CN106998666A - Transgenosis Canola low or without saturated fatty acid - Google Patents

Abstract

Composition and method are included in plant cell genetic coding and express a kind of new desaturase of Δ 9.In some embodiments, the method for express nucleic acid utilizes the activity of the desaturase of Δ 9 in plant cell so that the percentage composition reduction of plant seeds and aliphatic acid, while havingThe increase of 9 aliphatic acid.In other embodiments, amino acid sequence has the desaturase of Δ 9 activity.Method can relate to express the desaturase of Δ 9, the purpose for increasing the amount of monounsaturated fatty acids in whole plant, vegetable seeds and vegetable material such as seed in plant cell, vegetable material and whole plant.

Description

Transgenosis Canola low or without saturated fatty acid

Technical field

Some embodiments are usually directed to some desaturases of Δ -9, the nucleic acid of encoding such enzymes, and in plant cell The middle method for expressing the enzyme.Some embodiments relate to the use of the activity reduction vegetable material of some desaturases of Δ -9 (for example Seed) in saturated fatty acid percentage composition, and increase the percentage composition of ω -7 aliphatic acid.Other embodiments are related to The desaturase of Δ -9 is expressed in seed using seed specific promoters are preferential.There is disclosed herein pass through particular In the plant for preparing of method and vegetable material, and contain by those saturated fatty acid less than 3.5% or less than 2.7% The oil that plant produces.

Background

Plant source oil (vegetable-derived oils) gradually instead of the grease of animal origin as diet fat The main source of fat intake.However, the saturated fat intake of most industrial countries stills remain in total amount of heat consumption 15% to 20% or so.In order to promote more healthy lifestyles, United States Department of Agriculture (USDA) advises recently, saturated fat levels Less than the 10% of every daily caloric intake amount.In order to promote consumer to realize, the existing mark guide that United States Department of Agriculture promulgates is present It is required that total saturated fatty acid level can obtain " low saturation " mark less than every 14 grams 1.0 grams, it can be obtained less than every 14 grams 0.5 gram Obtain " no saturation " mark.This means vegetable oil will obtain " low saturation " or " no saturation " mark, saturated fatty acid content needs Respectively lower than 7% and 3.5%.Since these guides are issued, consumer swashs to the demand of " low saturation " and " no saturation " oil Increase.Up to the present, this demand is mainly what is met by canola oil, and sunflower oil and safflower oil are also contributed, but small It is many.

Although unsaturated fat (single unsaturated and how unsaturated) is beneficial (particularly in moderate consumption), saturation Fat and trans fats are not.Saturated fat and trans fats can increase undesired LDL-C water in blood It is flat.Dietary cholesterol can also increase LDL-C in the case of low-density lipoprotein is not improved, and can Heart disease can be caused.Therefore, it is suggested that selecting low saturated fat, low trans fatty and low cholesterol food as the one of health diet Part.

Oil, either plant or animal origin, its feature it is main by the carbon number and number of hydrogen atoms in oil molecule with And the quantity of the double bond included in fatty acid chain and position are determined.Most of oil from plant are by different amounts of palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) with leukotrienes (18:3) aliphatic acid is constituted.Generally, it is brown Palmitic acid acid and stearic acid are referred to as " saturation ", because their carbochain is by hydrogen atom saturation, therefore without double bond；They contain most The hydrogen atom of quantity possible greatly.However, oleic acid, linoleic acid plus linolenic acid is 18 with one, two and three double bond respectively Carbon fatty acid chain.Oleic acid is typically considered monounsaturated fatty acids, and linoleic acid plus linolenic acid is considered as how unsaturated fat Fat acid.The definition of United States Department of Agriculture " no saturation " oil product (means the oil production with less than 3.5% saturated fatty acid content Product) calculated with the saturated fatty acid weight content (compared with fatty acid total amount) of combination.

The saturated fatty acid level of canola oil is minimum in all vegetable oil." Canola " refers to such rape (Btassica), its sinapic acid (C22:1) content is at most 2 weight %, the total fatty acid content (preferably up to 0.5 based on seed Weight %, most preferably substantially 0 weight %), and produced after broken in degreasing (oil-free) dregs of rice containing less than 30 μm ol/g The air-dried dregs of rice of glucosinolate.The rape of these types has excellent edibility compared with the traditional category of the species.

