lowest TSFA are listed.
• Transgene copy number determination by hydrolysis probe assay was performed by real-time PCR using the LIGHTCYCLER ® 480 system (Roche Applied Science, Indianapolis, IN). Assays were designed for pat and an internal reference gene HMG1 (Weng et al. (2005). J. AOAC Int. 88(2):577-84) using the LIGHTCYCLER ® Probe Design Software 2.0. For amplification, LIGHTCYCLER ® 480 Probes Master mix (Roche Applied Science, Indianapolis, IN) was prepared at IX final concentration in a 10 ⁇ ⁇ volume multiplex reaction containing 0.4 ⁇ of each primer for AnD9DS and pat and 0.2 ⁇ of each probe (Table 3).
• a two-step amplification reaction was performed with an extension at 60°C for 40 seconds with fluorescence acquisition. All samples were run and the averaged Cycle threshold (Ct) values were used for analysis of each sample. Analysis of real time PCR data was performed using LIGHTCYCLER ® software release 1.5 using the relative quant module and is based on the AACt method. Controls included a sample of genomic DNA from a single copy calibrator and known two copy check that were included in each run. Table 4 lists the results of the hydrolysis probe assays. . Copy number was determined from N plants per Tl line (and averaged, giving the value in Table 4), using an qPCR Assay.
• Table 3 Primer and probe sequences used for hydrolysis probe assay of pat and internal reference (HMG1).
• Table 4 Copy amount results for the AnD9DS events (T2 plants) as determined using the hydrolysis probe assay.
• the results of the hydrolysis probe assay identified two lines (2182[12]- 138.Sx001.Sx094 and 2182[12]- 138.SX001.Sx090) which had a combination of relative standard deviation (shown in Table 4 as SD) and coefficient of variation (shown in Table 4 as CV %) that were comparable to the positive control plants (218- 11.30(HL)).
• the 218- 1 1.30(HL) control plants were previously identified to contain two fixed copies of the AnD9DS gene insertion (WO 2006042049).
• Southern blot analysis was used to establish the integration pattern of the inserted T- strand DNA fragment and identify canola lines which contained a full length AnD9DS gene expression cassette. Data were generated to demonstrate the integration and integrity of the transgene inserts within the canola genome. The detailed Southern blot analysis was conducted using a PCR amplified probe specific to the AnD9DS gene expression cassette.
• the hybridization of the probe with genomic DNA that had been digested with specific restriction enzymes identified genomic DNA fragments of specific molecular weights, the patterns of which were used to characterize the transgenic events for advancement to the next generation.
• Tissue samples were collected in 2 mL conical tubes and lyophilized for 2 days. Tissue maceration was performed with a KLECKOTM tissue pulverizer and tungsten beads. Following tissue maceration, the genomic DNA was isolated using a CTAB isolation procedure. The genomic DNA was further purified using the Qiagen Genomic TipsTM kit. Genomic DNA was quantified by Quant-IT Pico Green DNATM assay kit (Molecular Probes, Invitrogen, Carlsbad, CA). Quantified genomic DNA was adjusted to a consistent concentration.
• genomic DNA was thoroughly digested with the restriction enzyme BamHI (New England Biolabs, Beverley, MA). The digested DNA was concentrated by precipitation with Quick Precipitation SolutionTM (Edge Biosystems, Gaithersburg, MD) according to the manufacturer's suggested protocol. The genomic DNA was then resuspended in 25 i of water at 65°C for 1 hour. Resuspended samples were loaded onto a 0.8% agarose gel prepared in IX TAE and electrophoresed overnight at 1.1 V/cm in IX TAE buffer. The gel was sequentially subjected to denaturation (0.2 M NaOH / 0.6 M NaCl) for 30 minutes, and neutralization (0.5 M Tris-HCl (pH 7.5) / 1.5 M NaCl) for 30 minutes.
• BamHI New England Biolabs, Beverley, MA
• BamHI Quick Precipitation SolutionTM (Edge Biosystems, Gaithersburg, MD) according to the manufacturer's suggested protocol.
• the genomic DNA was then re
• Transfer of DNA fragments to nylon membranes was performed by passively wicking 20X SSC solution overnight through the gel onto treated IMMOBILONTM NY+ transfer membrane (Millipore, Billerica, MA) by using a chromatography paper wick and paper towels. Following transfer, the membrane was briefly washed with 2X SSC, cross-linked with the STRATALINKERTM 1800 (Stratagene, LaJolla, CA), and vacuum baked at 80°C for 3 hours.
