# Patent

## Title
Generation of transgenic canola with low or no saturated fatty acids

## Abstract

Compositions and methods include genetically encoding and expressing a novel delta-9 desaturase in plant cells. In some embodiments, methods of expressing nucleic acids in a plant cell to take advantage of the delta-9 desaturase enzyme's activity, such that the percent composition of saturated fatty acids in plant seeds is decreased and there is a concomitant increase in Δ9 fatty acids. In other embodiments, amino acid sequences have delta-9 desaturase activity. Methods can involve expression of delta-9 desaturase in plant cells, plant materials, and whole plants for the purpose of increasing the amount of mono unsaturated fatty acids in whole plants, plant seeds, and plant materials, for example, seeds.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application U.S. patent application Ser. No. 13/168,742, filed Jun. 24, 2011, which is a continuation-in-part of application U.S. patent application Ser. No. 11/576,750, which is a national phase entry of PCT International Patent Application No. PCT/US05/36052, filed Oct. 7, 2005, designating the United States of America, and published, in English, as PCT International Publication No. WO 2006/042049 A2 on Apr. 20, 2006. PCT International Patent Application No. PCT/US05/36052 is a continuation of U.S. Provisional Patent Application No. 60/617,532, filed Oct. 8, 2004. The contents of the entirety of each of the foregoing are hereby incorporated in their entireties herein by this reference.


FIELD OF THE INVENTION
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 ω-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. However, saturated fat intake in most industrialized nations has remained at about 15% to 20% of total caloric consumption. In efforts to promote healthier lifestyles, the United States Department of Agriculture (USDA) has recently recommended that saturated fats make up less than 10% of daily caloric intake. To facilitate consumer awareness, 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. This means that the saturated fatty acid content of plant oils needs to be less than 7% and 3.5% to receive the “low-sat” or “no-sat” label, respectively. Since issuance of these guidelines, there has been a surge in consumer demand for “low-sat” and “no-sat” oils. To date, this demand has been met principally with canola oil, and to a much lesser degree with sunflower and safflower oils.
While unsaturated fats (monounsaturated and polyunsaturated) are beneficial (especially when consumed in moderation), 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.
The characteristics of 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. Conventionally, 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. However, 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, whereas 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 (C22:1) 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 μmol/g of glucosinolates in defatted (oil-free) meal. These types of rapeseed are distinguished by their edibility in comparison to more traditional varieties of the species.
It is postulated that, in oilseeds, 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). While still in the plastid, 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. Specifically, stearate may be rapidly desaturated by a plastidial delta-9 desaturase enzyme to yield oleate (18:1). In fact, 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. Thus, the major products of fatty acid synthesis in the plastid are palmitate, stearate, and oleate. In most oils, 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.
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. Pat. Nos. 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. 44:3399-402; and Wang et al. (2001) Phytochemistry 58:227-32. While some increases in certain unsaturated fatty acids, and some decreases in certain saturated fatty acids, were reported for both tobacco and tomato using this yeast delta-9 desaturase gene, tobacco and tomato are clearly not oil crops. This yeast gene was also introduced into Brassica napus. U.S. Pat. No. 5,777,201.
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 A1. 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.
BRIEF SUMMARY OF THE INVENTION
Disclosed herein are novel fungal delta-9 desaturase enzymes; nucleic acids 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. Aspects of some embodiments are exemplified by 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:1, 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. In particular examples, 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:1, 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.
Also disclosed are methods of expressing at least one of the aforementioned nucleic acids and/or polypeptides in a plant cell. 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. In certain embodiments, ω-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. Such a method may comprise transforming a plant material (or plant cell) with a nucleic acid molecule encoding one or more delta-9 desaturase polypeptides, or one or more copies of a delta-9 desaturase polypeptide of the invention, and culturing the transformed plant material (or plant cell) to obtain a plant. In particular examples, a plant cell and/or plant material from an Arabidopsis sp. may be transformed with a nucleic acid molecule encoding a delta-9 desaturase polypeptide of the invention. In other particular examples, the two or more copies of the delta-9 desaturase gene may be transformed, where each delta-9 desaturase gene is controlled by a different promoter. In other particular examples, the two or more promoters are seed specific promoters.
The foregoing and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a plasmid map of pDAB7305.
FIG. 2 illustrates the distribution of TSFA (%) in bulk T2 seed from segregating T1 canola plants compared to NEXERA 710TM canola control plants and the positive control plants comprised of the 218-11.30HL transgenic canola plants.
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).



SEQUENCE LISTING
The 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. In the accompanying sequence listing:
SEQ ID NO:1 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:3 shows the nucleic acid sequence the first plant transcription unit (PTU) of pDAB7305.
SEQ ID NO:4 shows the nucleic acid sequence of the second PTU of pDAB7305.
SEQ ID NO:5 shows the nucleic acid sequence of the third PTU of pDAB7305.
SEQ ID NOS:6-11 show the sequence of primers and probes that may be useful in some embodiments.
SEQ ID NO:12 is an exemplary fragment of a M. grisea acyl-CoA delta-9 desaturase gene (referred to in some places as MgD9DS) that was amplified by PCR
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-1
SEQ ID NO:15 shows an exemplary nucleic acid sequence encoding a second exemplary L. nodorum acyl-CoA delta-9 desaturase, referred to in some places as LnD9DS-2
SEQ ID NO:16 shows a coding region from an exemplary native delta-9 desaturase gene from M. grisea (labeled as MgD9DS v1).
SEQ ID NO:17 shows a coding region from an exemplary native delta-9 desaturase gene from Helicoverpa zea (labeled as HzD9DS v/).
SEQ ID NO:18 shows a coding region from an exemplary native delta-9 desaturase (LnD9DS-2 v1) gene from L. nodorum. 
SEQ ID NO:19 shows the sequence of an exemplary canola-optimized delta-9 desaturase gene from M. grisea (MgD9DS v2).
SEQ ID NO:20 shows the sequence of an exemplary canola-optimized delta-9 desaturase gene from H. zea (HzD9DS v2).
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 desaturase.
SEQ ID NO:31 shows the amino acid sequence of an exemplary native delta-9 desaturase (ScOLE1) 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.
DETAILED DESCRIPTION
I. Overview of Several Embodiments
We previously introduced a fungal acyl-CoA delta-9 desaturase from Aspergillus nidulans into canola, thereby achieving reduced saturated fatty acid levels in seed oil. U.S. Patent Application Publication US 2008/0260933 A1. The A. nidulans delta-9 desaturase provided greater depletion of stearate (61-90%) than the more abundant palmitate fatty acids (36-49%) in the seed oil. It was discovered that providing multiple copies of the A. nidulans delta-9 desaturase was able to reduce the saturated fatty acid levels in canola below 3.5%.
Disclosed herein are 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. In some embodiments, the nucleic acid molecule may further comprise a gene regulatory element operably linked to the delta-9 desaturase polypeptide-encoding sequence. In particular embodiments, 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.
In some embodiments, there may be several copies of the nucleic acid molecules encoding a delta-9 desaturase polypeptide, and each may be under the regulatory control of a different set of regulatory elements. More specifically, 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. In other embodiments, the several copies of the nucleic acid encoding a delta-9 desaturase polypeptide (or multiple delta-9 destaurase polypeptides) 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 oleI 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 ORF1 3′ untranslated region, the Nicotiana tabacum RB7 Matrix Attachment Region, Overdrive, T-stand border sequences, the LfKCS3 promoter, FAE 1 promoter, a Myc tag, and a hemagglutin tag.
Also disclosed are 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 NO:1 as well as nucleic acid molecules encoding such delta-9 desaturase polypeptides, such as SEQ ID NO:2.
In some embodiments, 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.
II. Abbreviations