It is assumed that in oil seed, aliphatic acid synthesis is occurred mainly in plastid.Aliphatic acid synthesis primary product be Palmitic acid (16:0), it seems efficiently to be extended into stearic acid (18:0).Then, when it is also in plastid, saturated fat Fat acid can be by a kind of enzyme desaturation for being referred to as the desaturase of acyl-acp Δ -9, so as to introduce one or more carbon-to-carbon double bonds. Specifically, stearic acid can produce oleic acid (18 by the quick desaturation of the desaturase of plastid Δ -9:1).In fact, palmitic acid Palmitoleic acid (16 can also be turned into by the desaturase desaturation of plastid Δ -9:1), but in most plants oil, this fat Fat acid is only micro to have (0-0.2%).Therefore, the primary product of the aliphatic acid synthesis in plastid is palmitic acid, stearic acid and oil Acid.In most of oil, oleic acid is the main fatty acid of synthesis, because saturated fatty acid exists with much lower ratio.

The aliphatic acid newly synthesized is from plastid transit to cytoplasm.Then the vegetable fatty acid of desaturation seems in cytoplasm It is limited to oleic acid, it can be by acting on esterification to the microsome on the oleoyl or line base oil substrate of phosphatidyl choline (PC) Desaturase and by desaturation to linoleic acid (18:2) with leukotrienes (18:3).In addition, it is different according to plant, extension can be passed through (to 20:1、22:1 and/or 24:1) or by adding functional group oleic acid is further modified.Then these aliphatic acid, with palmitic acid With the saturated fatty acid such as stearic acid together, triglycerides is assembled into endoplasmic reticulum.

The desaturase of Plant acyl-ACP Δs -9 is solvable.It is located in plastid matrix, and using being esterified ACP's The aliphatic acid newly synthesized, mainly stearoyl-ACP, are used as substrate.This is compareed with the formation of other desaturases of Δ -9：Other Δs- 9 desaturases are located in endoplasmic reticulum (ER or microsome), using Co-A aliphatic acid is esterified as substrate, and make palm Two kinds of equal saturations of saturated fatty acid such as acid and stearic acid.United States Patent (USP) 5,723,595 and 6,706,950 is related to plant desaturation Enzyme.

The delta 8 desaturase genes of yeast Δ -9 are separated from saccharomyces cerevisiae, cloned and are sequenced.Stukey et al. (1989)J.Biol.Chem.264:16537-44；Stukey et al.(1990)J.Biol.Chem.265:20144-9.Should Yeast genes have been introduced into Leaf Explant of Nicotiana Tabacum L, and (Polashcok et al. (1991) FASEB is J.5:A1157；Polashok et al.(1992)Plant Physiol.100:In 894-901), and seem to express in the tissue.In addition, in tomato table The yeast genes are reached.Referring to Wang et al. (1996) J.Agric.Food Chem.44:3399-402；With Wang et al.(2001)Phytochemistry 58:227-32.Although using this desaturase of primary yeast Δ -9 in tobacco and tomato Gene all reports some increases of some unrighted acids and some reductions of some saturated fatty acids, but tobacco and kind Eggplant is apparently not oil crops.This yeast genes has also been introduced into colea.United States Patent (USP) 5,777,201.

Another different desaturase of fungi acyl-CoA Δ -9, from aspergillus nidulans, is introduced into Kano In drawing, the saturated fatty acid level of reduction is realized in seed oil.The A1 of patent application publication US 2008/0260933. The desaturase of aspergillus nidulans acyl-CoA Δ -9 is to the consumption (61-90%) of stearate than to richer palmitic acid aliphatic acid Consumption (36-49%) it is more.

It is open

Disclosed herein is the desaturase of novel fungitype Δ -9；Include the nucleotides sequence of at least one such desaturase of coding The nucleic acid of row；Include a kind of any of the above described plant, vegetable material (such as seed), plant part and plant commodity.Some realities The example for applying the aspect of scheme is from seat shell between grey big angle (Magnaporthe grisea), clever withered ball cavity bacteria The desaturase of fungi Δ -9 of (Leptosphaeria nodorum) and heliothis zea (Helicoverpa zea) separation.One A little examples include the desaturase of Δ -9 to palmitic acid or stearic acid with the natural of substrate preference and synthesis.

The nucleic acid molecules of the separation of some embodiments including the desaturase of coded delta -9, its include with selected from SEQ ID NO:1、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32 and SEQ ID NO:33 sequence at least 80% identical amino acid sequence.In instantiation, Nucleic acid molecules include with selected from SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24 and SEQ ID NO:25 at least 60% identical sequences. These embodiments and further embodiment can include the desaturase polypeptide of Δ -9 of separation, its include with selected from SEQ ID NO:1、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32 and SEQ ID NO:33 sequence at least 80% identical amino acid sequence.