• Blots were incubated with pre-hybridization solution (Perfect Hyb plus, Sigma, St. Louis, MO) for 1 hour at 65°C in glass roller bottles using a model 400 hybridization incubator (Robbins Scientific, Sunnyvale, CA). Probes were prepared from a PCR fragment containing the entire coding sequence. The PCR amplicon was purified using Q1AEX II gel extraction kitTM and labeled with DIG DNA Labeling KitTM (Roche Applied BioSciencse, Indianapolis, IN). Blots were hybridized overnight at 65°C with denatured probe added directly to hybridization buffer. Following hybridization, blots were sequentially washed at 65°C with 0.1X SSC / 0.1% SDS for 40 minutes. Finally, the blots were exposed to storage phosphor imaging screens and imaged using a Molecular Dynamics Storm 860TM. imaging system.
• Table 5 provides the banding profile of multiple T2 plants from selected lines based on the criteria defined above in Table 2. The control lines did not contain the selectable marker confirming the PCR data. Most of the lines selected from the three events show a homogeneous band pattern (number and size) except line from event 2182[12]- 125.Sx001.Sx014. All three T2 lines from event 2182[12]-138.Sx001 display T2 populations with consistent banding pattern.
• Table 5 Summary of Southern analysis completed on multiple T 2 canola lines from four transgenic events and the NEXERA 710TM canola control plants. The sizes of the observed bands for each sample were sized by comparison to a known standard run beside the samples on an agarose gel.
• T 2 canola lines Selected plants from the T 2 canola lines were grown to maturity in the greenhouse. Seed was harvested from the plants. The seed was cleaned and the yield of seed per T 2 canola line was detennined (Table 6). The yield of the seed from each line and compared to the yield of seed obtained from the untransformed control plants (NexeraTM 710GS) grown in the same conditions. Table 6 presents the yield results for the various plants which were obtained from each T 2 canola line. These results illustrate that the yield was variable for each plant and line tested.
• Example 7 FAME analysis of T 3 canola seeds obtained from transgenic pDAB7305 canola plants
• Both, single and bulked T3 canola seeds were analyzed via the previously described FAME analysis method to characterize the fatty acid profile of the lines to identify specific lines which resulted in a reduction in total saturated fatty acids as compared to control plants.
• the sum of the total saturated fatty acids were quantitated and compared to positive control and negative control plants.
• a sub-set of canola lines were used for seed FAME analysis to determine the lowest level of total saturated fatty acid and saturated fatty acid levels which could be obtained in a single canola seed.
• the single seed FAME analysis was completed on seeds obtained from the canola lines that were selected based on the lowest total saturated fatty acid of bulked seed and the high levels of plant yield. A total of 288 individual seeds were analyzed per line using the FAME method. The summary of the analysis is presented in Table 9. All single seeds from selected plants have a mean TSFA below 2.8%, which is significantly below the 3.5% TSFA level. The lowest TSFA level is 2.25% at the single seed canola level.
• Table 7 shows the distribution of T 2 mature seed bulk FAMEs analysis for five populations of genetically homogenous canola lines as compared to the untransformed NexeraTM 710G5 controls and transformed 218-11.30(HL) positive control plants. The average of all the individual measurement (N) were determined to represent TSFA and saturated percentage for the population of canola plants.
• Canola lines, 2182[12]- 138.Sx001.Sx085 and 2182[12]-138.Sx001.Sx094 are identified via bold print as these lines had an average TSFA percentage below 3.00 percent.
• Table 8 shows the lowest T3 mature seed bulk FAMEs and plant yield of single ⁇ progeny plants obtained from event 2182[12]- 138.Sx001as compared to NexeraTM 710G5 control. The results displayed are for percentage of oil, percentage of TSFA, percentage of saturated fatty acids (as determined by summing the palmitic and stearic acid content), and seed yield. Table 8
• Table 9 shows the distribution of T 3 single seed FAMEs analysis results from selected T2 lines.
• the table shows the average (Mean), minimum (Min), and maximum (Max) TSFA and saturated fatty acid percentage, as compared to a NexeraTM 710G5 control canola plants that grown in the same condition. There was a reduction of total saturated fatty acids (TSFA) and saturated fatty acid level in T3 seed of selected events as compared to a NexeraTM 710G5 control canola plants.
• TSFA total saturated fatty acids

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