    
    
        x:yΔz fatty acid containing x carbons and y double bonds in position z counting from the carboxyl end
        ACP acyl carrier protein
        CoA coenzyme A
        FA fatty acids
        FAS fatty acid synthase
        FAME fatty acid methyl ester
        KASII β-ketoacyl-ACP synthase II
        MUFA monounsaturated fatty acid
        PUFA polyunsaturated fatty acid
        WT wild type
    
    


III. Terms
Fatty acid: As used herein, the term “fatty acid” refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, for example, from about C12 to C22, although both longer and shorter chain-length acids are known. The structure of a fatty acid is represented by the notation, x:yΔz, where “x” is the total number of carbon (C) atoms in the particular fatty acid, and “y” is the number of double bonds in the carbon chain in the position “z,” as counted from the carboxyl end of the acid.
Metabolic pathway: The term, “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. Similarly, the product of one metabolic pathway may be a substrate for yet another metabolic pathway.
Metabolic engineering: For the purposes of the present invention, “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: As used herein, the term “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. Of particular interest in some embodiments are acyl-CoA delta-9 desaturases.
The delta-9-18:01-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: For the purposes of the present invention, “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: As used herein, the term “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 RNA, 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 duplexed, triplexed, hairpinned, circular and padlocked conformations.
Operably linked: 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. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. When recombinantly produced, 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: As used herein, the term “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-linked 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.
As used herein, the term “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: As used herein, the term “promoter” 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: The term “sequence identity” or “identity,” as used herein in the context of two nucleic acid or polypeptide sequences, may refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
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. Typically, 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, Calif.).
As used herein, 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.
Analogous position in an amino acid sequence: Nucleic acid and amino acid sequences may be aligned by the methods described in the following paragraphs. When aligned, a position in one sequence is in “an analogous position” with a position in the aligned sequence if the positions are identical within the consensus sequence.
Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-44, 1988; Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences 8:155-65, 1992; Pearson et al., Methods in Molecular Biology 24:307-31, 1994; Tatiana et al., FEMS Microbiol. Lett., 174:247-50, 1990; Altschul et al., J. Mol. Biol. 215:403-10, 1990 (detailed consideration of sequence-alignment methods and homology calculations).
The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) is available on the Internet (at blast.ncbi.nlm.nih.gov/Blast.cgi), for use in connection with sequence-analysis programs, for example, blastp and blastn. A description of how to determine sequence identity using this program is available on the Internet through NCBI at blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs.
For comparisons of amino acid sequences, 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: As used herein, the term “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.
IV. Metabolic Engineering Approaches to Decreasing Saturated Fatty Acids in a Host Cell, Tissue, or Organism
A. Overview
An embodiment of the invention includes introducing delta-9 desaturases with specific acyl-CoA preferences (for example, for palmitic or stearic acid) in plant seeds. The specific acyl-CoA preference of the delta-9 desaturase enables targeting of certain specific saturated fatty acid pools (e.g., palmitate for conversion to monounsaturated products). Acyl-CoA delta-9 desaturases were selected for lowering the saturated fatty acid content in plants as they are not normally produced in plant systems to any appreciable extent.
B. Polypeptides
Polypeptides according to some embodiments of the present invention comprise an amino acid sequence showing increasing percentage identities when aligned with a sequence selected from the group consisting of SEQ ID NO:1, 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. Specific amino acid sequences within these and other embodiments may comprise sequences having, for example, at least about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with the aforementioned sequences. In many embodiments, the amino acid sequence having the aforementioned sequence identity when aligned with the aforementioned sequences encode a peptide with enzymatic delta-9-18:0-ACP desaturase activity, or part of a such a peptide.
C. Nucleic acids
Some embodiments include nucleic acid molecules encoding a polypeptide described above. For example, nucleic acid sequences in some embodiments show increasing percentage identities when aligned with 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. Specific nucleic acid sequences within these and other embodiments may comprise sequences having, for example, at least about 60%, about 65%, about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with 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. It is understood by those of ordinary skill in the art that nucleic acid molecules may be modified without substantially changing the amino acid sequence of an encoded polypeptide, for example, by introducing permissible nucleotide substitutions according to codon degeneracy.
In some embodiments, 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.
Examples of useful tissue-specific, developmentally-regulated promoters include the β-conglycinin 7Sα promoter and seed-specific promoters. Plant functional promoters useful for preferential expression in seed plastid include those from proteins involved in fatty acid biosynthesis in oilseeds and from plant storage proteins. Examples of such promoters include the 5′ regulatory regions from such transcribable nucleic acid molecule sequences as phaseolin, napin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and oleosin. Another exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue.
More specifically, promoters may include the Phaseolus vulgaris Phaseolin promoter, (alone or in conjunction with the Phaseolus vulgaris Phaseolin 3′ untranslated region and the Phaseolus vulgaris Phaseolin 3′ Matrix Attachment Region), the Lesquerella fendleri KCS3 promoter, or the Agrobacterium tumefaciens Mannopine Synthase promoter.
Other useful promoters include the nopaline synthase, mannopine synthase, and octopine synthase promoters, which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens; the cauliflower mosaic virus (CaMV) 19S and 35S promoters; the enhanced CaMV 35S promoter; the Figwort Mosaic Virus 35S promoter; the light-inducible promoter from the small subunit of ribulose-1,5-bisphosphate carboxylase (ssRUBISCO); the EIF-4A promoter from tobacco (Mandel et al. (1995) Plant Mol. Biol. 29:995-1004); corn sucrose synthetase; corn alcohol dehydrogenase I; corn light harvesting complex; corn heat shock protein; the chitinase promoter from Arabidopsis; the LTP (Lipid Transfer Protein) promoters; petunia chalcone isomerase; bean glycine rich protein 1; potato patatin; the ubiquitin promoter; and the actin promoter. Useful promoters are preferably seed-selective, tissue selective, or inducible. Seed-specific regulation is discussed in, for example, EP 0 255 378.
To obtain higher expression of 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.