Also disclose the method that at least one of above-mentioned nucleic acid and/or polypeptide are expressed in plant cell.It is specific real Apply the activity that scheme utilizes the desaturase of Δ -9 so that in the plant comprising the plant cell, vegetable material (such as seed) And/or plant part, and/or the percentage composition by the saturated fatty acid in a kind of plant commercial product of any of the above described production It can reduce.In certain embodiments, the ω -7 in the plant, vegetable material, plant part and/or plant commercial product Aliphatic acid can increase simultaneously.Other embodiments further reduce saturated fat in seed oil using seed-specific expression The level of fat acid.

Some embodiments include reducing plant, vegetable material, the saturated fat of plant part and/or plant commercial product The method of the amount of acid, methods described includes being made with the nucleic acid molecules conversion plant cell present invention of the desaturase polypeptide of coded delta -9 The amount for obtaining the saturated fatty acid in the cell is reduced.Some embodiments include the method for being used to produce genetically engineered plant, The plant includes the saturated fatty acid of decrement compared with the wild-type plant of same species in the plant.Such method The nucleic acid molecules with the one or more desaturase polypeptides of Δ -9 of coding or the desaturase polypeptide of Δ -9 of the present invention can be included One or more copies conversion vegetable material (or plant cell), and cultivate conversion vegetable material (or plant cell) obtain Plant.In instantiation, arabidopsis can be come from the nucleic acid molecules conversion of the desaturase polypeptide of Δ -9 of the coding present invention The plant cell and/or vegetable material of species.In other instantiations, two of the delta 8 desaturase genes of Δ -9 can be converted Or more a copy, wherein each delta 8 desaturase genes of Δ -9 are controlled by a different promoter.In other instantiations, Described two or more promoters are seed specific promoters.

From the detailed description of the several embodiments carried out below with reference to accompanying drawing, above and other feature will become more clear It is clear.

Brief description of the drawings

Fig. 1 shows pDAB7305 plasmid map.

Fig. 2 is shown and NEXERA 710^TMCanola check plant and by 218-11.30HL transgenosis canola plant groups Into positive control plant compare, the T of separation₁Large quantities of T of canola plant₂TSFA (%) distribution in seed.

Fig. 3 is shown and negative control NEXERA 710^TMCanola plant is compared, the transgenic event from three selections T₂TSFA distribution in seed populations.Dim spot represents seed progeny's (concealed wire) that TSFA is less than 3.5%.As illustrated, TSFA Plant less than 3.5% produces different amounts of yield, and with the pat transgenosis of 2 to 10 copies, these transgenosis are included With in AnD9DS transgenosis identical T chain intergrants.

Fig. 4 shows TSFA distributions and saturated fatty acid percentage (wild type control plants in the single seed of Canola It is excluded, so that the figure shows the TSFA values of transgenosis Canola event).

Sequence table

Represent to arrange in appended sequence table using the standard letter abbreviation of the nucleotide base defined in 37C.F.R. § 1.822 The nucleotide sequence gone out.Only show a chain of each nucleotide sequence, but complementary strand is understood to refer to shown by any Chain includes.In the sequence table enclosed：

SEQ ID NO:The desaturase of 1 display aspergillus nidulans acyl-CoA Δ -9 (being referred to as AnD9DS in some places) egg The amino acid sequence of white matter.

SEQ ID NO:2 show that the delta 8 desaturase genes of aspergillus nidulans acyl-CoA Δ -9 (are referred to as in some places AnD9DS the nucleotide sequence of v3).

SEQ ID NO:The nucleotide sequence of 3 display pDAB7305 the first plant transcription unit (PTU).

SEQ ID NO:4 display pDAB7305 the 2nd PTU nucleotide sequence.

SEQ ID NO:5 display pDAB7305 the 3rd PTU nucleotide sequence.

SEQ ID NO:6-11 shows the sequence of the primer and probe come in handy in some embodiments.

SEQ ID NO:12 be the delta 8 desaturase genes of seat shell acyl-CoA Δ -9 between the grey big angle expanded by PCR The exemplary fragment of (being referred to as MgD9DS in some places).

SEQ ID NO:13 be exemplary intronless MgD9DS clones.

SEQ ID NO:14 show the first clever withered desaturase of ball cavity bacteria acyl-CoA Δ -9 of coding (at some Place be referred to as LnD9DS-1) Exemplary nucleic acid sequences.

SEQ ID NO:15 show second of clever withered desaturase of ball cavity bacteria acyl-CoA Δ -9 of coding (at some Place be referred to as LnD9DS-2) Exemplary nucleic acid sequences.

SEQ ID NO:16 show the exemplary natural delta 8 desaturase genes of Δ -9 (mark of the seat shell between grey big angle For MgD9DS v1) code area.

SEQ ID NO:17 show that the exemplary natural delta 8 desaturase genes of Δ -9 from heliothis zea (are labeled as HzD9DS v1) code area.

