JP2017537633A - Generation of low-saturated or unsaturated fatty acid transgenic canola - Google Patents

Abstract

The compositions and methods include genetically encoding and expressing a novel delta-9 desaturase in plant cells. In some embodiments, a method of expressing nucleic acid in a plant cell to utilize the activity of a delta-9 desaturase enzyme, thereby reducing the compositional percentage of saturated fatty acids in the plant seed and concomitantly increasing Δ9 fatty acids. In other embodiments, the amino acid sequence has delta-9 desaturase activity. The method may involve expressing delta-9 desaturase in plant cells, plant material, and whole plant for the purpose of increasing the amount of monounsaturated fatty acids in the whole plant, plant seed, and plant material, eg seed.

Description

  Some embodiments generally relate to certain delta-9 desaturase enzymes, nucleic acids encoding these enzymes, and methods for expressing these enzymes in plant cells. Some embodiments relate to utilizing the activity of certain delta-9 desaturase enzymes to reduce the composition percentage of saturated fatty acids and increase the composition percentage of omega-7 fatty acids in plant material (eg, seeds). A further embodiment relates to the preferential expression of delta-9 desaturase enzyme in seeds utilizing a seed specific promoter. Also disclosed herein are plants and plant materials produced by the methods in certain embodiments, and those plants containing less than 3.5% or less than 2.7% saturated fatty acids. It is produced oil.

  Plant-derived oils are gradually replacing animal-derived oils and fats as the main source of fat intake in the diet. However, saturated fat intake in most developed countries remains at about 15% to 20% of total calorie consumption. As part of an attempt to promote a healthier lifestyle, the US Department of Agriculture (USDA) has recently recommended that saturated fat be less than 10% of daily caloric intake. To make it easier for consumers to recognize, the current labeling guidelines published by the USDA now show that the total saturated fatty acid level is less than 1.0 g per 14 g offer for “low-saturated fatty acid” labeling. The “unsaturated fatty acid” label requires less than 0.5 g per 14 g serving. This means that the saturated fatty acid content of the vegetable oil needs to be less than 7% and 3.5%, respectively, in order to indicate “low saturated fatty acid” or “unsaturated fatty acid”. The issuance of these guidelines has increased consumer demand for “low-saturated fatty acid” and “unsaturated fatty acid” oils. To date, this demand has been largely met with canola oil and little with sunflower and safflower oil.

  Unsaturated fats (monounsaturated fats and polyunsaturated fats) are advantageous (especially when moderately consumed) while saturated fats and trans fats are not. Saturated and trans fats raise unwanted LDL cholesterol levels in the blood. Dietary cholesterol also raises LDL cholesterol and can cause heart disease even without raising LDL. Therefore, it is desirable to select foods that are low in saturated fat, trans fat, and cholesterol as part of a healthy diet.

  Whether plant or animal, the characteristics of the oil are mainly determined by the number of carbon and hydrogen atoms in the oil molecule and the number and position of double bonds contained in the fatty acid chain. Most plant-derived oils contain varying amounts of palmitic fatty acid (16: 0), stearic fatty acid (18: 0), oleic fatty acid (18: 1), linoleic fatty acid (18: 2), and linolenic fatty acid (18: 3 )including. Traditionally, palmitic acid and stearic acid are called “saturated fatty acids” because their carbon chains are saturated with hydrogen atoms and therefore do not have double bonds. These contain as many hydrogen atoms as possible. However, oleic acid, linoleic acid, and linolenic acid are 18 carbon fatty acid chains with 1, 2, and 3 double bonds, respectively. Oleic acid is typically considered to be a monounsaturated fatty acid, while linoleic acid and linolenic acid are considered to be polyunsaturated fatty acids. The USDA definition of an "unsaturated fatty acid" oil product, meaning one having a saturated fatty acid content of less than 3.5%, is as combined saturated fatty acid content per weight (compared to total fatty acid) Calculated.

  Canola oil has the lowest saturated fatty acid level of all vegetable oils. “Canola” refers to rapeseed (Brassica), which is based on the total fatty acid content of the seed, up to 2% by weight (preferably up to 0.5% by weight, most preferably essentially 0% by weight). ) Erucic acid (C22: 1) content, and after squeezing, an air-dried powder containing less than 30 μmol / g glucosinolate is produced in the defatted (oil-free) powder. These types of rapeseed are distinguished in that they are edible compared to various more traditional species.

  In oil seeds, it is hypothesized that fatty acid synthesis occurs mainly within plastids. The main product of fatty acid synthesis is palmitic acid ester (16: 0), which appears to be efficiently extended to stearic acid ester (18: 0). While remaining in the plastid, the saturated fatty acid can then be desaturated by an enzyme known as acyl-ACP delta-9 desaturase to introduce one or more carbon-carbon double bonds. Specifically, stearates can be rapidly desaturated by plastid delta-9 desaturase enzymes to yield oleates (18: 1). In fact, palmitate can also be desaturated to palmitoleate (16: 1) by plastid delta-9 desaturase, but this fatty acid is only a small amount (0-0.2%) in most vegetable oils. It seems not to exist. Thus, the main products of fatty acid synthesis within plastids are palmitate, stearate, and oleate. In most oils, saturated fatty acids are present in very small proportions, so oleate is the main fatty acid synthesized.

  Newly synthesized fatty acids are transported from the plastids to the cytoplasm. Subsequent desaturation of plant fatty acids in the cytoplasm appears to be limited to oleates, which are microsomal desaturases that act on oleoyl or linoleoyl substrates that are esterified to phosphatidylcholine (PC), Can be unsaturated to linoleic acid ester (18: 2) and linolenic acid ester (18: 3). Furthermore, depending on the plant, the oleate can be further modified by extension (20: 1, 22: 1, and / or 24: 1) or by addition of functional groups. These fatty acids are then assembled into triglycerides within the reticular inner membrane, along with the saturated fatty acids palmitic and stearic esters.

  The plant acyl-ACP delta-9 desaturase enzyme is soluble. It is located in the plastid stroma and uses as a substrate newly synthesized fatty acids that are esterified to ACP, mainly stearyl-ACP. It is located within the endoplasmic reticulum membrane (ER or microsome), uses fatty acids esterified to Co-A as a substrate, and desaturates both the palmitic and stearic esters that are saturated fatty acids. In contrast to other delta-9 desaturase enzymes. US Pat. No. 5,723,595 and US Pat. No. 6,706,950 relate to plant desaturases.

  The yeast delta-9 desaturase gene has been isolated, cloned and sequenced from Saccharomyces cerevisiae. 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 is evident in this tissue. Expressed. Furthermore, this yeast gene was also expressed in tomato. See Wang et al. (1996) J. Agric. Food Chem. 44: 3399-402; and Wang et al. (2001) Phytochemistry 58: 227-32. Using this yeast delta-9 desaturase gene, some increase in certain unsaturated fatty acids and some decrease in certain saturated fatty acids have been reported in both tobacco and tomato, but tobacco and tomato are clearly oil crops is not. This yeast gene was also introduced into Brassica napus. US Pat. No. 5,777,201.

  Different fungal acyl-CoA delta-9 desaturases from Aspergillus nidulans have been introduced into canola, thereby successfully reducing saturated fatty acid levels in oil seeds. US Patent Application Publication No. 2008/0260933. Aspergillus nidulans acyl-CoA delta-9 desaturase reduced stearic fatty acids (61-90%) in oil seeds more than abundant palmitic fatty acids (36-49%).

DISCLOSURE Disclosed herein are novel fungal delta-9 desaturase enzymes, nucleic acids comprising at least one nucleotide sequence encoding such desaturases, as well as plants, plant materials (eg, seeds), plant parts And any plant product comprising any of the foregoing. Aspects of some embodiments are exemplified by fungal delta-9 desaturase enzymes isolated from rice blast fungus (Magnaporthe grisea), Leptosphaeria nodorum, and Helicoverpa zea. Some examples include native and synthetic delta-9 desaturases that prefer palmitic acid or stearic acid as a substrate.

  Some embodiments are 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. An isolated nucleic acid molecule encoding a delta-9 desaturase enzyme comprising an amino acid sequence that is at least 80% identical to the sequence. In particular examples, the nucleic acid molecule comprises 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, It comprises a sequence that is at least 60% identical to a sequence selected from the group consisting of 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 are 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. An isolated delta-9 desaturase polypeptide comprising an amino acid sequence at least 80% identical to the sequence.

  Also disclosed is a method for expressing in a plant cell at least one of the aforementioned nucleic acids and / or polypeptides. Certain embodiments utilize the activity of the delta-9 desaturase enzyme to determine the percent composition of saturated fatty acids of plants, plant materials (eg, seeds), and / or plant parts that include plant cells, and / or those described above. It can be reduced in plant products produced from either. In certain embodiments, omega-7 fatty acids can be incidentally increased in plants, plant materials, plant parts, and / or plant products. Further embodiments utilize seed specific expression to further reduce saturated fatty acid levels in oil seeds.

  Some embodiments provide a method for reducing the amount of saturated fatty acids in a plant, plant material, plant part, and / or plant product, a nucleic acid encoding a delta-9 desaturase polypeptide of the invention. It includes a method of transforming with a molecule to reduce the amount of saturated fatty acids in the cell. Some embodiments include a method for generating a genetically engineered plant that has a reduced amount of saturated fatty acid in the plant compared to a wild-type plant of the same species. Such methods involve transforming plant material (or plant cells) 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. Transforming and culturing the transformed plant material (or plant cells) to obtain a plant. In a particular example, plant cells and / or plant material obtained from Arabidopsis sp. Can be transformed with a nucleic acid molecule encoding a delta-9 desaturase polypeptide of the invention. In other specific examples, two or more copies of the delta-9 desaturase gene can be transformed, each delta-9 desaturase gene being controlled by a different promoter. In other specific examples, the two or more promoters are seed-specific promoters.