Due to the plasticity afforded by the redundancy/degeneracy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes in different organisms or classes of organisms has resulted in differential usage of synonymous codons. This “codon bias” is reflected in the mean base composition of protein coding regions. For example, organisms having genomes with relatively low G+C contents utilize more codons having A or T in the third position of synonymous codons, whereas those having higher G+C contents utilize more codons having G or C in the third position. Further, it is thought that the presence of “minor” codons within an mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this reasoning is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mRNAs having high relative contents of minor codons in a particular expression host would have correspondingly low translation rates. This rate may be reflected by correspondingly low levels of the encoded protein.
In engineering optimized genes encoding a delta-9 desaturase for expression in canola or Arabidopsis (or other plants, such as rice, tobacco, maize, cotton or soybean), it is helpful if the codon bias of the prospective host plant(s) has been determined. Multiple publicly-available DNA sequence databases exist wherein one may find information about the codon distribution of plant genomes or the protein coding regions of various plant genes.
The codon bias is the statistical distribution of codons that the expression host (e.g., a plant such as canola or Arabidopsis) uses for coding the amino acids of its proteins. The codon bias can be calculated as the frequency at which a single codon is used relative to the codons for all amino acids. Alternatively, the codon bias may be calculated as the frequency at which a single codon is used to encode a particular amino acid, relative to all the other codons for that amino acid (synonymous codons).
In designing optimized coding regions for plant expression of delta-9 desaturase genes, the primary (“first choice”) codons preferred by the plant should be determined, as well as the second, third, fourth etc. choices of preferred codons when multiple choices exist. A new DNA sequence can then be designed which encodes the amino sequence of the delta-9 desaturase gene, wherein the new DNA sequence differs from the native DNA sequence (encoding the desaturase) by the substitution of expression host-preferred (first preferred, second preferred, third preferred, or fourth preferred, etc.) codons to specify the amino acid at each position within the amino acid sequence. The new sequence is then analyzed for restriction enzyme sites that might have been created by the modifications. The identified putative restriction sites are further modified by replacing these codons with a next-preferred codon to remove the restriction site. Other sites in the sequence which may affect transcription or translation of heterologous sequence are exon:intron junctions (5′ or 3′), poly-A addition signals, and/or RNA polymerase termination signals. The sequence may be further analyzed and modified to reduce the frequency of TA or CG doublets. In addition to these doublets, sequence blocks that have more than about six G or C nucleotides that are the same may also adversely affect transcription or translation of the sequence. Therefore, these blocks are advantageously modified by replacing the codons of first or second choice, etc. with the next-preferred codon of choice.
The method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in PCT application WO 97/13402. Thus, optimized synthetic genes that are functionally equivalent to desaturases/genes of some embodiments may be used to transform hosts, including plants. Additional guidance regarding the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.
Once a plant-optimized DNA sequence has been designed on paper or in silico, actual DNA molecules can be synthesized in the laboratory to correspond in sequence precisely to the designed sequence. Such synthetic DNA molecules may be cloned and otherwise manipulated exactly as if they were derived from natural or native sources.
D. Methods for Genetic Transformation of Plant Material
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. Such nucleic acid molecules may also comprise, for example, non-coding regulatory elements, such as promoters. Other sequences may also be introduced into the cell along with the non-coding regulatory elements and transcribable nucleic acid molecule sequences. These other sequences may include 3′ transcriptional terminators, 3′ poly-adenylation signals, other untranslated sequences, transit or targeting sequences, selectable markers, enhancers, and operators.
A method of transformation generally comprises the steps of selecting a suitable host cell, transforming the host cell with a recombinant vector, and obtaining the transformed host cell. Technology for introduction of DNA into cells is well-known to those of skill in the art. These methods can generally be classified into five categories: (1) chemical methods (Graham and Van der Eb (1973) Virology 54(2):536-9; Zatloukal et al. (1992) Ann. N.Y. Acad. Sci. 660:136-53); (2) physical methods such as microinjection (Capechi (1980) Cell 22(2):479-88), electroporation (Wong and Neumann (1982) Biochim. Biophys. Res. Commun. 107(2):584-7; Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82(17):5824-8; U.S. Pat. No. 5,384,253), and particle acceleration (Johnston and Tang (1994) Methods Cell Biol. 43(A):353-65; Fynan et al. (1993) Proc. Natl. Acad. Sci. USA 90(24):11478-82; (3) viral vectors (Clapp (1993) Clin. Perinatol. 20(1):155-68; Lu et al. (1993) J. Exp. Med. 178(6):2089-96; Eglitis and Anderson (1988) Biotechniques 6(7):608-14); (4) receptor-mediated mechanisms (Curiel et al. (1992) Hum. Gen. Ther. 3(2):147-54; Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89(13):6099-103); and (5) bacterial-mediated mechanisms, such as with Agrobacterium. Alternatively, nucleic acids may be directly introduced into pollen by directly injecting a plant's reproductive organs. Zhou et al. (1983) Methods in Enzymology 101:433; Hess (1987) Intern. Rev. Cytol. 107:367; Luo et al. (1988) Plant Mol. Biol. Reporter 6:165; Pena et al. (1987) Nature 325:274. Other transformation methods include, for example, protoplast transformation as illustrated in U.S. Pat. No. 5,508,184. Nucleic acid molecules may also be injected into immature embryos. Neuhaus et al. (1987) Theor. Appl. Genet. 75:30.
The most commonly used methods for transformation of plant cells are: the Agrobacterium-mediated DNA transfer process (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803) (as illustrated in U.S. Pat. No. 5,824,877; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,981,840; and U.S. Pat. No. 6,384,301) and the biolistics or microprojectile bombardment-mediated process (i.e., the gene gun) (such as described in U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 6,160,208; U.S. Pat. No. 6,399,861; and U.S. Pat. No. 6,403,865). Typically, nuclear transformation is desired, but where it is desirable to specifically transform plastids, such as chloroplasts or amyloplasts, plant plastids may be transformed utilizing a microprojectile-mediated delivery of the desired nucleic acid molecule in certain plant species, such as for example, Arabidopsis, tobacco, potato, and Brassica species.
Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. Several Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA,” which can be genetically engineered to carry any desired piece of DNA into many plant species. The major events marking the process of T-DNA mediated pathogensis are: induction of virulence genes, and processing and transfer of T-DNA. This process is the subject of many reviews. See, e.g., Ream (1989) Ann. Rev. Phytopathol. 27:583-618; Howard and Citovsky (1990) Bioassays 12:103-8; Kado (1991) Crit. Rev. Plant Sci. 10:1-32; Zambryski (1992) Annual Rev. Plant PhysioL Plant Mol. Biol. 43:465-90; Gelvin (1993) in Transgenic Plants, Kung and Wu eds., Academic Press, San Diego, Calif., pp. 49-87; Binns and Howitz (1994) In Bacterical Pathogenesis of Plants and Animals, Dang, ed., Berlin: Springer Verlag., pp. 119-38; Hooykaas and Beijersbergen (1994) Ann. Rev. Phytopathol. 32:157-79; Lessl and Lanka (1994) Cell 77:321-4; and Zupan and Zambryski (1995) Annual Rev. Phytopathol. 27:583-618.