SEQ ID NO:18 show the exemplary natural desaturase of Δ -9 (LnD9DS- from clever withered ball cavity bacteria 2v1) the code area of gene.

SEQ ID NO:19 show the exemplary card Nola optimization of the seat shell (MgD9DS v2) between grey big angle The sequence of the delta 8 desaturase genes of Δ -9.

SEQ ID NO:20 show the delta 8 desaturase genes of Δ -9 of the exemplary card Nola optimization from heliothis zea The sequence of (HzD9DS v2).

SEQ ID NO:21 show the desaturase base of Δ -9 of the exemplary card Nola optimization from clever withered ball cavity bacteria Because of the sequence of (LnD9DS-2v2).

SEQ ID NO:22 show the desaturation of Δ -9 of the another exemplary Canola optimization from clever withered ball cavity bacteria The sequence of enzyme gene (LnD9DS-2v3).

SEQ ID NO:23 show the desaturation of Δ -9 of the Canola optimization of the another exemplary from heliothis zea The sequence of enzyme gene (HzD9DS v3).

SEQ ID NO:24 show a kind of Exemplary nucleic acid sequences for encoding the desaturase of aspergillus nidulans Δ -9 (at some Place is referred to as AnD9DS v2).

SEQ ID NO:25 show the Exemplary nucleic acid sequences of second of desaturase of coding aspergillus nidulans Δ -9 (one A little places are referred to as AnD9DS v3).

SEQ ID NO:26 show the exemplary natural desaturase of Δ -9 of the seat shell (MgD9DS) between grey big angle Amino acid sequence.

SEQ ID NO:27 show the ammonia of the exemplary natural desaturase of Δ -9 (HzD9DS) from heliothis zea Base acid sequence.

SEQ ID NO:28 show the exemplary natural desaturase of Δ -9 (LnD9DS-2) from clever withered ball cavity bacteria Amino acid sequence.

SEQ ID NO:29 show by SEQ ID NO:The amino acid sequence of nucleic acid coding illustrated in 24-25 (AnD9DS) Row.

SEQ ID NO:The amino acid sequence of 30 display another exemplary AnD9DS desaturases.

SEQ ID NO:The amino acid of 31 exemplary natural Δ -9 desaturases (ScOLE1) of the display from saccharomyces cerevisiae Sequence.

SEQ ID NO:32 show the residue (1-68) of N- ends 68 of exemplary AnD9DS desaturases.

SEQ ID NO:33 show 175 residues (281-455) of C-terminal of exemplary AnD9DS desaturases.

Embodiment (S)

I. the general introduction of several embodiments

The desaturase of fungi acyl-CoA Δ -9 from aspergillus nidulans was introduced rapeseed by us in the past, so as to plant The saturated fatty acid level of reduction is realized in seed oil.Patent application publication US 2008/0260933A1.Aspergillus nidulans Δ -9 The stearic acid that desaturase is provided exhausts the exhaustion (36- of (61-90%) the palmitic acid aliphatic acid more more rich than in seed oil 49%) it is more.It has been found that can be by the saturated fat in Canola there is provided the desaturase of aspergillus nidulans Δ -9 of multiple copies Sour water is flat to be reduced to less than 3.5%.

Disclosed herein is the nucleic acid molecules of the desaturase polypeptide of coded delta -9, its include with selected from SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24 and SEQ ID NO:25 sequence at least 60% is identical Nucleotide sequence.In some embodiments, nucleic acid molecules can also be more comprising the desaturase of Δ -9 is operably connected to The gene regulatory elements of peptide-coding sequence.In specific embodiments, gene regulatory elements can be phaseolin promoter, dish Legumin 5' non-translational regions, Phaseolin 3' non-translational regions (" UTR "), Agrobacterium tumefaciens ORF1 3' non-translational regions, cassava leaves Vein mosaic virus promoter, tobacco RB7 matrix attachment regions, T chains border sequence, LfKCS3 promoters and the promoters of FAE 1.

In some embodiments, there may be several copies of the nucleic acid molecules of the desaturase polypeptide of coded delta -9, and And each copy is likely to be under the regulation control of a different set of regulating element.More specifically, gene regulatory elements can To be phaseolin promoter and Phaseolin 5'UTR, and Lesquerella fenderi LfKCS3 promoters, so as to have two The AND9DS of individual copy is present, and a copy is controlled by phaseolin promoter and 5'UTR, and second copy is started by LfKCS3 Son control.In other embodiments, the nucleic acid of the desaturase of coded delta -9 polypeptide (or the multiple desaturase of Δ -9 polypeptides) Several copies can be under the control of other regulating elements, including the desaturase promoter of saccharomyces cerevisiae Δ -9, the desaturation of Δ -9 Enzyme 3'UTR/ terminators, ole1 gene promoters, bean phaseolin 3' non-translational regions, bean phaseolin matrix attachment regions, Agrobacterium tumefaciens mannopine synthase promoter, Agrobacterium tumefaciens ORF23 3' non-translational regions, cassava vein mosaic virus Promoter, Agrobacterium tumefaciens ORF1 3' non-translational regions, tobacco RB7 matrix attachment regions, overdrive (Overdrive), T- chains side Boundary's sequence, LfKCS3 promoters, the promoters of FAE 1, Myc labels and Hemagluttinin tags.