  The foregoing and other features are expected to become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

It is a figure which shows the plasmid map of pDAB7305. NEXERA710 (TM) compared to the positive control plants consisting of canola control plants and 218-11.30HL transgenic canola plants is a diagram showing the distribution of TSFA (%) in _{T 2} Seeds of separation _{T 1} of the canola plant bulk. FIG. ₂ shows the distribution of TSFA in a T2 seed population obtained from three selected transgenic events compared to a negative control NEXERA 710 ™ canola plant. Black dots represent seed progeny with a TSFA less than 3.5% (black line). As shown in the graph, plants with a TSFA of less than 3.5% produce varying amounts of yield and 2 to 10 copies of the pat transgene contained on the same T chain component as the AnD9DS transgene. Had a number. FIG. 3 shows the distribution of TSFA and saturated fatty acid percentages in single canola grains (graph shows TSFA values for transgenic canola events since wild type control plants have been excluded). FIG. 3 shows the distribution of TSFA and saturated fatty acid percentages in single canola grains (graph shows TSFA values for transgenic canola events since wild type control plants have been excluded).

Sequence Listing Nucleic acid sequences listed in the attached Sequence Listing are 37C. F. R. Indicated using standard abbreviations for nucleotide bases as defined in § 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 attached sequence listing:
SEQ ID NO: 1 shows the amino acid sequence of an Aspergillus nidulans acyl-CoA delta-9 desaturase protein (sometimes called AnD9DS).
SEQ ID NO: 2 shows the nucleic acid sequence of v3 of the Aspergillus nidulans acyl-CoA delta-9 desaturase gene (referred to in part as AnD9DS).
SEQ ID NO: 3 shows the nucleic acid sequence of 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 primer and probe sequences that may be useful in some embodiments.
SEQ ID NO: 12 is a typical fragment of the rice blast fungus (M. grisea) acyl-CoA delta-9 desaturase gene (referred to in some places as MgD9DS) amplified by PCR.
SEQ ID NO: 13 is a typical 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 the coding region of a typical non-denatured delta-9 desaturase gene (denoted MgD9DS v1) of rice blast fungus (M. grisea).
SEQ ID NO: 17 shows the coding region of a typical native delta-9 desaturase gene (denoted HzD9DS v1) of Helicoverpa zea.
SEQ ID NO: 18 shows the coding region of a typical native delta-9 desaturase (LnD9DS-2 v1) gene of L. nodorum.
SEQ ID NO: 19 shows the sequence of a typical canola-optimized delta-9 desaturase gene (MgD9DS v2) of rice blast fungus (M. grisea).
SEQ ID NO: 20 shows the sequence of a typical canola-optimized delta-9 desaturase gene (HzD9DS v2) of American tobacco moth (H. zea).
SEQ ID NO: 21 shows the sequence of a typical canola-optimized delta-9 desaturase gene (LnD9DS-2 v2) of L. nodorum.
SEQ ID NO: 22 shows the sequence of a further exemplary canola-optimized delta-9 desaturase gene (LnD9DS-2 v3) of L. nodorum.
SEQ ID NO: 23 shows the sequence of a further exemplary canola-optimized delta-9 desaturase gene (HzD9DS v3) from H. zea.
SEQ ID NO: 24 shows an exemplary nucleic acid sequence encoding an Aspergillus nidulans delta-9 desaturase, in some places called AnD9DS v2.
SEQ ID NO: 25 shows a second exemplary nucleic acid sequence that encodes an A. nidulans delta-9 desaturase, in some places called AnD9DS v3.
SEQ ID NO: 26 shows the amino acid sequence of a typical non-denaturing delta-9 desaturase (MgD9DS) of rice blast fungus (Mgrisea).
SEQ ID NO: 27 shows the amino acid sequence of a typical non-denaturing delta-9 desaturase (HzD9DS) of American tobacco moth (H. zea).
SEQ ID NO: 28 shows the amino acid sequence of a typical native delta-9 desaturase (LnD9DS-2) of L. nodorum.
SEQ ID NO: 29 shows the amino acid sequence encoded by the nucleic acid (AnD9DS) exemplified by SEQ ID NOs: 24-25.
SEQ ID NO: 30 shows the amino acid sequence of another exemplary AnD9DS desaturase.
SEQ ID NO: 31 shows the amino acid sequence of a typical non-denaturing delta-9 desaturase (ScOLE1) of Saccharomyces cerevisiae.
SEQ ID NO: 32 shows the N-terminal 68 residues (1-68) of a typical AnD9DS desaturase.
SEQ ID NO: 33 shows the C-terminal 175 residues (281-455) of a typical AnD9DS desaturase.

DETAILED DESCRIPTION OF THE INVENTION I. Overview Overview of some embodiments We previously introduced a fungal acyl-CoA delta-9 desaturase from Aspergillus nidulans into canola, thereby reducing saturated fatty acid levels in oil seeds I succeeded in making it happen. US Patent Application Publication No. 2008/0260933. Aspergillus nidulans delta-9 desaturase reduced stearic fatty acids (61-90%) in oil seeds more than abundant palmitic fatty acids (36-49%). It has been discovered that by providing multiple copies of Aspergillus nidulans delta-9 desaturase, the saturated fatty acid level in canola can be reduced to less than 3.5%.

  Disclosed herein are 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, A nucleotide sequence that is at least 60% identical to a sequence selected from the group consisting of 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 A nucleic acid molecule encoding a delta-9 desaturase polypeptide. In some embodiments, the nucleic acid molecule can further comprise a gene regulatory element operably linked to a sequence encoding a delta-9 desaturase polypeptide. In certain embodiments, the gene regulatory element comprises a phaseolin promoter, phaseolin 5 ′ untranslated region, phaseolin 3 ′ untranslated region (“UTR”), 3 ′ untranslated of Agrobacterium tumefaciens ORF1 The region, cassava bean mosaic virus promoter, Nicotiana tabacum RB7 matrix attachment region, T chain border sequence, LfKCS3 promoter, and FAE1 promoter.

  In some embodiments, there may be several copies of a nucleic acid molecule encoding a delta-9 desaturase polypeptide, each of which may be under regulatory control of a different set of regulatory elements. More specifically, the gene regulatory elements can be the phaseolin promoter and phaseolin 5'UTR, and the Lesquerella fendleri LfKCS3 promoter, so that there are two copies of AND9DS, one copy Is controlled by the phaseolin promoter and 5′UTR and the second copy is controlled by the LfKCS3 promoter. In other embodiments, several copies of a nucleic acid encoding a single delta-9 desaturase polypeptide (or multiple delta-9 desaturase polypeptides) are obtained from the Saccharomyces cerevisiae delta-9 desaturase promoter, delta -9 desaturase 3'UTR / terminator, ole1 gene promoter, Phaseolus vulgaris phaseolin 3 'untranslated region, phaseolus vulgaris phaseolin matrix attachment region, Agrobacterium tumefaciens mannopine synthesis Enzyme promoter, 3 'untranslated region of Agrobacterium tumefaciens ORF23, cassava bain mosaic virus promoter, Agrova Other regulation, including 3 'untranslated region of Agrobacterium tumefaciens ORF1, Nicotiana tabacum RB7 matrix attachment region, overdrive, T chain boundary sequence, LfKCS3 promoter, FAE1 promoter, Myc tag, and hemagglutinin tag It can be under the control of the element.

  Also disclosed are delta-9 desaturase polypeptides comprising an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1, and such delta-, such as SEQ ID NO: 2. A nucleic acid molecule encoding a 9 desaturase polypeptide.

  In some embodiments, the nucleic acid molecule and the delta-9 desaturase polypeptide reduce the amount of saturated fatty acids in the plant material, cell, tissue, or whole plant as compared to the amount found in wild-type plants of the same species. As such, it can be expressed in plant material, cells, tissues, or whole plants. Alternative embodiments of the present invention include methods for reducing the amount of saturated fatty acids in plant material, cells, tissues, or whole plants. Such a method involves transforming a plant material, cell, tissue or whole plant with at least one of the aforementioned nucleic acid molecules to reduce the amount of saturated fatty acids in the plant material, cell, tissue or whole plant. Can be included. Certain embodiments include a method for preferentially reducing palmitic and / or stearic fatty acids in plant material, cells, tissues, or whole plants.

  The methods disclosed herein can be performed, for example, on plant material derived from plants or plants (eg, plants of the genus Arabidopsis, or canola). Certain embodiments provide a method, plant cell or plant material for generating or regenerating a genetically engineered plant having a reduced amount of saturated fatty acid in the plant compared to a wild-type plant of the same species, the nucleic acid molecule described above. And a method comprising culturing transformed plant material to obtain a plant. Also disclosed are plants, plant materials, plant cells, and seeds obtained by any of the foregoing methods.

II. Abbreviated x: yΔ ^z x carbons and y pieces in position z counted from the carboxyl terminal double bond and a fatty acid containing ACP acyl carrier protein CoA coenzyme A
FA Fatty acid 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. Terminology Fatty acid: As used herein, the term “fatty acid” refers to long chain aliphatic acids (alkanoic acids) of various chain lengths, eg, about C12 to C22, but of longer and shorter chain lengths. Acids are known. Structure of fatty acids, notation x: is represented by Waideruta ^z, where "x" is the total number of carbon (C) atoms in the particular fatty acid, "y", the position counted from the carboxyl terminus of the acid " z "is the number of double bonds in the carbon chain.

  Metabolic pathway: The term “metabolic pathway” refers to a series of chemical reactions occurring within a cell that are catalyzed by an enzyme to achieve the formation of a metabolite or the initiation of another metabolic pathway. Metabolic pathways can involve several or many steps and can compete with different metabolic pathways for specific reaction substrates. Similarly, the product of one metabolic pathway can be a substrate for yet another metabolic pathway.