To select or score for transformed plant cells regardless of transformation methodology, the DNA introduced into the cell may contain a gene that functions in a regenerable plant tissue to produce a compound that confers upon the plant tissue resistance to an otherwise toxic compound. Genes of interest for use as a selectable, screenable, or scorable marker include, but are not limited to, β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase, and antibiotic or herbicide tolerance genes. Examples of antibiotic resistance genes include genes conferring resistance to the penicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate (and trimethoprim); chloramphenicol; and tetracycline. For example, glyphosate resistance may be conferred by a herbicide resistance gene. Della-Cioppa et al. (1987) Bio/Technology 5:579-84. 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. 4:111-7), and are considered within the scope of embodiments of the present invention.
The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, may then be allowed to mature into plants.
The presently disclosed methods may be used with any transformable plant cell or tissue. Transformable cells and tissues, as used herein, includes but is not limited to those cells or tissues that are capable of further propagation to give rise to a plant. Those of skill in the art recognize that a number of plant cells or tissues are transformable in which after insertion of exogenous DNA and appropriate culture conditions the plant cells or tissues can form into a differentiated plant. Tissue suitable for these purposes can include but is not limited to immature embryos, scutellar tissue, suspension cell cultures, immature inflorescence, shoot meristem, nodal explants, callus tissue, hypocotyl tissue, cotyledons, roots, and leaves.
The regeneration, development, and cultivation of plants from transformed plant protoplast or explants are known in the art. Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, (Eds.) Academic Press, Inc., San Diego, Calif.; Horsch et al. (1985) Science 227:1229-31. This regeneration and growth process typically includes the steps of selecting transformed cells and culturing those cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. In this method, transformants are generally cultured in the presence of a selective media which selects for the successfully transformed cells and induces the regeneration of plant shoots. Fraley et al. (1993) Proc. Natl. Acad. Sci. USA 80:4803. These shoots are typically obtained within two to four months. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Cells that survive the exposure to a selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. The shoots may then be transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Many of the shoots will develop roots. These are then transplanted to soil or other media to allow the continued development of roots. The method, as outlined above, will generally vary depending on the particular plant strain employed, and particulars of the methodology are therefore within the discretion of one of skill in the art.
The regenerated transgenic plants may be self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated transgenic plants may be crossed with non-transgenic plants, preferably inbred lines of agronomically important species. Conversely, 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.
The progeny from these plants may be evaluated, among other things, for gene expression. The gene expression may be detected by several common methods such as western blotting, northern blotting, immunoprecipitation, and ELISA (Enzyme-Linked ImmunoSorbent Assay). The transformed plants may also be analyzed for the presence of the introduced DNA and the expression level and/or fatty acid profile conferred by the nucleic acid molecules and amino acid molecules of the present invention. Those of skill in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blots or northern blots, PCR-based approaches, biochemical assays, phenotypic screening methods, field evaluations, and immunodiagnostic assays.
Methods for specifically transforming dicots are well-known to those skilled in the art. Transformation and plant regeneration using these methods have been described for a number of crops including, but not limited to, members of the genus Arabidopsis, cotton (Gossypium hirsutum), soybean (Glycine max), peanut (Arachis hypogaea), and members of the genus Brassica. Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants have been published for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135; U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe et al. (1988) Biotechnology 6:923; Christou et al. (1988) Plant Physiol. 87:671-4); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. (1996) Plant Cell Rep. 15:653-7; McKently et al. (1995) Plant Cell Rep. 14:699-703); papaya; and pea (Grant et al. (1995) Plant Cell Rep. 15:254-8).
Methods for transforming monocots are also well-known in the art. Transformation and plant regeneration using these methods have been described for a number of crops including, but not limited to, barley (Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass (Dactylis glomerate); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festuca arundinacea); turfgrass species (e.g., Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticum aestivum); and alfalfa (Medicago sativa). It is apparent to those of skill in the art that a number of transformation methodologies can be used and modified for production of stable transgenic plants for any number of target crops of interest.
Any plant may be chosen for use in the presently disclosed methods. Preferred plants for modification according to the present invention include, for example and without limitation, oilseed plants, Arabidopsis thaliana, borage (Borago spp.), canola (Brassica spp.), castor (Ricinus communis), cocoa bean (Theobroma cacao), corn (Zea mays), cotton (Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.), Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.), Oenothera spp., olive (Olea spp.), palm (Elaeis spp.), peanut (Arachis spp.), rapeseed, safflower (Carthamus spp.), soybean (Glycine and Soja spp.), sunflower (Helianthus spp.), tobacco (Nicotiana spp.), Vernonia spp., wheat (Triticum spp.), barley (Hordeum spp.), rice (Oryza spp.), oat (Avena spp.) sorghum (Sorghum spp.), and rye (Secale spp.) or other members of the Gramineae. 
It is apparent to those of skill in the art that a number of transformation methodologies can be used and modified for production of stable transgenic plants from any number of target crops of interest.
E. Transgenic Seeds
In some embodiments, 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:1, 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. In these and other embodiments, the transgenic seed may comprise a nucleic acid 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. In certain embodiments, a transgenic seed may exhibit decreased levels of saturated fatty acids (for example, palmitic fatty acids and/or stearic fatty acids). The seeds may be harvested from a fertile transgenic plant, and may be used to grow progeny generations of transformed plants, including hybrid plant lines comprising at least one nucleic acid sequence as set forth above, and optionally at least one additional gene or nucleic acid construct of interest.
Each document, patent, and reference cited herein is herein incorporated by its entirety.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.
EXAMPLES
Example 1
Construct Design of pDAB7305