Also disclose comprising with selected from SEQ ID NO:The Δ -9 of 1 sequence at least 80% identical amino acid sequence goes to satisfy With enzyme polypeptide and the nucleic acid molecules of this desaturase of Δ -9 polypeptide of coding, such as SEQ ID NO:2.

In some embodiments, nucleic acid molecules and the desaturase of Δ -9 polypeptide can vegetable material, cell, tissue or Expressed in whole plant, with reduce vegetable material, cell, tissue or entirely the saturated fatty acid in plant amount (relative to The amount observed in the wild-type plant of same species).The present invention alternate embodiment include reduce vegetable material, cell, The method of the amount of tissue or the saturated fatty acid in whole plant.Such method can be included with least one above-mentioned nucleic acid Molecule conversion vegetable material, cell, tissue or whole plant so that the saturation in vegetable material, cell, tissue or whole plant The amount of aliphatic acid is reduced.Specific embodiment includes being used to preferentially reduce vegetable material, cell, the palm fibre in tissue or whole plant Palmitic acid is sour and/or stearic method.

Method disclosed herein can be for example in plant or plant from plant (such as Arabidopsis plant or Canola) Carried out in thing material.One specific embodiment be related to for produce or secondary gene engineered plant method, the plant with The wild-type plant of same species compares the saturated fatty acid that decrement is included in plant, and methods described is included with least one Above-mentioned nucleic acid molecules conversion plant cell or material；And cultivate inverted vegetable material to obtain plant.Also disclose logical Cross plant, vegetable material, plant cell and seed that any of above method is obtained.

II. abridge

x:yΔ^zContain x carbon atom and the aliphatic acid positioned at the y double bond of z from c-terminus number

ACP acyl carrier proteins

CoA coacetylases

FA aliphatic acid

FAS lipase synthase

FAME fatty acid methyl esters

KASII β -one acyl-ACP synthases II

MUFA monounsaturated fatty acids

PUFA polyunsaturated fatty acids

WT wild types

III. term

Aliphatic acid：As used in this article, term " fatty acid " " refers to the long-chain of the different chain length for example from about C12 to C22 Aliphatic acid (alkanoic acid), although both acid of longer and shorter chain length is known.The structure of aliphatic acid is with symbol x:yΔ^z Represent, wherein " x " is the sum of carbon (C) atom in special fatty acid, and " y " be in carbochain such as since the sour c-terminus number Number of double bonds in " z " position risen.

Metabolic pathway：Term " metabolic pathway " refer to intracellular presence, by enzymatic to realize metabolite conversion or another The series of chemical that one metabolic pathway starts.Metabolic pathway can involve several perhaps multi-steps, and can be with difference Metabolic pathway competes specific reaction substrate.Similarly, a kind of product of metabolic pathway can be the substrate of another metabolic pathway.

Metabolic engineering：For purposes of the present invention, " metabolic engineering " refers to the one or more metabolic pathways changed in cell, Initial substrate is progressively modified to desired essence so that being realized in the overall plan of the total metabolism approach run in cell The rationality strategy design of the product of true chemical constitution.

Desaturase：As used in this article, term " desaturase " refers to can go to satisfy in one or more aliphatic acid (that is, introduce double bond) is to generate the polypeptide of aliphatic acid interested or precursor.Plant solubility fatty acid desaturase enzyme can So that double bond regiospecificity is introduced into the acyl-acp substrate of saturation.Acyl-CoA desaturase draws double bond regiospecificity In the fatty acyl group-CoA substrates for entering saturation.The reaction involves to be coordinated by constructing the four-helix bundle of core by forming desaturase Two electron reduction Xing Ertie centers activate molecular oxygens.It is particularly interesting that acyl group-CoA delta-9 in some embodiments Desaturase.

delta-9-18:0¹- ACP desaturases are that all plants maintain membrane fluidity needs.Although this enzyme mainly makes Stearoyl-ACP desaturations, but it is also active in small degree in the case of palmityl-ACP.