  Metabolic manipulation: For the purposes of the present invention, “metabolic manipulation” refers to the first substance in the overall scheme of all metabolic pathways that can function in a cell by modifying one or more metabolic pathways in the cell. Refers to a rational strategic design to achieve stepwise modification from 1 to a product with the desired exact chemical structure.

  Desaturase: As used herein, the term “desaturase” refers to a polypeptide that can be desaturated within one or more fatty acids (ie, the introduction of a double bond) to produce a fatty acid or precursor of interest. Point to. Plant soluble fatty acid desaturase enzymes can introduce double bonds regiospecifically into saturated acyl-ACP substrates. Acyl-CoA desaturases introduce double bonds regiospecifically into saturated fatty acyl-CoA substrates. The reaction involves the activation of molecular oxygen by a two-electron reduced diiron center coordinated by a bundle of four helices that form the nucleus of the desaturase architecture. Of particular interest in some embodiments is acyl-CoA delta-9 desaturase.

Delta-9-18: 0 ¹ -ACP desaturase is required for all plants to maintain membrane fluidity. This enzyme first desaturates stearoyl-ACP, which is also slightly active against palmitoyl-ACP.

  Progeny plant: For purposes of the present invention, a “progeny plant” refers to any plant or plant material obtained therefrom that can be obtained by a plant crossing method. Plant mating methods are well known in the art and include natural mating, artificial mating, selective mating including DNA molecular marker analysis, transgenic, and commercial mating.

  Plant material: As used herein, the term “plant material” refers to any cell or tissue obtained from a plant.

  Nucleic acid molecules: RNA, cDNA, polymeric forms of nucleotides that can contain both sense and antisense strands of genomic DNA, and synthetic and mixed polymers of the above. Nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. As used herein, “nucleic acid molecule” is synonymous with “nucleic acid” and “polynucleotide”. The term includes single and double stranded forms of DNA. Nucleic acid molecules can include either or both natural and modified nucleotides linked together by natural and / or non-natural nucleotide linkages.

  Nucleic acid molecules can be chemically or biochemically modified or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution with one or more natural nucleotide analogs, internucleotide modifications, such as uncharged linkages (eg, methylphosphonates, phosphotriesters, amides). Phosphites, carbamates, etc.), charged linkages (eg, thiophosphates, dithiophosphates, etc.), pendant moieties (eg, peptides), intercalators (eg, acridine, psoralen, etc.), chelating agents, alkylating agents , And modified linkages such as alpha anomeric nucleic acids. The term “nucleic acid molecule” also includes any single-stranded, double-stranded, partially duplexed, tripled, hairpinned, circular, and padlock-type conformations. A topological solid structure is also included.

  Operably linked: a first nucleic acid sequence is operably linked to a second nucleic acid sequence if the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter acts on the transcription or expression of the coding sequence. When produced recombinantly, the operably linked nucleic acid sequences are usually contiguous, and when necessary to link two protein coding regions, they are performed in the same reading frame. However, the 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 that has gene regulatory activity, ie, a nucleic acid molecule that has the ability to affect transcription or translation of an operably linked transcribable nucleic acid molecule. Point to. Regulatory elements such as promoters, leaders, introns, and transcription termination regions are non-coding nucleic acid molecules with gene regulatory activity that play an essential role in the overall expression of genes in living cells. Isolated regulatory elements that function in plants are therefore useful for modifying plant phenotypes through molecular engineering techniques. “Regulatory element” means a series of nucleotides that determines whether, when, and the level of a particular gene is expressed. Regulatory DNA sequences interact specifically with regulatory proteins or other proteins.

  As used herein, the term “gene regulatory activity” refers to a nucleic acid molecule that can affect the transcription or translation of a operably linked nucleic acid molecule. An isolated nucleic acid molecule having gene regulatory activity can result in transient or spatial expression or can regulate the level and rate of expression of a operably linked nucleic acid molecule. An isolated nucleic acid molecule with gene regulatory activity can include a promoter, intron, leader, or 3 'transcription termination region.

  Promoter: As used herein, the term “promoter” is involved in the recognition and binding of other proteins such as RNA polymerase II or transcription factors (trans-acting protein factors that regulate transcription) and are operably linked. A nucleic acid molecule that initiates transcription of a gene The promoter itself may contain subelements such as cis elements or enhancer domains that affect the transcription of genes that are operably linked. A “plant promoter” is a non-denatured or denatured promoter that is functional in plant cells. Plant promoters can be used as 5 'regulatory elements to regulate the expression of one or more genes that are operably linked. Plant promoters can be defined by their transient, spatial or developmental expression patterns. The nucleic acid molecules described herein can include a nucleic acid sequence that includes a promoter.

  Sequence identity: The term “sequence identity” or “identity” as used herein in the context of two nucleic acid or polypeptide sequences is identical when aligned with the greatest correspondence over a particular comparison window. It may refer to residues within a sequence.

  When sequence identity percentages are used with respect to proteins, residue positions that are not identical often differ by conservative amino acid substitutions, but amino acid residues have similar chemical properties (eg, charge, hydrophobicity, or stericity). It will be appreciated that substitution with other amino acid residues having a chemical effect) will thus not alter the functional properties of the molecule.

  Thus, if the sequences differ by conservative substitutions, the percent sequence identity can be adjusted up to correct the conservative nature of the substitution at the site of non-identical residues. 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 skilled in the art. Typically, such techniques involve scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. For example, if the same amino acid is given a score between 0 and 1, and a non-conservative substitution is given a score of 0, the conservative substitution is given a score between 0 and 1. Conservative substitution scoring can be calculated, for example, to be performed in the program PC / GENE (Intelligenetics, Mountain View, CA).

  As used herein, the term “percent sequence identity” can refer to a value determined by comparing two optimally aligned sequences over a comparison window, and in an optimal alignment of two sequences, The portion of the sequence within the comparison window may contain additions or deletions (ie gaps) compared to a reference sequence (not including additions or deletions). The percentage determines the number of positions where the same nucleotide or amino acid residue occurs in both sequences to obtain the number of match positions, divides the number of match positions by the total number of positions in the comparison window and multiplies the result by 100. Calculated as a percentage of sequence identity.

  Similar positions in amino acid sequences: Nucleic acid sequences and amino acid sequences can be aligned by the methods described in the following paragraphs. Once aligned, if the position is the same in the consensus sequence, the position in one sequence is “similar to the position” in the aligned sequence.

  Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described below. 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 discussion on sequence alignment methods and homology calculations).

  A Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) for use in combination with sequence analysis programs such as blastp and blastn is available on the Internet (blast.ncbi.nlm.nih.gov). /Blast.cgi). For a description of how to determine sequence identity using this program, see blast. ncbi. nlm. nih. gov / Blast. cgi? CMD = Web & PAGE_TYPE = BlastDocs is available on the Internet via NCBI.

  For comparison of amino acid sequences, the “Blast2 sequence” function (bl2seq) of the BLAST program is employed with default parameters. For example, specific parameters for giving a penalty for a mismatch or a reward for a match can be adjusted within the discretion of one of ordinary skill in the art.

  Transformed: As used herein, the term “transformed” refers to a cell, tissue, organ, or organism into which a foreign nucleic acid molecule, such as a construct, has been introduced. The introduced nucleic acid molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism so that the introduced polynucleotide molecule is inherited by subsequent progeny. A “transgenic” or “transformed” cell or organism also contains foreign nucleic acid molecules produced from a mating program that uses the cell or organism's progeny and such transgenic plants as parents, for example, in crosses Also included are progeny that exhibit altered phenotypes.

IV. A metabolic engineering approach to reduce saturated fatty acids in host cells, tissues, or organisms. Overview Embodiments of the invention include introducing a delta-9 desaturase in preference to a specific acyl-CoA (eg, palmitic acid or stearic acid) into plant seeds. The preference for specific acyl-CoA of delta-9 desaturase makes it possible to target specific specific saturated fatty acid pools (e.g. palmitate esters for conversion to monounsaturated products). Since acyl-CoA delta-9 desaturases are rarely produced in plant systems, they were selected to reduce the saturated fatty acid content in plants.

B. Polypeptides according to some embodiments of the invention are 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 It comprises an amino acid sequence that exhibits an increase in percent identity when aligned with a sequence selected from the group consisting of SEQ ID NO: 33. Specific amino acid sequences within these and other embodiments may be, for example, at least about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86% with the aforementioned sequences. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence having identity May be included. In many embodiments, an amino acid sequence having the aforementioned sequence identity when aligned with the aforementioned sequence comprises a peptide having enzymatic delta-9-18: 0-ACP desaturase activity or a portion of such a peptide. Code.

C. Nucleic acids Some embodiments include nucleic acid molecules that encode the polypeptides described above. For example, the nucleic acid sequence in some embodiments is 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 percent identity when aligned with a sequence selected from the group consisting of SEQ ID NO: 25 Increase. Specific nucleic acid sequences within these and other embodiments are 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 a sequence selected from the group consisting of SEQ ID NO: 25, 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. One skilled in the art will appreciate that a nucleic acid molecule can be modified without substantially changing the amino acid sequence of the encoded polypeptide, eg, by introduction of permissible nucleotide substitutions according to codon degeneracy. .

  In some embodiments, the nucleic acid molecules of the invention include gene regulatory elements (eg, promoters). The promoter can be selected based on the cell type into which the vector construct is inserted. Promoters that function in bacteria, yeast, and plants are well known in the art. Promoters can also be selected based on their regulatory characteristics. Examples of such features include enhanced transcriptional activity, inducibility, tissue specificity, and developmental stage specificity. In plants, inducible, viral or synthetically derived, constitutively active, temporally regulated and spatially regulated promoters have been described. See, for example, 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, salicylic acid or polyacrylic acid induced promoters, heat shock promoters, spinach nitrate reductase transcription induced by the application of safeners (substituted benzenesulfonamide herbicides). Nitrate-inducible promoters derived from possible nucleic acid molecule sequences, hormone-inducible promoters, and light-inducible promoters with small subunits of RuBP carboxylase and LHCP family are included.