The Aspergillus nidulans Δ-9 Desaturase (AnD9DS) enzyme was previously disclosed in U.S. Patent App. No. 2008/0260933, herein incorporated by reference in its entirety, and is presented herein as SEQ ID NO:1. A polynucleotide sequence (SEQ ID NO:2) comprising the AnD9DS v3 coding sequence was synthesized and incorporated into plasmid construct pDAB7305 (FIG. 1) for Agrobacterium-mediated plant transformation. The resulting construct contained three plant transcription units (also described and used interchangeably as a gene expression cassette). The first plant transcription unit (PTU) (SEQ ID NO:3) was comprised of the RB7 Matrix Attachment Region (RB7 MAR; International Patent App. No. WO9727207), Phaseolus vulgaris Phaseolin promoter (Pv Phas promoter; U.S. Pat. No. 5,504,200), AnD9DS coding sequence (An delta 9 desaturase v3), Phaseolus vulgaris Phaseolin 3′ untranslated region (Pv Phas 3′UTR; U.S. Pat. No. 5,504,200), and the Phaseolus vulgaris Phaseolin 3′ Matrix Attachment Region (Pv Phas 3′ MAR; U.S. Pat. No. 5,504,200). The second PTU (SEQ ID NO:4) was comprised of the Lesquerella fendleri KCS3 promoter (LfKCS3 promoter; U.S. Pat. No. 7,253,337), AnD9DS coding sequence (An delta 9 desaturase v3), and Agrobacterium tumefaciens ORF 23 3′ untranslated region (AtuORF23 3′UTR; U.S. Pat. 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. D., (1983), a polynucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955, Plant Molecular Biology, 2(6), 335-50), phosphinothricin acetyl transferase gene (PAT; Wohlleben et al., (1988) Gene, 70: 25-37), and Agrobacterium tumefaciens ORF 1 3′ untranslated region (AtuORF1 3′UTR; Huang et al., (1990) J. Bacteriol., 172:1814-1822). The construct was confirmed via restriction enzyme digestion and sequencing. Finally, the construct was transformed into Agrobacterium tumefaciens and stored as a glycerol stock.
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. The scraped Agrobacterium strain containing the pDAB7305 binary plasmid was then inoculated into 150 mL modified YEP liquid with streptomycin (100 mg/ml) and spectinomycin (50 mg/ml) into sterile 500 mL baffled flask(s) and shaken at 200 rpm at 28° C. The cultures were centrifuged and resuspended in M-medium (LS salts, 3% glucose, modified B5 vitamins, 1 μM kinetin, 1 μM 2,4-D, pH 5.8) and diluted to the appropriate density (50 Klett Units as measured using a spectrophotometer) prior to transformation of Canola hypocotyls.
Canola Transformation
Seed Germination:
Canola seeds (var. NEXERA 710TM) were surface-sterilized in 10% CloroxTM for 10 minutes and rinsed three times with sterile distilled water (seeds are contained in steel strainers during this process). Seeds were planted for germination on 1⁄2 MS Canola medium (1⁄2 MS, 2% sucrose, 0.8% agar) contained in PhytatraysTM (25 seeds per PhytatrayTM) and placed in a PercivalTM growth chamber with growth regime set at 25° C., photoperiod of 16 hours light and 8 hours dark for 5 days of germination.
Pre-Treatment:
On day 5, hypocotyl segments of about 3 mm in length were aseptically excised, the remaining root and shoot sections were discarded (drying of hypocotyl segments was prevented by immersing the hypocotyls segments into 10 mL of sterile milliQTM water during the excision process). Hypocotyl segments were placed horizontally on sterile filter paper on callus induction medium, MSK1D1 (MS, 1 mg/L kinetin, 1 mg/L 2,4-D, 3.0% sucrose, 0.7% phytagar) for 3 days pre-treatment in a PercivalTM growth chamber with growth regime set at 22-23° C., and a photoperiod of 16 hours light, 8 hours dark.
Co-Cultivation with Agrobacterium: 
The day before Agrobacterium co-cultivation, flasks of YEP medium containing the appropriate antibiotics, were inoculated with the Agrobacterium strain containing the pDAB7305 binary plasmid. Hypocotyl segments were transferred from filter paper callus induction medium, MSK1D1 to an empty 100×25 mm PetriTM dishes containing 10 mL of liquid M-medium to prevent the hypocotyl segments from drying. A spatula was used at this stage to scoop the segments and transfer the segments to new medium. The liquid M-medium was removed with a pipette and 40 mL of Agrobacterium suspension was added to the PetriTM dish (500 segments with 40 mL of Agrobacterium solution). The hypocotyl segments were treated for 30 minutes with periodic swirling of the PetriTM dish so that the hypocotyl segments remained immersed in the Agrobacterium solution. At the end of the treatment period, the Agrobacterium solution was pipetted into a waste beaker; autoclaved and discarded (the Agrobacterium solution was completely removed to prevent Agrobacterium overgrowth). The treated hypocotyls were transferred with forceps back to the original plates containing MSK1D1 media overlaid with filter paper (care was taken to ensure that the segments did not dry). The transformed hypocotyl segments and non-transformed control hypocotyl segments were returned to the PercivalTM growth chamber under reduced light intensity (by covering the plates with aluminum foil), and the treated hypocotyl segments were co-cultivated with Agrobacterium for 3 days.
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 AgNO3, 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.
Selection and Shoot Regeneration:
After 7 days on callus induction medium, the callusing hypocotyl segments were transferred to Shoot Regeneration Medium 1 with selection, MSB3Z1H1 (MS, 3 mg/L BAP, 1 mg/L zeatin, 0.5 gm/L MES, 5 mg/L AgNO3, 300 mg/L TimentinTM, 200 mg/L carbenicillin, 1 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar). After 14 days, the hypocotyl segments which had developed shoots were transferred to Regeneration Medium 2 with increased selection, MSB3Z1H3 (MS, 3 mg/L BAP, 1 mg/L Zeatin, 0.5 gm/L MES, 5 mg/L AgNO3, 300 mg/1 TimentinTM, 200 mg/L carbenicillin, 3 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar) with growth regime set at 22-26° C.
Shoot Elongation:
After 14 days, the hypocotyl segments that had developed shoots were transferred from Regeneration Medium 2 to shoot elongation medium, MSMESH5 (MS, 300 mg/L TimentinTM, 5 mg/l HerbiaceTM, 2% sucrose, 0.7% TC Agar) with growth regime set at 22-26° C. Shoots that were already elongated were isolated from the hypocotyl segments and transferred to MSMESH5. After 14 days the remaining shoots which had not elongated in the first round of culturing on shoot elongation medium were transferred to fresh shoot elongation medium, MSMESH5. At this stage all remaining hypocotyl segments which did not produce shoots were discarded.
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.
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 T0 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. The seeds obtained from theses transgenic T0 canola plants, i.e., T1 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 T1 Canola Seeds Obtained from Transgenic pDAB7305 Canola Plants
Segregating T1 canola seeds were analyzed via a FAME analysis method to identify T0 canola events which produced T1 canola seeds comprising a reduction in total saturated fatty acids (C14:0, C16:0, C18:0, 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 T1 canola seed. A total of 24 single T1 canola seed from each individual canola T0 event were assayed and the TSFA results from each single were quantitated.
Single canola seed samples were homogenized in heptane containing triheptadecanoin (Nu-Chek prep) as a triacylglycerol internal standard, using steel ball mill. Prior to homogenization, a solution of 0.25 M of freshly prepared sodium methoxide (Sigma-Aldrich, St. Louis, Mo.) in methanol was added. Extraction was conducted at 40° C. with constant shaking. Endogenous fatty acid recoveries were normalized by the recovery of the methylated surrogate C17 fatty acid. Extraction of FAMEs (fatty acid methyl esters) was repeated three times and the heptane layers were pooled prior to analysis. The resulting FAMEs were analyzed by GC-FID using a capillary column BPX 70 from SGE (15 m×0.25 mm×0.25 μm). 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 Minn.).
The bulk seed analysis consisted of 50 mg aliquot (10 to 15 seeds combined) and followed the same protocol described above with a slight modification. In order to drive the reaction of derivatization to completeness, the oil was first extracted three times with heptane. Then an aliquot of the combined oil extract, corresponding to 1 seed, was derivatized in FAMEs as described in the single seed protocol above. The completeness of the reaction was verified by checking for the presence of endogenous FAMEs in a fourth extraction/derivatization.
Three transgenic canola events (Event 2182[12]-138.001, Event 2182[12]-125.001, and Event 2182[12]-156.001) were identified and selected for advancement to the T1 generation based on the FAME results which indicated a significant reduction in TSFA as compared to control canola plants. Two additional categories of plant fatty acid content were assayed. These categories included the Mono Unsaturated Fatty Acid (MUFA: C16:1, C18:1 and C20:1) and Poly Unsaturated Fatty Acid (PUFA: C18:2 and C18:3) concentrations, are listed to show the effect of lowering TSFA in T1 seed. (Table 1).