Progeny plants：For the purpose of the present invention, " progeny plants " refer to any plant that can be obtained by plant breeding method Thing, or the vegetable material obtained from it.Plant breeding method is it is well known in the art that and including natural breeding, manually educate Selection and use, transgenics and the business breeding plant, involved DNA molecular MARKER ASSAYS.

Vegetable material：As used in this article, term " vegetable material " refers to any cell or tissue obtained from plant.

Nucleic acid molecules：The polymerized form of nucleotides, it can include both RNA sense and antisense chains, cDNA, genome The polymer of the synthesized form and mixing of DNA and above-mentioned substance.Nucleotides refers to ribonucleotide, deoxynucleotide or any sort Nucleotides through modified forms.As used in this article, " nucleic acid molecules " and " nucleic acid " and " polynucleotides " are synonymous.The art Language includes DNA single-stranded and double chain form.Nucleic acid molecules can be included by naturally occurring and/or non-naturally occurring nucleosides Naturally occurring and through modification the nucleotides that acid connection links together it is any or both.

Nucleic acid molecules can be by chemistry or biochemical modification, or can contain non-natural or derivatization nucleosides Soda acid base, as those of ordinary skill in the art are comprehensible.Such modification includes such as label, methylates, uses analog Replace between one or more naturally occurring nucleotides, nucleotides modify, such as it is uncharged connection (for example, methyl-phosphonate, Phosphotriester, phosphoramidate, carbamate, etc.), electrically charged connection is (for example, thiophosphate, phosphordithiic acid Ester, etc.), pendency module (moiety) (for example, peptide), inserting agent (for example, acridine, psoralen, etc.), chelating agent, Alkylating agent (alkylator) and the connection (for example, different head nucleic acid of alpha, etc.) through modification.Term " nucleic acid molecules " is also wrapped Include any topology conformation, including single-stranded, double-strand, partial duplex, triplet, hair clip, annular and padlocked conformation.

It is operatively connected：When the first nucleotide sequence is in the functional relationship with second nucleotide sequence, the first nucleic acid Sequence is operatively connected with second nucleotide sequence.If for example, promoter influence coded sequence transcription or expression, promoter with Coded sequence is operatively connected.When recombinating generation, the nucleotide sequence being operatively connected is usually continuous, and must connected During two protein coding regions, in same reading frame.However, nucleic acid needs not be continuous to be operatively connected.

Regulating element：As used in this article, term " regulating element " refers to the nucleic acid molecules with gene modulatory activity；I.e. The nucleic acid molecules of the ability of transcription or the translation of the transcribed nucleic acid molecules being operatively connected with influence.Regulating element is such as opened Mover, lead, introne and transcription termination region are the non-coding nucleic acid molecules with gene modulatory activity, and it is in living cells Indispensable effect is played in general gene expression.Therefore, the regulating element of the separation of function can be used in plant Via molecular engineering technology modified plant phenotype." regulating element " means to decide whether expression specific gene, when expresses spy Determine gene and a series of nucleotides with which kind of horizontal expression specific gene.Adjust DNA sequence dna and regulatory protein or other albumen Matter specificity interaction.

As used in this article, term " gene modulatory activity " refers to influence the transcription for the nucleic acid molecules being operatively connected Or the nucleic acid molecules of translation.The nucleic acid molecules of separation with gene modulatory activity can provide the nucleic acid molecules being operatively connected Time or space expression or regulating and expressing level and speed.The nucleic acid molecules of separation with gene modulatory activity can include Promoter, introne, lead or 3 ' transcription termination regions.

Promoter：As used in this article, term " promoter ", which refers to, involves rna plymerase ii or other oroteins such as turn The identification of the record factor (trans-acting protein factor of regulation transcription) and the core for combining the genetic transcription being operatively connected to start Acid molecule.Promoter can be contained with itself causes the subcomponent such as cis element or enhancing for the genetic transcription being operatively connected Subdomain." plant promoter " is functional natural or non-natural promoter in plant cell.Plant can be used to open Mover is as 5 ' regulating elements to regulate and control one or more gene expressions being operatively connected.Plant promoter can be with that time Between, space or growth expression pattern limit.Nucleic acid molecules described herein can include the nucleic acid sequence containing promoter Row.

Sequence identity：As used in the background of two kinds of nucleic acid or peptide sequence, " sequence is same for term herein Property " or " homogeneity " can refer in two kinds of sequences it is defined compare compared in window in order to realize maximum correspondence when identical Residue.

Similitude between two kinds of nucleotide sequences or between two kinds of amino acid sequences is according to the sequence identity water shared between sequence It is flat to represent.Sequence identity is generally represented according to percentage identity；Percentage is higher, and two kinds of sequences are more similar.For comparing The method of comparative sequences is described in detail below.