  Examples of useful tissue specific, developmentally regulated promoters include the β-conglycinin 7Sα promoter and the seed specific promoter. Plant functional promoters useful for preferential expression in seed plastids include promoters of proteins involved in fatty acid biosynthesis in oil seeds and promoters of plant conserved proteins. Examples of such promoters include 5 'regulatory regions of transcribable nucleic acid molecule sequences such as phaseolin, napin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and oleosin. Another typical tissue specific promoter is a lectin promoter specific for seed tissue.

  More specifically, the promoter includes a bean (Phaseolus vulgaris) phaseolin promoter (alone or in combination with a phaseolus vulgaris phaseolin 3 'untranslated region and a phase bean (Phaseolus vulgaris) phaseolin 3' matrix attachment region. ), The Lesquerella fendleri KCS3 promoter, or the Agrobacterium tumefaciens mannopine synthase promoter.

  Other useful promoters include nopaline synthase, mannopine synthase, and octopine synthase promoters on Agrobacterium tumefaciens tumor-inducing plasmids; cauliflower mosaic virus (CaMV) 19S and 35S promoters; An enhanced CaMV 35S promoter; sesame mosaic virus 35S promoter; a light-inducible promoter of ribulose-1,5-niphosphate carboxylase small subunit (ssRUBISCO); tobacco EIF-4A promoter (Mandel et al. (1995) Plant Mol. Biol. 29: 995-1004); corn sucrose synthase; corn alcohol dehydrogenase 1; corn condensing complex; corn heat shock protein; These include the Arabidopsis chitinase promoter; LTP (lipid transfer protein) promoter; petunia chalcone isomerase; bean glycine rich protein 1; potato patatin; ubiquitin promoter; Useful promoters are preferably seed selective, tissue selective, or inducible. Seed specific regulation is discussed, for example, in EP 0255378.

  In order to express higher levels of the heterologous gene, it is preferable to reengineer the gene to express it more efficiently in expression host cells (eg plant cells such as canola, rice, tobacco, corn, cotton, and soybean). There is a case. Thus, an optimal further step in the design of a gene encoding a delta-9 desaturase for plant expression (ie, in addition to providing one or more gene regulatory elements) would be the region encoding a heterologous gene protein. Re-manipulate for optimal expression. Certain embodiments have increased expression levels in transgenic canola plant cells or Arabidopsis plant cells than in canola plant cells or Arabidopsis plant cells transformed with native heterologous gene sequences ( Ie, redesigned genes that are optimized to produce more protein).

  Due to the flexibility provided by the redundancy / degeneracy of the genetic code (ie, some amino acids are specified by more than one codon), the evolution of the genome in different organisms or classes of organisms is synonymous Codons are used differentially. This “codon bias” is reflected in the average base composition of the protein coding region. For example, organisms with relatively low G + C content genomes utilize more codons with A or T at the third position of the synonymous codon, while organisms with higher G + C content use G or C. Use more codons at the third position. Further, it is believed that the presence of a “minor” codon in the mRNA can reduce the complete translation rate of that mRNA, particularly when the relative abundance of charged tRNA corresponding to that minor codon is small. An extension of this reasoning is that the reduction in translation rate due to individual minor codons will be at least additive to multiple minor codons. Thus, mRNA with a high relative content of minor codons in a particular expression host will have a correspondingly low translation rate. This translation rate can be reflected by the correspondingly low level of the encoded protein.

  In the manipulation of optimized genes encoding delta-9 desaturase for expression in canola or Arabidopsis (or other plants such as rice, tobacco, corn, cotton, or soybean) It is useful if the codon bias of a certain host plant has been determined. There are several publicly available DNA sequence databases in which information about the codon distribution of the plant genome or protein coding regions of various plant genes can be known.

  Codon bias is the statistical distribution of codons used by expression hosts (eg, plants such as canola or Arabidopsis) to encode the amino acids of the protein. Codon bias can be calculated as the frequency with which a single codon is used for all amino acid codons. Alternatively, codon bias can be calculated as the frequency with which a single codon is used to encode that amino acid for all other codons (synonymous codons) for that particular amino acid.

  In designing an optimized coding region for plant expression of the delta-9 desaturase gene, the first ("first option") codon preferred by the plant is determined, and if multiple options exist, Preferred codon second, third, fourth, etc. options are also determined. A new DNA sequence encoding the amino sequence of the delta-9 desaturase gene can then be designed, where the new DNA sequence comprises a non-denaturing DNA sequence (encoding the desaturase) and an amino acid at each position within the amino acid sequence. Depending on the substitution of codons preferred by the expression host to identify (such as the first preferred, second preferred, third preferred or fourth preferred codon). The new sequence is then analyzed for restriction enzyme sites that may have been generated by the modification. The identified putative restriction sites are further modified by replacing these codons with the next preferred codon to remove the restriction sites. Other sites in the sequence that can affect transcription or translation of heterologous sequences are exons: intron junctions (5 'or 3'), poly A addition signals, and / or RNA polymerase termination signals. The sequence can be further analyzed and modified to reduce the frequency of TA or CG doublets. In addition to these doublets, sequence blocks having about 7 or more identical G or C nucleotides can also adversely affect the transcription or translation of the sequence. Thus, these blocks are advantageously modified by replacing codons such as the first or second option with the next preferred codon option.

  The above method allows a person skilled in the art to modify a foreign gene for a specific plant and optimally express that gene in the plant. This method is further described in PCT application WO 97/13402. Thus, an optimized synthetic gene functionally equivalent to the desaturase / gene of some embodiments can be used to transform a host, including plants. Further guidance regarding the production of synthetic genes can be found, for example, in US Pat. No. 5,380,831.

  Once the plant-optimized DNA sequence is designed on the desk or in silico, the actual DNA molecule can be synthesized in the laboratory so that the sequence corresponds exactly to the designed sequence. Such synthetic DNA molecules can be cloned and otherwise manipulated exactly as if they were derived from natural or non-denaturing sources.

D. Methods of Genetic Transformation of Plant Material Some embodiments include 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, at least 60% of the sequence selected from the group consisting of 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 relates to a method for producing transformed cells comprising one or more nucleic acid molecules comprising identical nucleic acid sequences. Such nucleic acid molecules can also include non-coding regulatory elements such as, for example, a promoter. Other sequences can also be introduced into cells along with non-coding regulatory elements and transcribable nucleic acid molecule sequences. These other sequences can include 3 ′ transcription terminators, 3 ′ polyadenylation signals, other untranslated sequences, transit or targeting sequences, selectable markers, enhancers, and operators.

  The method of transformation usually comprises selecting an appropriate host cell, transforming the host cell with a recombinant vector, and obtaining a transformed host cell. Techniques for introducing DNA into cells are well known to those skilled in the art. These methods can usually be classified into the following five categories. (1) Chemical method (Graham and Van der Eb (1973) Virology 54 (2): 536-9; Zatloukal et al. (1992) Ann. NY Acad. Sci. 660: 136-53), (2) Micro Injection (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; US 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) Lu et al. (1993) J. Exp. Med. 178 (6): 2089-96; Eglitis and Anderson (1988) Biotechniques 6 (7): 608-14), (4) receptor mediated Sexual mechanism (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) A mechanism mediated by bacteria such as Agrobacterium. Alternatively, the nucleic acid can be introduced directly into the pollen by being directly injected into the reproductive organs of the plant. 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 described in US Pat. No. 5,508,184. Nucleic acid molecules can also be injected into immature embryos. Neuhaus et al. (1987) Theor. Appl. Genet. 75:30.

  The most commonly used method for plant cell transformation is the Agrobacterium-mediated DNA transfer process (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80: 4803). Biolistics or microprojectile bombardment (as described in US Pat. No. 5,824,877, US Pat. No. 5,591,616, US Pat. No. 5,981,840, and US Pat. No. 6,384,301). Interventional processes (ie gene guns) (US Pat. No. 5,550,318, US Pat. No. 5,538,880, US Pat. No. 6,160,208, US Pat. No. 6,399,861, and US Pat. No. 6,403,865) As described in). Typically, nuclear transformation is desirable, but when it is desirable to specifically transform plastids such as chloroplasts or amyloplasts, within a particular plant species, such as Arabidopsis, tobacco, Microprojectile-mediated delivery of the desired nucleic acid molecule into potato and Brassica species can be used to transform plant plastids.

  Agrobacterium-mediated transformation is performed through the use of genetically engineered soil bacteria belonging to the genus Agrobacterium. Several Agrobacterium species mediate specific DNA imports known as “T-DNA” that can be genetically engineered to carry any desired piece of DNA into many plant species. . The main events that characterize the process of T-DNA-mediated pathogenesis are the induction of virulence genes, and the processing and transfer of T-DNA. This process is the subject of many book reviews. For example, 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 Weds., Academic Press, San Diego, CA, 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.

  Regardless of the method of transformation, in order to select or score for transformed plant cells, the DNA introduced into the cells will produce a compound that confers resistance to other toxic compounds on the plant tissue. It may contain genes that function in renewable plant tissues. Genes of interest for use as selectable, screenable, or scorable markers include, but are not limited to, β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase, and antibiotics or herbicides Resistance genes are included. Examples of antibiotic resistance genes include genes that confer resistance to penicillin, kanamycin (and neomycin, G418, bleomycin), methotrexate (and trimethoprim), chloramphenicol, and tetracycline. For example, glyphosate tolerance can be conferred by a herbicide tolerance gene. Della-Cioppa et al. (1987) Bio / Technology 5: 579-84. Other selection devices can also be implemented including, but not limited to, phosphinothricin, resistance to bialaphos, and positive selection mechanisms (Joersbro et al. (1998) Mol. Breed. 4: 111-7). Are considered to be within the scope of embodiments of the present invention.