TABLE 1







Summary composition of single T1 seed TSFA, MUFA and PUFA accumulations


obtained from three transgenic canola events as compared to several


NEXERA 710 TM non-transformed control plants.











TSFA (%)
MUFA (%)
PUFA (%)

















Event
N*
Mean
Min
Max
Mean
Min
Max
Mean
Min
Max




















2182[12]-125.001
23
3.16
2.30
4.30
82.74
74.00
90.20
14.09
6.50
22.20


2182[12]-138.001
24
3.40
2.70
4.70
84.10
80.40
88.90
12.50
8.00
16.30


2182[12]-156.001
23
3.49
2.80
5.00
87.30
83.00
90.70
9.23
6.00
12.50


NEXERA 710.1.16
24
6.96
6.10
8.00
80.23
76.90
82.40
12.82
10.50
16.20


NEXERA 710.1.22
24
6.94
5.90
8.40
79.91
77.40
82.60
13.15
10.60
16.40


NEXERA 710.1.29
24
7.06
6.10
8.50
77.33
73.20
81.70
15.64
11.50
19.40


NEXERA 710.1.5—
24
6.99
6.20
7.80
82.71
78.50
84.40
10.28
8.20
13.90





N* indicates the number of individual T1 seed analyzed for each plant progeny.






The mean 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 (C18:1 and C16:1) was observed. The increase in MUFA content is the direct result from the over-expression of the AnD9DS introducing a double bond at the 9th carbon (Δ9) from the carboxylic function of saturated fatty acid. Interestingly, the PUFA content did not increase with the accumulation of MUFA substrate of phosphoglycerolipid desaturase FAD2 synthesizing C18:2.
T1 canola plant events were grown in a greenhouse and self-fertilized to fix the introgressed transgene in progeny plants. InvaderTM quantitative PCR assays were completed on multiple T1 canola plants from each transgenic event. These results indicated that the T1 canola plants obtained from each of the three events contained about 2 or 3 copies of the pDAB7305 T-strand integration (FIG. 3 top panel). A specific determination of copy number was unobtainable, as the pDAB7305 T-strand integrant segregated at varying copy numbers across the three events. The T1 canola plant events were grown in the greenhouse to maturity and self-fertilized. The resulting T2 canola seed was harvested for fatty acid profile analysis via FAME assay.
Example 4
FAME Analysis of T2 Canola Seeds Obtained from Transgenic pDAB7305 Canola Plants
Bulked T2 canola seeds were analyzed via the previously described FAME analysis method to identify T1 canola plant lines which comprised a reduction in total saturated fatty acids as compared to control plants (NEXERA 710TM). (FIG. 2). The yield of each plant was recorded as gram of seed per plant. The yield results were compared to the yield of a transgenic positive control event, 218-11.30 (also described herein as 218-11.30HS50 or 218-11.30HL) containing a stably integrated Aspergillus nidulans delta-9 desaturase transgene (See, WO 2006/042049) that was grown alongside the transgenic plants of the subject disclosure. Both the 218-11.30 plants and the transgenic canola plants of the subject disclosure express a similar transgene. However, the construct used in the transformation of the 2182[12]-125.Sx001, 2182[12]-138.Sx001 and 2182[12]-156.Sx00 canola plants in the subject disclosure is different, as it contains a second PTU comprised of a Lesquerella fendleri KCS3 promoter driving expression of a AnD9DS coding sequence, and flanked by the Agrobacterium tumefaciens ORF 23 3′ untranslated region.
The TSFA (%) of the transgenic plants of the subject disclosure were quantitated and compared to the TSFA (%) obtained from the positive control, 218-11.30HL 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. These results confirmed that the total saturated fatty acid content of the bulked T2 canola seed was reduced below 3.5% in identified T1 canola plant.
The T2 canola lines were further analyzed to determine which canola lines contained low copy numbers of the pDAB7305 T-strand integrant and produced the high T2 seed yield. (FIG. 3).
Individual canola plants of the subject disclosure were selected which contained less than 3.5% TSFA, produced more than 10 g of yield, and contained the lowest T-strand copy number. Based on these three criteria (levels of TSFA below 3.5%, high seed yield, and low copy number) seven canola plants were selected and advanced for further characterization of the TSFA profile. Copy number was determined from one plant per T1 line, using an Invader Assay. Table 2. These T2 canola plant lines were transferred to the greenhouse, grown to maturity and self-fertilized. The T2 canola plant lines were further analyzed molecularly and the T3 seed was harvested for fatty acid profile analysis via FAME assay.