When referring to that protein uses Percentage of sequence identity, accreditation, the resi-dues differed are often differed Conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, wherein amino acid residue, which are used, has similar chemical characteristic (for example, electric charge, hydrophobicity or three-dimensional effect) The substitution of other amino acid residues, and therefore do not change the functional characteristic of molecule.

Therefore, sequence differ conservative replacement when, Percent sequence identity can be adjusted up with correct differ it is residual The conservative property of Ji Weidianchu substitutions.The sequence for differing such conservative replacement is said to be with " sequence similarity " or " similar Property ".Technology for carrying out this regulation is known to a person of ordinary skill in the art.Generally, such technology involves takes conservative Generation scoring is part, rather than completely matching, thus increases Percent sequence identity.For example, to identical amino Acid gives 0-1 score, and in the case of giving 0 score to non-conservative substitutions, 0-1 score is given to conservative replacement.Can be with The score of conservative replacement is calculated, such as performed in program PC/GENE (Intelligenetics, Mountain View, CA) 's.

As used herein, term " Percentage of sequence identity " can refer to compares two optimal ratios in comparison window The numerical value determined during to sequence, wherein for the optimal comparison of two sequences, comparing the part of sequence and canonical sequence (its in window Not comprising addition or missing) compare to include and add or missing (that is, breach).There is phase same core by determining in two sequences The number of the position of thuja acid or amino acid residue by the number of matched position divided by compares window middle position to produce matched position number The sum put, and result is multiplied by 100 to produce Percentage of sequence identity to calculate percentage.

Similar position in amino acid sequence：Nucleic acid and amino acid can be compared by the method described in aforementioned paragraphs Sequence.When comparing, if each position is identical in consensus sequence, the position in position and aligned sequences in a sequence Put in " similar position ".

Method for comparing comparative sequences is as known in the art.Various programs and alignment algorithm are recorded in：Smith And Waterman, Adv.Appl.math.2:482,1981；Needleman and Wunsch, J.Mol.Biol.48:443,1970； Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444,1988；Higgins and Sharp, Gene 73: 237-44,1988；Higgins and Sharp, CABIOS 5:151-3,1989；Corpet etc., Nucleic Acids Research 16:10881-10890,1988；Huang, etc. Computer Applications in the Biosciences 8:155-65,1992；Pearson etc., Methods in Molecular Biology 24:307-31, 1994；Tatiana etc., FEMS Microbiol.Lett., 174:247-50,1990.Altschul etc., J.Mol.Biol.215:403-10,1990 (on being discussed in detail that sequence alignment method and homology are calculated).

American National Biotechnology Information center (National Center for Biotechnology Information, NCBI) basic Local Alignment Search Tool (BLAST) can obtain on the internet (in Blast.ncbi.nlm.nih.gov/Blast.cgi), itself and sequence analysis programs, such as blastp and blastn, which are combined, to be made With.How using description as described in this program determination sequence identity on the internet via NCBI in blast.ncbi.nlm.nih.gov/Blast.cgiCMD=Web＆PAGE_TYPE=BlastDocs can be obtained.

For comparing amino acid sequence, " 2 kinds of sequence (Blast of Blast of blast program are used using default parameter 2sequence) " function (bl2seq).Special parameter can be adjusted in the judgement of those skilled in the art, for example to provide mistake With point penalty or matching bonusing.

Inverted：As used in this article, term " inverted ", which refers to, has received foreign nucleic acid molecules, such as builds Cell, tissue, organ or organism that body is imported.The nucleic acid molecules of importing can be integrated into and receive cell, tissue, organ Or in the genomic DNA of organism so that the polynucleotide molecule of importing obtains follow-up offspring and inherited." transgenosis " or " through turning Change " cell or organism are also included in the cell or the offspring of organism and comfortable such as hybridization using such genetically modified plants The offspring of the phenotype from the change that there are foreign nucleic acid molecules is generated and shown as the procedure of breeding of parent.

IV. the metabolic engineering method of saturated fatty acid in host cell, tissue or organism is reduced

A. summarize

One embodiment of the invention, which is included in vegetable seeds to introduce, has specific acyl group-CoA preferences (for example, right In palmitic acid or stearic acid) delta-9 desaturases.Specific acyl group-CoA the preferences of delta-9 desaturases are realized to certain The targeting of a little specific saturated fatty acid set (for example, palmitate, for changing into single unsaturated product).Select acyl group-CoA Delta-9 desaturases reduce the saturated fatty acid content in plant, because they are not generally with any perceptible degree Generated in botanical system.