  Transformed cells identified by selection or screening and cultured in a suitable medium that supports regeneration can then be matured in plants.

  The methods disclosed herein can be used with any transformable plant cell or tissue. As used herein, transformable cells and tissues include, but are not limited to, cells or tissues capable of further growth to give rise to plants. One skilled in the art will recognize that many plant cells or tissues can be transformed in which, after insertion of exogenous DNA and appropriate culture conditions, the plant cells or tissues can be formed into differentiated plants. doing. Suitable tissues for these purposes include, but are not limited to, immature embryos, scutellum tissues, suspension cell cultures, immature inflorescences, shoot meristems, nodal explants, callus tissues, hypocotyl tissues, cotyledons, Roots and leaves can be included.

  The regeneration, development, and culture of plants from protoplasts or explants of transformed plants is known in the art. Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, (Eds.) Academic Press, Inc., San Diego, CA; Horsch et al. (1985) Science 227: 1229-31. This regeneration and growth process typically includes the steps of selecting transformed cells and culturing these cells through the normal embryo growth stage through the rooted plantlet stage. Transgenic embryos and seeds are regenerated as well. In this method, transformants are usually cultured in the presence of a selective medium that selects for successfully transformed cells and induces regeneration of plant shoots. Fraley et al. (1993) Proc. Natl. Acad. Sci. USA 80: 4803. These shoots are typically obtained within 2 to 4 months. The resulting rooted transgenic shoots are then planted in a suitable plant growth medium such as soil. Cells that have survived exposure to the selective agent, or cells that have been positively scored in the screening assay, can be cultured in a medium that supports plant regeneration. The shoots can then be transferred to a suitable rooting induction medium containing a selective agent and an antibiotic to prevent bacterial growth. Many of the shoots grow roots. These are then transplanted into soil or other media to allow continuous root growth. Methods such as those outlined above usually vary depending on the particular plant strain used, and methodological details are therefore within the discretion of one of ordinary skill in the art.

  Regenerated transgenic plants can be self-pollinated to produce homozygous transgenic plants. Alternatively, pollen obtained from regenerated transgenic plants can be crossed with non-transgenic plants, preferably inbred lines of agronomically important species. Conversely, pollen from non-transgenic plants can be used to pollinate regenerated transgenic plants.

  A transgenic plant can transmit a transformed nucleic acid sequence to its progeny. The transgenic plant is preferably homozygous for the transformed nucleic acid sequence and transfers that sequence during sexual reproduction and as a result of sexual reproduction to all of its progeny. Offspring can grow from seeds produced by the transgenic plants. These additional plants can then be self-pollinated to produce a true mating system of the plants.

  The progeny of these plants can be evaluated for gene expression among others. Gene expression can be detected by several common methods such as Western blot, Northern blot, immunoprecipitation, and ELISA (enzyme linked immunosorbent assay). Transformed plants can also be analyzed for the presence of the introduced DNA and the expression levels and / or fatty acid profiles conferred by the nucleic acid and amino acid molecules of the present invention. The person skilled in the art knows many methods available for the analysis of transformed plants. For example, plant analysis methods include, but are not limited to, Southern or Northern blots, PCR-based approaches, biochemical assays, phenotypic screening methods, field assessments, and immunodiagnostic assays.

  Methods for specifically transforming dicotyledonous plants are well known to those skilled in the art. Transformation and plant regeneration using these methods include, but are not limited to, members of the genus Arabidopsis, cotton (Gossypium hirsutum), soybean (Glycine max), peanut (Arachis hypogaea), and Brassica Many crops have been described, including Methods for transforming dicotyledonous plants to obtain transgenic plants mainly by using Agrobacterium tumefaciens are described in cotton (US Pat. No. 5,004863, US Pat. No. 5,159,135, US Pat. No. 5,518,908), soybean (US Pat. No. 5,569,834, US Pat. No. 5,461,611, McCabe et al. (1988) Biotechnology 6: 923; Christou et al. (1988) Plant Physiol. 87 : 671-4), Brassica (US 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 peas (Grant et al. (1995) Plant Cell Rep. 15: 254-8).

  Methods for transforming monocotyledonous plants are also well known in the art. Transformation and plant regeneration using these methods include, but are not limited to, barley (Hordeum vulgarae), maize (Zea mays), oats (Avena sativa), orchard grass (Dactylis glomerata), rice (indica and japonica types) Oryza sativa, Sorghum bicolor, sugar cane (Saccharum sp), tall fescue (Festuca arundinacea), turfgrass species (eg, Agrostis stolonifera), prickly grass (Poa pratensis), cent (Stenotaphrum secundatum), wheat (Triticum aestivum), and alfalfa (Medicago sativa) have been described for many crops. It will be apparent to those skilled in the art that many transformation methods are available and can be modified to produce stable transgenic plants for any number of target crops of interest.

  Any plant can be selected for use in the methods disclosed herein. Preferred plants for modification according to the present invention include, but are not limited to, oil seed plants, Arabidopsis thaliana, borage (Borago spp.), Canola (Brassica spp.). ), Castor bean (Ricinus communis), cacao bean (theobroma cacao), maize (Zea mays), cotton (Gossypium spp)), hamana (Crambe spp.), Kuphea spp (Cuphea spp) .), Flax (Linum spp.), Lesquerella and Limnanthes species, Linola, Nosenhallen (Tropaeolum spp.), Oenothera species spp.), olives (Olea spp.), palm (Elaeis spp.), peanuts (Arachis spp.), rapeseed, safflower Bana species (Carthamus spp.), Soybeans (Glycine, Soja species), sunflowers (Helianthus spp.), Tobacco (Nicotiana spp.)) , Vernonia spp., Wheat (Triticum spp.), Barley (Hordeum spp.), Rice (Oryza spp.), Oat ( Other members include Avena spp.), Sorghum (Sorghum spp.), And Rye (Secale spp.), Or other members of the Gramineae family.

  It will be apparent to those skilled in the art that many transformation methods are available and can be modified to produce stable transgenic plants for any number of target crops of interest.

E. Transgenic Seeds In some embodiments, the transgenic seeds are 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: A delta-9 desaturase polypeptide comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of 33 may be included. In these and other embodiments, the transgenic seed is 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, At least 60% identical to a sequence selected from the group consisting of 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 Nucleic acid sequences can be included. In certain embodiments, the transgenic seed may exhibit reduced saturated fatty acid (eg, palmitic and / or stearic fatty acid) levels. Seed can be harvested from a reproductive transgenic plant and comprises a hybrid plant system comprising a hybrid plant system comprising at least one nucleic acid sequence as indicated above and optionally at least one further gene or nucleic acid construct of interest. Can be used to grow offspring generations.

The following examples are provided to illustrate specific features and / or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.
[Example]

Construct Design of pDAB7305 The Aspergillus nidulans Δ-9 desaturase (AnD9DS) enzyme was previously disclosed in US Patent Application No. 2008/0260933 and is designated herein as SEQ ID NO: 1 . A polynucleotide sequence (SEQ ID NO: 2) containing the AnD9DS v3 coding sequence was synthesized and incorporated into the plasmid construct pDAB7305 (FIG. 1) for Agrobacterium-mediated plant transformation. The resulting construct contained three plant transcription units (also described as gene expression cassettes and used without distinction). The first plant transcription unit (PTU) (SEQ ID NO: 3) consists of an RB7 matrix attachment region (RB7 MAR, International Publication No. WO9727207), Phaseolus vulgaris phaseolin promoter (Pv Phas promoter, US Pat. No. 5,504,200). No.), AnD9DS coding sequence (Delta 9 desaturase v3), common bean (Phaseolus vulgaris) phaseolin 3 ′ untranslated region (Pv Phase 3′UTR, US Pat. No. 5,504,200), and common bean (Phaseolus vulgaris) phaseolin 3 It consisted of a 'matrix adhesion region (Pv Phase 3' MAR, US Pat. No. 5,504,200). The second PTU (SEQ ID NO: 4) consists of the Lesquerella fendleri KCS3 promoter (LfKCS3 promoter, US Pat. No. 7,253,337), the AnD9DS coding sequence (Delta 9 desaturase v3), and Agrobacterium tumefaciens ( Agrobacterium tumefaciens) ORF23 3 ′ untranslated region (AtuORF23 3′UTR, US Pat. No. 5,428,147). The third PTU (SEQ ID NO: 5) is an Agrobacterium tumefaciens mannopine synthase promoter (AtuMas promoter, Barker, RF, Idler, KB, Thompson, DV, Kemp, JD, (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 acetyltransferase gene (PAT, Wohlleben et al., (1988) Gene, 70 : 25-37), and Agrobacterium tumefaciens ORF1 3 ′ untranslated region (AtuORF1 3′UTR, Huang et al., (1990) J. Bacteriol., 172: 1814-1822) Met. The construct was confirmed via restriction enzyme digestion and sequencing. Finally, the construct was transformed into Agrobacterium tumefaciens and stored as a glycerol stock.

Agrobacterium-mediated transformation of canola (Brassica napus) hypocotyl Agrobacterium preparation Agrobacterium strain containing the pDAB7305 binary plasmid, streptomycin (Agrobacterium) 100 mg / ml) and spectinomycin (50 mg / mL) on YEP medium (Bacto Peptone ™ 20.0 gm / L and yeast extract 10.0 gm / L) and streaked on plate for 2 days Incubated at 0 ° C. The grown Agrobacterium strain containing the pDAB7305 binary plasmid was scraped from the streaked plate for 2 days using a sterile inoculation loop. The scraped Agrobacterium strain containing the pDAB7305 binary plasmid is then modified to 150 mL with streptomycin (100 mg / ml) and spectinomycin (50 mg / ml) in a sterile 500 mL baffled flask. The YEP solution was inoculated and shaken at 200 rpm at 28 ° C. The culture is centrifuged and resuspended in M medium (LS salt, 3% glucose, modified B5 vitamins, 1 μM kinetin, 1 μM 2,4-D, pH 5.8) and the appropriate density (spectroscopic). When measured using a photometer, it was diluted to 50 kret units), and then the canola hypocotyl was transformed.