TABLE 2







The T2 canola lines which were selected based on highest yield (seed weight), 


lowest PAT copy number (T1 plants) and lowest TSFA are listed.













Seed Weight 
PAT 



Event
Plant Line
(g)
(Copy Number)
TSFA (%)














2182[12]-138.001
2182[12]-138.Sx001.sSx085
16.37
2.6
3.03



2182[12]-138.Sx001.Sx090
16.58
2.2
3.11



2182[12]-138.Sx001.Sx094
12.47
2.3
3.02



2182[12]-138.Sx001.Sx029
18
3.0
3.31



2182[12]-138.Sx001.Sx084
15.59
3.1
3.08


2182[12]-125.001
2182[12]-125.Sx001.Sx014
11.26
2.5
3.27


2182[12]-156.001
2182[12]-156.Sx001.Sx049
14.44
2.3
3.28









Example 5
Molecular Confirmation of T2 Canola Lines
Selected T2 canola events which contained the pat gene expression cassette (and the closely linked AnD9DS gene expression cassettes) were characterized for their molecular integration pattern using quantitative PCR and Southern blot analysis.
AnD9DS Integration Confirmation via Hydrolysis Probe Assay
The presence of the AnD9DS gene expression cassette was confirmed via hydrolysis probe assays. Isolated T2 canola plants were initially screened via a hydrolysis probe assay, analogous to TAQMANTM, to confirm the presence of the pat transgenes. The data generated from these studies was used to determine the transgene copy number and used to select transgenic canola events for back crossing and advancement to subsequent generations.
Tissue samples were collected in 96-well plates, tissue maceration was performed with a KLECOTM tissue pulverizer and stainless steel beads (Hoover Precision Products, Cumming, Ga.), in Qiagen RLTTM buffer. Following tissue maceration, the genomic DNA was isolated in high-throughput format using the Biosprint 96TM Plant kit (Qiagen, Germantown, Md.) according to the manufacturer's suggested protocol. Genomic DNA was quantified by Quant-IT Pico Green DNA assay KitTM (Molecular Probes, Invitrogen, Carlsbad, Calif.). Quantified genomic DNA was adjusted to around 2 ng/μL for the hydrolysis probe assay using a BIOROBOT3000TM automated liquid handler (Qiagen, Germantown, Md.). Transgene copy number determination by hydrolysis probe assay, analogous to TAQMAN® assay, was performed by real-time PCR using the LIGHTCYCLER® 480 system (Roche Applied Science, Indianapolis, Ind.). 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, Ind.) was prepared at 1× final concentration in a 10 μL volume multiplex reaction containing 0.4 μM of each primer for AnD9DS and pat and 0.2 μM 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 ΔΔCt 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 T1 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).









Gene
Forward
Reverse











Detected
Primer
Primer
Probe
Label





PAT v5
SEQ ID NO: 6
SEQ ID NO: 7
SEQ ID NO: 8
FAM



acaagagtgga
ctttgatgccta
ccagcgtaagca




ttgatgatcta
tgtgacacgtaa
ataccagccaca




gagaggt
acagt
acacc



 


HMG1
SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11
HEX



cctctctacca
gatctggccgga
cgctcctcagct




ccgtctcacat
ctgtttcaca
accacctcaac




g
















TABLE 4







Copy amount results for the AnD9DS events (T2 plants) as determined


using the hydrolysis probe assay.









PAT gene











Event/Line
N
mean
SD
CV (%)














218-11.30(HL)
12
3.61
0.28
7.82


2182[12]-125.Sx001.Sx014
35
5.93
0.98
16.48


2182[12]-138.Sx001.Sx085
34
9.40
1.03
11.00


2182[12]-138.Sx001.Sx090
33
7.69
0.35
4.54


2182[12]-138.Sx001.Sx094
35
9.98
0.79
7.93


2182[12]-156.Sx001.Sx049
33
4.35
0.48
11.12









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 218-11.30(HL) control plants were previously identified to contain two fixed copies of the AnD9DS gene insertion (WO 2006042049). By comparing the selected canola lines (2182[12]-138.Sx001.Sx094 and 2182[12]-138.SX001.Sx090) to the 218-11.30(HL) control plants, specific canola lines were identified which would contain fixed copies of the pDAB7305 T-strand integrants.
AnD9DS Genomic Integration Confirmation via Southern Blot Analysis.
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, Calif.). Quantified genomic DNA was adjusted to a consistent concentration.
For each sample, 4 μg of genomic DNA was thoroughly digested with the restriction enzyme BamHI (New England Biolabs, Beverley, Mass.). 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 μL of water at 65° C. for 1 hour. Resuspended samples were loaded onto a 0.8% agarose gel prepared in 1×TAE and electrophoresed overnight at 1.1 V/cm in 1×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.
Transfer of DNA fragments to nylon membranes was performed by passively wicking 20×SSC solution overnight through the gel onto treated IMMOBILONTM NY+ transfer membrane (Millipore, Billerica, Mass.) by using a chromatography paper wick and paper towels. Following transfer, the membrane was briefly washed with 2×SSC, cross-linked with the STRATALINKERTM 1800 (Stratagene, LaJolla, Calif.), 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, Calif.). Probes were prepared from a PCR fragment containing the entire coding sequence. The PCR amplicon was purified using QIAEX II gel extraction KitTM and labeled with DIG DNA Labeling KitTM (Roche Applied BioSciencse, Indianapolis, Ind.). 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 0.1×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.
The Southern blot analyses completed in this study were used to determine the copy number and confirm that selected events contained the AnD9DS gene expression cassette within the genome of canola. 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 T2 canola lines from four transgenic


events and the NEXERA 710 TM 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.










Event- T2 Line
Number of Plants
Band Numbers
Size Observed (Kb)





NEXERA 710 TM
2
0
NA


218-11.30(HL)
2
4
5.8, 3.5, 1, 0.35*


2182[12]-125.Sx001.Sx014
8
3 bands (2 plants) and
9.2, 4.4, 3.5, 2.6, 0.5




4 bands (6 plants)



2182[12]-138.Sx001.Sx085
8
4
9.2, 5.8, 3.5, 0.7


2182[12]-138.Sx001.Sx090
8
4
9.2, 5.1, 2.5, 0.6


2182[12]-138.Sx001.Sx094
8
4
9.2, 5.1, 2.5, 0.6


2182[12]-156.Sx001.Sx049
8
2
4.5, 3.9









Example 6
T3 Seed Yield of Selected Canola Lines
Selected plants from the T2 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 T2 canola line was determined (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 T2 canola line. These results illustrate that the yield was variable for each plant and line tested. But that the average amounts of yield of the T2 canola lines (2182[12]-125.Sx001.Sx014, 2182[12]-138.Sx001.Sx085, 2182[12]-138.Sx001.5x090, 2182[12]-138.Sx001.5x094, and 2182[12]-156.Sx001.Sx049) were relatively similar and did not significantly deviate from the control plants (NexeraTM 710G5 and 218-11.30(HL)).