Canola Transformation Seed Germination: The surface of canola seed (var. NEXERA 710 ™) was sterilized in 10% Clorox ™ for 10 minutes and rinsed 3 times with sterile distilled water (seeds were treated during this process). In a steel rod). Seeds were sown for germination on 1 / 2MS canola medium (1 / 2MS, 2% sucrose, 0.8% agar) placed in Physatrays ™ (25 seeds per Physatray ™) Placed in a Percival ™ growth chamber with a photoperiodic growth regime setting at 25 ° C., 16 hours light period and 8 hours dark period, and allowed to germinate for 5 days.

  Pretreatment: On day 5, approximately 3 mm long hypocotyl sections were aseptically removed and the remaining root and shoot parts were discarded (drying of hypocotyl sections was performed by removing 10 mL of hypocotyl sections during the excision process. Prevented by soaking in sterile milliQ ™ water). Hypocotyl sections were obtained from a callus induction medium in a Percival ™ growth chamber, MSK1D1 (MS, 1 mg / L kinetin, 1 mg / L 2,4-D, 3.0% sucrose, 0.7% phytagar) The above sterile filter paper was placed horizontally and pretreated for 3 days at a growth regime setting of 22-23 ° C. and 16 hours light period, 8 hours dark period photoperiod.

  Co-culture with Agrobacterium: On the day before Agrobacterium co-culture, the Agrobacterium strain containing the pDAB7305 binary plasmid was placed in a flask of YEP medium containing the appropriate antibiotic. Vaccinated. To prevent desiccation of hypocotyl sections, hypocotyl sections were transferred from filter paper callus induction medium MSK1D1 to an empty 100 × 25 mm Petri ™ dish containing 10 mL of liquid M medium. A spatula was used at this stage to scrape the sections and the sections were transferred to fresh medium. Liquid M medium was removed with a pipette and 40 mL of Agrobacterium suspension was added to the Petri ™ dish (500 sections and 40 mL of Agrobacterium solution). The hypocotyl slices were processed for 30 minutes by swirling the Petri ™ dish intermittently so that the hypocotyl slices remained immersed in the Agrobacterium solution. At the end of the treatment period, the Agrobacterium solution was pipetted into a waste beaker and discarded by autoclaving (to prevent Agrobacterium solution from growing abnormally, the Agrobacterium solution must be completely removed. Removed). Treated hypocotyls were returned with forceps to the original plate covered with filter paper containing MSK1D1 medium (note that the sections were not dry). Transformed hypocotyl sections and non-transformed control hypocotyl sections were returned to the Percival ™ growth chamber with reduced light intensity (by covering the plates with aluminum foil) and treated hypocotyls. Sections were co-cultured with Agrobacterium for 3 days.

Callus induction in selective medium: After 3 days of co-culture, hypocotyl slices with forceps and callus induction medium MSK1D1H1 (MS, 1 mg / L kinetin, 1 mg / L 2,4 with a growth regime setting of 22-26 ° C. -D, MES of 0.5 mg / L, AgNO of 5mg / L _3, 300mg / L of Timentin (TM), carbenicillin 200 mg / L, of 1mg / L Herbiace (R), 3% sucrose, 0.7 % Phytagar) individually. The hypocotyl slice was fixed in the medium, but not deeply embedded in the medium.

Selection and regeneration of shoots: After 7 days on callus induction medium, callus-forming hypocotyl sections were taken from shoot regeneration selection medium 1, MSB3Z1H1 (MS, 3 mg / L BAP, 1 mg / L zeatin, 0.5 mg / L of MES, 5 mg / L AgNO ₃ of, 300 mg / L of Timentin (TM), carbenicillin 200 mg / L, of 1mg / L Herbiace (R), 3% sucrose, transferred to 0.7% Phytagar). After 14 days, hypocotyl slices from which shoots were germinated were set at a growth regime of 22-26 ° C., regeneration-enhanced selective medium 2, MSB3Z1H3 (MS, 3 mg / L BAP, 1 mg / L zeatin, 0.5 mg / L MES, 5 mg / L AgNO ₃ , 300 mg / L Timentin ™, 200 mg / L carbenicillin, 3 mg / L Herbiace ™, 3% sucrose, 0.7% phytagar).

  Elongation of shoots: After 14 days, hypocotyl slices from which shoots were germinated were grown from regeneration medium 2 to shoot elongation medium MSMESH5 (MS, 300 mg / L of Timentin ™, 5 mg / l) at a growth regime setting of 22-26 ° C. Herbiace ™, 2% sucrose, 0.7% TC Agar). Already growing shoots were isolated from hypocotyl sections and transferred to MSMESH5. After 14 days, the remaining shoots that did not grow in the first culture in the shoot elongation medium were transferred to the fresh shoot elongation medium MSMESH5. At this stage, all remaining hypocotyl sections that did not produce shoots were discarded.

  Root induction: After 14 days of culture in shoot elongation medium, isolated shoots were cultured at 22-26 ° C. in MSMEST medium (MS, 0.5 g / L MES, 300 mg / L for root induction. Timentin ™, 2% sucrose, 0.7% TC Agar). Any shoots that did not give rise to roots after incubation in the first transfer to MSMEST medium were transferred to a second or third incubation in MSMEST medium until the shoots gave rise to roots.

  PCR analysis: Transplanted canola hypocotyl sections regenerated into shoots containing roots were further analyzed via PCR molecular confirmation assay. Leaf tissue was obtained from green shoots and tested for the presence of the pat selectable marker gene via PCR. Any dwarf shoots were discarded and no PCR analysis was performed. Samples identified as positive for the presence of the pat selectable marker gene were maintained and cultured in MSMEST medium to continue germination and elongation of shoots and roots. Samples that were negative by PCR analysis and that did not contain the pat selectable marker gene were discarded.

Transformed canola plants containing shoots and roots that were PCR positive for the presence of the pat selectable marker gene were transplanted to soil in the greenhouse. After establishment of canola plants in soil, the canola plants were further analyzed to determine the copy number of the pat gene expression cassette via Invader ™ quantitative PCR assay and Southern blot. Transgenic T ₀ canola plants were confirmed to contain at least one copy of pat gene expression cassette was proceeded for further analysis of the seed fatty acid profile. Seeds obtained from these transgenic T ₀ canola plants, ie T ₁ canola seeds, were analyzed via the FAME analysis method to identify events in which total saturated fatty acids were reduced compared to control plants ( The total saturated fatty acid content was determined by summing all of the saturated fatty acids including single and long chain fatty acids).

FAME analysis of T ₁ canola seeds obtained from transgenic pDAB7305 canola plants. Isolated T ₁ canola seeds were analyzed via the FAME analysis method and compared to seeds obtained from control plants grown under the same conditions. A T ₀ canola event was identified that produced T ₁ canola seeds with reduced saturated fatty acids (C14: 0, C16: 0, C18: 0, C20: 0, C22: 0, C24: 0). The sum of all total saturated fatty acids (TSFA) was quantified and compared to negative control plants. FAME analysis was performed using the protocol described below for single T ₁ canola seeds. A total of 24 single T ₁ canola seeds from each canola T ₀ event were assayed and the TSFA results for each single seed were quantified.

  Single canola seed samples were homogenized using a steel ball mill in heptane containing triheptadecanoin (Nu-Chek prep) as a triacylglycerol internal standard. Prior to homogenization, 0.25 M freshly prepared sodium methoxide solution in methanol (Sigma-Aldrich, St. Louis, MO) was added. Extraction was performed at 40 ° C. with continuous shaking. Endogenous fatty acid recovery was normalized by recovery of methylated alternative C17 fatty acids. The extraction of FAME (fatty acid methyl ester) was repeated three times and the heptane layer was pooled and subsequently analyzed. The obtained FAME was analyzed by GC-FID using an SGE capillary column BPX70 (15 m × 0.25 mm × 0.25 μm). By identifying each FAME by retention time, adding the appropriate long chain fatty acid (Nu-Chek Prep, Elysian MN) and injecting a rapeseed oil reference mixture of Matreya LLC (Pleasant Gap, PA) as a calibration standard Quantified.

  Bulk seed analysis consisted of 50 mg aliquots (10 to 15 seeds combined) and followed the same protocol described above with slight modifications. To complete the derivatization reaction, the oil was first extracted three times with heptane. An aliquot of the combined oil extract corresponding to one seed was then derivatized in FAME as described in the single seed protocol above. The integrity of the reaction was verified by checking for the presence of endogenous FAME in the fourth extraction / derivatization.

Three transgenic canola events (event 2182 [12] -138.001, event 2182 [12] -125.001, and event 2182 [12] -156.001) were significantly associated with TSFA compared to control canola plants. based on the results of FAME exhibit reduced were identified and selected to advance the T ₁ generation. Two further categories of plant fatty acid content were assayed. These categories include concentrations of monounsaturated fatty acids (MUFA: C16: 1, C18: 1, and C20: 1) and polyunsaturated fatty acids (PUFA: C18: 2 and C18: 3), It listed to show the effect of reducing the TSFA of T ₁ in seeds (Table 1).

  The average TSFA of transgenic canola events is significantly reduced compared to NEXERA 710 ™ non-transformed control plants. Along with the reduction in TSFA, an increase in MUFA content (C18: 1 and C16: 1) was also observed. The increase in MUFA content is a direct result of the overexpression of AnD9DS that introduces a double bond at the ninth carbon (Δ9) from the carboxyl group of the saturated fatty acid. Interestingly, PUFA content did not increase with the accumulation of MUFA substrate of glycerophospholipid desaturase FAD2, which synthesizes C18: 2.

T ₁ canola plant events were grown in the greenhouse, self-pollinated, and the transferred transgene was fixed in the progeny plants. Invader ™ quantitative PCR assays were performed on multiple T ₁ canola plants for each transgenic event. These results indicated that the T ₁ canola plants obtained from each of the three events contained about two or three copies of pDAB7305 T chain incorporation (upper panel in FIG. 3). A specific determination of copy number is not possible because the T chain components of pDAB7305 were separated at different copy numbers in the three events. The T ₁ canola plant events were grown to maturity in the greenhouse, were self-pollinated. The resulting T ₂ canola seeds were harvested for fatty acid profile analysis via FAME assay.

The _{T 2} canola seed _{T 2} canola seeds FAME analysis bulk obtained from transgenic pDAB7305 canola plants, and analyzed via FAME analysis method described above, in comparison with the control plants (NEXERA710 (TM)) A T ₁ canola plant line with reduced total saturated fatty acids was identified (FIG. 2). The yield of each plant was recorded as grams of seed per plant. Yield results are stable for an Aspergillus nidulans delta-9 desaturase transgene (see WO 2006/042049) grown in parallel with the transgenic plants to be disclosed. Compared to the yield of the integrated transgenic positive control event 218-11.30 (also described herein as 218-11.30HS50 or 218-11.30HL). Both 218-11.30 plants and the disclosed transgenic canola plants express similar transgenes. However, 2182 [12] -125. Sx001, 1182 [12] -138. Sx001, and 2182 [12] -156. The construct used in the transformation of Sx00 canola plants is different, which consists of the Lesquerella fendleri KCS3 promoter-driven expression of the AnD9DS coding sequence and the 3 ′ of Agrobacterium tumefaciens ORF23. This is because the non-translated region is adjacent and contains the second PTU.

The TSFA (%) of the disclosed transgenic plants was quantified and compared to the TSFA (%) obtained from the positive control 218-11.30 HL transgenic canola plant and the negative control NEXERA 710 ™ plant. A small number of disclosed transgenic plants were identified as containing high levels of TSFA, similar levels to the negative control NEXERA710 ™ plants. These plants are sibling nulls as a result of segregation of the transgene during self-pollination and do not contain any expressed copy of the disclosed transgene activity . These results confirmed that the total saturated fatty acid content of bulk T ₂ canola seeds was reduced to less than 3.5% in the identified T ₁ canola plants.

The T ₂ canola line was further analyzed to determine which canola line contained the low copy number pDAB7305 T chain component and produced a high T ₂ seed yield (FIG. 3).

Individual canola plants to be disclosed were selected that produced less than 3.5% TSFA, produced yields greater than 10 g, and contained the lowest T chain copy number. Based on these three criteria (less than 3.5% TSFA, high seed yield, and low copy number level), seven canola plants were selected to proceed to further characterization of the TSFA profile. Copy number was determined from one plant per T1 line using an invader assay. Table 2. Transfer these T ₂ canola plant in a greenhouse, grown to maturity and allowed to self-pollinate. T ₂ canola plant molecularly analyzed further, the T ₃ seeds were taken for fatty acid profile analysis via FAME assay.

The T ₂ canola-based molecular confirmation pat gene expression cassette T ₂ canola events selected contained (and closely linked AnD9DS gene expression cassette) of, using quantitative PCR and Southern blot analysis, the molecules The incorporation pattern was characterized.

Confirmation of AnD9DS integration via hydrolysis probe assay The presence of the AnD9DS gene expression cassette was confirmed via hydrolysis probe assay. Isolated T ₂ canola plants, first screened through a similar hydrolysis probe assay to TAQMAN (TM), confirmed the presence of pat transgene. The data obtained from these studies was used to determine transgene copy number and to select transgenic canola events for backcrossing and proceeding to subsequent generations.

  Tissue samples were collected in 96-well plates and the tissues were macerated in a Qiagen RLT ™ buffer using a KLECO ™ tissue grinder and stainless steel beads (Hoover Precision Products, Cumming, GA). Following tissue maceration, genomic DNA was isolated in a high-throughput format using the Bioprint 96 ™ Plant kit (Qiagen, Germantown, MD) according to the manufacturer's suggested protocol. Genomic DNA was quantified by the Quant-IT Pico Green DNA assay kit ™ (Molecular Probes, Invitrogen, Carlsbad, CA). Quantified genomic DNA was adjusted to approximately 2 ng / μL for hydrolysis probe assay using a BIOROBOT 3000 ™ automated liquid handler (Qiagen, Germany, MD). Determination of transgene copy number by a hydrolysis probe assay similar to the TAQMAN® assay was performed by real-time PCR using the LIGHTCYCLER® 480 system (Roche Applied Science, Indianapolis, Ind.). The assay was designed for pat and the 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, a LIGHTCYCLER® 480 probe master mix (Roche Applied Science, Indianapolis, IN) was multiplexed into a 10 μL volume containing 0.4 μM of each primer for AnD9DS and pat and 0.2 μM of each probe. (Multiplex) Prepared at 1x final concentration in reaction (Table 3). A two-step amplification reaction was performed at 60 ° C. for 40 seconds of extension and fluorescence acquisition. All samples were run and the average cycle threshold (Ct) was used for analysis of each sample. Analysis of real-time PCR data is performed using the LIGHTCYCLER® software release 1.5 using a relative quantification module and is based on the ΔΔCt method. The control included a genomic DNA sample obtained from a single copy calibrator and two known copy checks included in each run. Table 4 lists the results of the hydrolysis probe assay. Copy number was determined from N plants per T1 line using a qPCR assay (and averaged to give the values in Table 4).

  The results of the hydrolysis probe assay are relative standard deviation (shown as SD in Table 4) and coefficient of variation (shown as CV% in Table 4) comparable to the positive control plant (218-11.30 (HL)). Two systems (2182 [12] -138.Sx001.Sx094 and 2182 [12] -138.SX001.Sx090) having a combination of: The 218-11.30 (HL) control plant has previously been identified as containing two fixed copies of the AnD9DS gene insertion (WO 2006/042049). By comparing selected canola lines (2182 [12] -138.Sx001.Sx094 and 2182 [12] -138.SX001.Sx090) with 218-11.30 (HL) control plants, the T chain of pDAB7305 A specific canola system was identified that would contain a fixed copy of the component.

Confirmation of AnD9DS genomic integration via Southern blot analysis.
Southern blot analysis was used to establish the integration pattern of the inserted T-strand DNA fragment and to identify a canola system containing the full-length AnD9DS gene expression cassette. Data was generated to demonstrate integration and integrity of the transgene insert within the canola genome. Detailed Southern blot analysis was performed using a PCR amplified probe specific for the AnD9DS gene expression cassette. Hybridization of the probe with genomic DNA digested with specific restriction enzymes identified genomic DNA fragments of specific molecular weight, and their patterns were used to characterize 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 KLECKO ™ tissue grinder and tungsten beads. After tissue maceration, genomic DNA was isolated using the CTAB isolation procedure. Genomic DNA was further purified using a Qiagen Genomic Tips ™ kit. Genomic DNA was quantified by the Quant-IT Pico Green DNA ™ assay kit (Molecular Probes, Invitrogen, Carlsbad, CA). Quantified genomic DNA was adjusted to a consistent concentration.

  In each sample, 4 μg of genomic DNA was completely digested with the restriction enzyme BamHI (New England Biolabs, Beverley, Mass.). The digested DNA was concentrated by precipitation using Quick Precipitation Solution ™ (Edge Biosystems, Gaithersburg, MD) according to the manufacturer's suggested protocol. Genomic DNA was then resuspended in 25 μL of water at 65 ° C. for 1 hour. The resuspended sample was loaded onto a 0.8% agarose gel prepared in 1 × TAE and electrophoresed at 1.1 V / cm overnight in 1 × TAE buffer. The gel was denatured continuously for 30 minutes (0.2 M NaOH / 0.6 M NaCl) and neutralized for 30 minutes (0.5 M Tris-HCl (pH 7.5) /1.5 M NaCl).

  Transfer the DNA fragment to the nylon membrane using a chromatography paper wick and paper towel on a treated IMMOBILON ™ NY + transfer membrane (Millipore, Billerica, Mass.) Over 20 × SSC solution via gel overnight. Done by sucking passively. After transfer, the membrane was briefly washed with 2 × SSC, crossed with STRATALINKER ™ 1800 (Stratagene, LaJolla, Calif.) And vacuum baked at 80 ° C. for 3 hours.

  Blots were pre-hybridized (Perfect Hybrid plus, Sigma, St. Louis, MO) with a model 400 hybridization incubator (Robbins Scientific, Sunnyvale, Calif.) For 1 hour at 65 ° C. in a glass roller bottle. Incubated. The probe was prepared from a PCR fragment containing the entire coding sequence. PCR amplicons were purified using QIAEX II gel extraction kit ™ and labeled with DIG DNA Labeling Kit ™ (Roche Applied BioScience, Indianapolis, IN). The blot was hybridized overnight at 65 ° C. with denatured probe added directly to the hybridization buffer. After hybridization, the blots were washed sequentially with 0.1 × SSC / 0.1% SDS at 65 ° C. for 40 minutes. Finally, the blot was exposed to a storage phosphor imaging screen and imaged using a Molecular Dynamics Storm 860 ™ imaging system.

Southern blot analysis performed in this study was used to determine copy number and to confirm that the selected event contained the AnD9DS gene expression cassette within the canola genome. Table 5 is based on the criteria defined above in Table 2 shows the banding profiles for T ₂ plants of the selected system. The control system did not contain a selectable marker that supported the PCR data. Most of the systems selected from the three events are events 2182 [12] -125. Sx001. Except for the Sx014 system, a homogeneous band pattern (number and size) is shown. Event 2182 [12] -138. All three T2 lines of Sx001 show a T2 population with a consistent banding pattern.