TABLE 6







ANNOVA analysis of the seed yield, measured as total grams, from canola transgenic lines 


and the untransformed Nexera TM 710GS control plants. Yield results are not significantly


different (p < 0.05) for plants that are connected by the same letter in parenthesis.










Event/Line
Plant Count
Ratio
Yield (g)














218-11.30(HL)
12
0.06186
5.7233333
(A, B)


2182[12]-125.Sx001.Sx014
35
0.18041
5.9874286
(A, B)


2182[12]-138.Sx001.Sx085
34
0.17526
6.8394118
(A)


2182[12]-138.Sx001.Sx090
33
0.17010
6.4887879
(A, B)


2182[12]-138.Sx001.Sx094
35
0.18041
5.0302857
(B)


2182[12]-156.Sx001.Sx049
33
0.17010
6.3715152
(A, B)


Nexera TM 710G5
12
0.06186
7.1666667
(A, B)










Total
194
1.00000
—









Example 7
FAME Analysis of T3 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. The results confirmed that the total saturated fatty acid and saturated fatty acid (as determined from the sum of palmitic and stearic acid content) content of the single and bulked T3 canola seed was reduced below 3.5% in the selected canola plant lines.
Surprisingly, two lines, 2182[12]-138.Sx001.Sx085 and 2182[12]-138.Sx001.Sx094, accumulated TSFA levels that averaged under 3.0%. Described for the first time are canola lines which are comprised of bulked seed that contains less than 3.0% saturated fatty acid. Table 7 and Table 8.
In addition, 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. This is significantly lower than the TSFA level of 5.11% which was obtained in the NexeraTM 710G5 control plants. Finally, the maximum TSFA percentage in the transgenic canola lines does not excede 3.5% and the mean saturated level in single seed is 2.52% which is well below 3.5%. Table 9 and FIG. 4.
Table 7 shows the distribution of T2 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 7







T2 parent line
N
TSFA (%)
Saturated (%)














—
—
Mean
Min
Max
Mean
Min
Max





218-11.30(HL)
12
3.94
3.75
4.21
3.39
3.19
3.65


2182[12]-125.Sx001.Sx014
35
3.43
2.59
4.24
3.08
2.34
3.74


2182[12]-138.Sx001.Sx085
34
2.92
2.53
3.65
2.68
2.31
3.36


2182[12]-138.Sx001.Sx090
33
3.24
2.66
3.87
2.93
2.37
3.59


2182[12]-138.Sx001.Sx094
35
2.98
2.50
3.52
2.68
2.24
3.21


2182[12]-156.Sx001.Sx049
33
3.47
2.50
4.21
3.15
2.26
3.73


Nexera 710G5
12
6.43
6.14
6.69
5.19
4.89
5.40









Table 8 shows the lowest T3 mature seed bulk FAMEs and plant yield of single T2 progeny plants obtained from event 2182[12]-138.Sx0001 as 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









TSFA






Saturated
Reduction



Event/Line (T2)
Oil (%)
TSFA (%)
(%)
(%)
Yield (g)




















2182[12]-138.Sx001.Sx094.Sx112
34.5
2.5
2.24
61.12
4.16


2182[12]-138.Sx001.Sx085.Sx070
38.7
2.53
2.31
60.65
6.05


2182[12]-138.Sx001.Sx085.Sx076
37.8
2.57
2.33
60.03
7.72


2182[12]-138.Sx001.Sx094.Sx122
39.3
2.65
2.41
58.79
5.73


2182[12]-138.Sx001.Sx090.Sx062
37.4
2.66
2.37
58.63
4.44


Nexera 710GS.Sx552
43.7
6.28
5.15
2.33
6.97









Table 9 shows the distribution of T3 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.










TABLE 9







Events/Line
N
TSFA (%)
Saturated (%)














—
—
Mean
Min
Max
Mean
Min
Max





2182[12]-138.Sx001.Sx085.Sx070
43
2.60
2.27
3.02
2.33
2.05
2.64


2182[12]-138.Sx001.Sx085.Sx076
48
2.57
2.27
3.12
2.40
2.16
2.85


2182[12]-138.Sx001.Sx090.Sx062
48
2.66
2.36
3.16
2.52
2.26
2.99


2182[12]-138.Sx001.Sx094.Sx112
48
2.51
2.25
2.78
2.38
2.13
2.61


2182[12]-138.Sx001.Sx094.Sx122
37
2.73
2.44
3.46
2.46
2.13
3.18


Nexera 710GS.Sx552
48
6.34
5.71
7.52
5.11
4.73
5.90

## Claims

1. A method for decreasing the amount of saturated fatty acids in a plant cell, the method comprising:
transforming a plant cell with a nucleic acid molecule comprising a heterologous gene regulatory element operably linked to a polynucleotide encoding a delta-9 desaturase enzyme comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:1,
wherein the plant cell also comprises an extraplastidial desaturase selected from the group consisting of LnD9DS desaturase, HzD9DS desaturase, and MgD9DS desaturase,
such that the amount of saturated fatty acids in the plant cell is decreased.

2. A plant seed which expresses an extraplastidial desaturase selected from the group consisting of LnD9DS desaturase, HzD9DS desaturase, and MgD9DS desaturase, and comprises a nucleic acid molecule comprising a polynucleotide encoding a delta-9 desaturase enzyme comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:1.

3. The plant seed of claim 2, wherein the extraplastidial desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:26-28.

4. An oil seed plant comprising a polynucleotide encoding a delta-9 desaturase enzyme comprising an amino acid sequence that is at least 80% identical to SEP ID NO:1, and further comprising an extraplastidial desaturase selected from the group consisting of LnD9DS desaturase, HzD9DS desaturase, and MgD9DS desaturase.

5. A plant material comprising a polynucleotide encoding a delta-9 desaturase enzyme comprising an amino acid sequence that is at least 80% identical to SEP ID NO:1, and further comprising an extraplastidial desaturase selected from the group consisting of LnD9DS desaturase, HzD9DS desaturase, and MgD9DS desaturase.

6. The plant material of claim 5, wherein the extraplastidial desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:26-28.