Yield of selected canola line T ₃ seeds Selected plants of the T ₂ canola line were grown to maturity in the greenhouse. Seeds were collected from the plants. The seeds were cleaned and the seed yield per T ₂ canola line was determined (Table 6). The seed yield of each line was compared to the seed yield obtained from a non-transformed control plant (Nexera ™ 710GS) grown under the same conditions. Table 6 presents the results of yield for various plants obtained from each T ₂ canola system. These results indicate that the yield varied for each plant and line tested. However, the T ₂ canola system (2182 [12] -125.Sx001.Sx014, 2182 [12] -138.Sx001.Sx085, 2182 [12] -138.Sx001.Sx090, 2182 [12] -138.Sx001.Sx094 , And 2182 [12] -156.Sx001.Sx049) are relatively similar and significantly deviate from the control plants (Nexera ™ 710G5 and 218-11.30 (HL)) There wasn't.

Both FAME analysis single and bulk of _{T 3} canola seed of _{T 3} canola seeds obtained from transgenic pDAB7305 canola plants, and analyzed via FAME analysis method described above, characterized fatty acid profile of the system Thus, a specific system with reduced total saturated fatty acids compared to control plants was identified. The total total saturated fatty acids were quantified and compared to positive and negative control plants. The result shows that the total saturated and saturated fatty acid (determined from the sum of palmitic and stearic acid contents) content of single and bulk T ₃ canola seeds is less than 3.5% in the selected canola plant line It was confirmed that it was reduced.

  Surprisingly, two systems, 2182 [12] -138. Sx001. Sx085 and 2182 [12] -138. Sx001. Sx094 accumulated an average TSFA level of less than 3.0%. For the first time a canola line comprising bulk seeds containing less than 3.0% saturated fatty acids is described. Table 7 and Table 8.

  In addition, a subset of canola lines was used for FAME analysis of seeds to determine the lowest levels of total and saturated fatty acids that could be obtained with a single canola seed. Single seed FAME analysis was performed on seeds obtained from canola lines selected based on the lowest total saturated fatty acids and high levels of plant yield in bulk seeds. A total of 288 seeds per line were analyzed using the FAME method. A summary of the analysis is shown in Table 9. All single seeds of the selected plants have an average 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 5.11% TSFA level obtained in the Nexera ™ 710G5 control plant. Finally, the maximum TSFA percentage in transgenic canola lines does not exceed 3.5% and the average saturated fatty acid level in a single seed is 2.52%, well below 3.5%. Table 9 and FIG.

Table 7 shows the T ₂ mature seed bulk for five populations of genetically homogeneous canola lines compared to non-transformed Nexera ™ 710G5 control and transformed 218-11.30 (HL) positive control plants. The distribution of FAME analysis is shown. The average of all individual measurements (N) was determined to represent the percentage of TSFA and saturated fatty acids for the population of canola plants. Canola system 2182 [12] -138. Sx001. Sx085 and 2182 [12] -138. Sx001. Sx094 is identified in bold because these systems have an average TSFA percentage below 3.00 percent.

Table 8, Nexera (TM) 710G5 compared to control, FAME and event 2182 of the lowest _{T 3} mature seeds bulk [12] -138. It shows a plant yield of single _{T 2} progeny plants obtained from Sx001. The results shown are for oil percentage, TSFA percentage, saturated fatty acid percentage (determined by summing palmitic acid and stearic acid contents), and seed yield.

Table 9 shows the FAME analysis of the results the distribution of _{T 3} single seeds _{T 2} system selected. The table shows the mean (Mean), minimum (Min), and maximum (Max) TSFA and saturated fatty acid percentages compared to Nexera ™ 710G5 control canola plants grown under the same conditions. Total saturated fatty acid (TSFA) and saturated fatty acid levels were reduced in T ₃ seeds of selected events compared to Nexera ™ 710G5 control canola plants.

Claims (28)

  An isolated nucleic acid molecule encoding a delta-9 desaturase enzyme comprising an amino acid sequence at least 80% identical to SEQ ID NO: 2.   2. The nucleic acid molecule of claim 1 further comprising at least one gene regulatory element.   The gene regulatory elements include Saccharomyces cerevisiae delta-9 desaturase promoter, delta-9 desaturase 3'UTR / terminator, ole1 gene promoter, phaseolus vulgaris phaseolin promoter, phaseolus vulgaris phaseolin 5 'Untranslated region, common bean (Phaseolus vulgaris) phaseolin 3' Untranslated region, common bean (Phaseolus vulgaris) phaseolin matrix attachment region, Lesquerella fendleri KCS3 promoter, Agrobacterium tumefaciens mannopine Enzyme promoter, Agrobacterium tumefaciens ORF23 3 'untranslated region, cassavabe Mosaic virus promoter, Agrobacterium tumefaciens ORF1 3 ′ untranslated region, Nicotiana tabacum RB7 matrix attachment region, overdrive, T chain boundary sequence, LfKCS3 promoter, FAE1 promoter, Myc tag, and hemagglutinin 3. A nucleic acid molecule according to claim 2, selected from the group consisting of tags.   A construct comprising the first nucleic acid molecule of claim 3 and the second nucleic acid molecule of claim 3, wherein the first nucleic acid molecule further comprises a first gene regulatory element, and the second A construct in which the nucleic acid molecule comprises a second gene regulatory element.   5. The construct of claim 4, wherein the first or second nucleic acid molecule contains at least two gene regulatory elements. A method for reducing the amount of saturated fatty acids in a cell,
A method comprising transforming a cell with the nucleic acid molecule of claim 1 to reduce the amount of saturated fatty acid in the cell. A method for reducing the amount of saturated fatty acids in a cell,
A method comprising transforming a cell with the construct of claim 5 to reduce the amount of saturated fatty acid in the cell.   8. The method of claim 7, wherein the cell is a yeast cell.   The method according to claim 7 or 8, wherein the cell is a plant cell.   10. The method of claim 9, comprising transforming the plant cell with two or more nucleic acid molecules of claim 1.   10. The method of claim 9, wherein transformation of the plant cell introduces means to the plant cell to reduce the level of 16: 0-CoA in the plant cell.   12. The method of claim 11, wherein the means for reducing the level of 16: 0-CoA in the plant cell is an extraplastid desaturase.   13. The method of claim 12, wherein the extraplastidic desaturase is a desaturase selected from the group consisting of LnD9DS desaturase, AnD9DS desaturase, HzD9DS desaturase, and MgD9DS desaturase.   13. The method of claim 12, wherein the extraplastidic desaturase is AnD9DS desaturase.   The plant cells are Arabidopsis, Borago, Canola, Ricinus, Theobroma, Zea, Gossypium, Crambe, Quafe Genus (Cuphea), flax genus (Linum), lesquerella genus (Limnanthes), linola (Linola), genus genus (Topaeolum), evening primrose (Oenothera), olive genus (Olea), oil genus (Elaeis) ), Peanut genus (Arachis), rapeseed, safflower (Carthamus), soybean genus (Glycine), soya subgenus (Soja), sunflower genus (Helianthus), tobacco genus (Nicotiana), Drosophila genus (Vernonia), wheat Genus (Triticum), barley (Hordeum), rice (Oryza), oat (Avena), sorghum (Secale), rye (Secale), and rice Obtained from a plant selected from the genera selected from the group consisting of other members of the (Gramineae), The method of claim 9.   An oilseed plant comprising the nucleic acid sequence of claim 1.   An oilseed plant comprising the construct according to claim 5 or 6.   Sequence number 2, Sequence number 14, Sequence number 15, Sequence number 16, Sequence number 17, Sequence number 18, Sequence number 19, Sequence number 19, Sequence number 21, Sequence number 21, Sequence number 22, Sequence number 23, Sequence number 24, Sequence number 25, a plant seed expressing an extraplastidic desaturase selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.   A seed of a transgenic Brassica napus line, wherein the level of saturated fatty acid is reduced as compared to the isogenic type of the transgenic Brassica napus line. A method for producing a genetically engineered plant with a reduced amount of saturated fatty acids in the plant compared to a wild type plant, comprising:
A method comprising the steps of transforming a plant material with the nucleic acid molecule of claim 1 and culturing the transformed plant material to obtain a plant. A method for producing a genetically engineered plant with a reduced amount of saturated fatty acids in the plant compared to a wild type plant, comprising:
A method comprising transforming plant material with the construct of claim 4, and culturing the transformed plant material to obtain a plant.   The plant is in the genus Arabidopsis, Borago, Canola, Ricinus, Theobroma, Zea, Gossypium, Crambe, Kuffer (Cuphea), flax genus (Linum), Lesquerella genus (Limnanthes), linola (Linola), genus genus (Topaeolum), evening primrose (Oenothera), olive genus (Olea), oil genus (Elaeis) , Peanut genus (Arachis), rapeseed, safflower (Carthamus), soybean genus (Glycine), soya subgenus (Soja), sunflower (Helianthus), tobacco genus (Nicotiana), Drosophila (Vernonia), wheat genus (Triticum), Hordeum, Oryza, Ovena, Sorghum, Secale, and Gramineae (G 24. The method of claim 21, wherein the method is selected from a genus selected from the group consisting of other members of ramineae).   A plant obtained by the method of claim 21.   A plant material obtained from the plant of claim 23.   24. The plant material according to claim 23, wherein the plant material is a seed.   24. A vegetable oil according to claim 23.   24. The plant oil of claim 23, wherein the oil contains less than 3.5% saturated fatty acids.   24. The plant oil of claim 23, wherein the oil contains less than 3.0% saturated fatty acids.