B. polypeptide

Polypeptide according to some embodiments of the present invention includes following amino acid sequence, and it is in the sequence with being selected from the group Row show increased percentage identity when comparing：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.These Can be included with the specific amino acid sequence in other embodiments has for example, at least about 70%, about 75% with foregoing sequences, About 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, The sequence of 95%96%, 97%, 98%, 99% or 100% homogeneity.In many embodiments, compared with foregoing sequences When have foregoing sequences homogeneity amino acid sequence coding have delta-9-18:The peptide of 0-ACP desaturase enzymatic activities, Or the part of such peptide.

C. nucleic acid

Some embodiments include encoding the nucleic acid molecules of polypeptide as described above.For example, in some embodiments Nucleotide sequence is the increased percentage identity of display in the sequence alignment with being selected from the group：SEQ ID NO:2、SEQ ID NO: 3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO: 15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24 and SEQ ID NO:25.These and other embodiment The sequence that interior specific nucleic acid sequence can include with being selected from the group has 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%, The sequence of 94%, 95%96%, 97%, 98%, 99% or 100% homogeneity：SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:8,SEQ ID NO:9,SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:44,SEQ ID NO:45,SEQ ID NO:48 and SEQ ID NO:49.Ability Domain those of ordinary skill understands, for example can be substituted in not by introducing admissible nucleotides according to Codon degeneracy Modified nucleic acid molecule in the case of the amino acid sequence of material alterations coded polypeptide.

In some embodiments, nucleic acid molecules of the invention include gene regulatory elements (for example, promoter).Promoter It can be selected based on the cell type that can receive vector construct insertion.The startup of function in bacterium, yeast and plant Son is as known in the art.Promoter can also adjust feature based on it and be selected.The example of this category feature includes enhancing Transcriptional activity, inducibility, tissue specificity and stage of development specificity.In plant, it has been described that virus is synthesized Induction type, composition activity, time adjustment and the Space adjustment promoter in source.See such as Poszkowski (1989) EMBO J.3:2719；Odell etc. (1985) Nature 313:810；And (1989) Science 244 such as Chau:174-81).

Useful inducible promoter includes inducing for example by application safener (substituted benzenesulfonamide herbicide) Salicylic acid or the promoter of polyacrylic acid induction, heat-shock promoters, from the transcribed nucleic acid molecules of spinach nitrate reductase Nitrate inducible promoter, hormone inducible promoter and the small subunit with RuBP carboxylases and LHCP family derived from sequence The light-inducible promoter that race combines.

Useful tissue specificity, the example of growth adjustment promoter include beta-conglycinin (conglycinin) 7S α promoters and seed specific promoters.Available for the plant function that precedence table reaches in seed plastid Property promoter include those from involving protein that fatty acid biological in oily seed synthesizes and from vegetative storage protein. The example of such promoter includes coming from transcribed sequence of nucleic acid molecules such as Phaseolin, rapeseed protein, the molten egg of corn alcohol In vain, 5 ' regulatory regions of soybean trypsin inhibitor, ACP, stearyl-ACP desaturase and oleosin.It is another to illustrate The tissue-specific promoter of property is the specific agglutinin promoter of seed tissue.

More specifically, promoter can include Kidney bean (Phaseolus vulgaris) phaseolin promoter (individually Or be combined with bean phaseolin 3' non-translational regions and bean phaseolin 3' Matrix-attachment regions), Lesquerella Fendleri KCS3 promoters or the graceful mannopine synthase promoter of Agrobacterium tumefaciens.

Other useful promoters include nopaline synthase, mannopine synthase and octopine synthase promoter (its Carried on the tl plasmid of Agrobacterium tumefaciens (Agrobacterium tumefaciens))；Cauliflower mosaic virus (CaMV) 19S and 35S promoter；Enhanced CaMV 35S promoters；Radix scrophulariae mosaic virus 35 S promoter；From ribulose-1,5-bisphosphate, The light-inducible promoter of 5- diphosphonic acid carboxylases (ssRUBISCO) small subunit；EIF-4A promoters (Mandel from tobacco Deng (1995) Plant Mol.Biol.29:995-1004)；Corn sucrose synthetase；Corn alcohol dehydrogenase I；Corn light is harvested Compound (light harvesting compolex)；Maize Heat Shock Protein；Chitinase promoter from arabidopsis； LTP (lipid transfer protein) promoter；Petunia chalcone isomerase；Beans are rich in the albumen 1 of glycine；Potato patatin； Ubiquitin promoter；And actin promoter.Preferably, useful promoter be seed selective, tissue selectivity or Induction type.Seed specific regulation is discussed in such as EP 0 255 378.

In order to obtain higher allogeneic gene expression, it may be preferable that reengineering modifying gene so that it is in expression More effectively expressed in host cell (for example, plant cell, for example, Canola, rice, tobacco, corn, cotton and soybean).Cause This, optional additional step (that is, is providing a kind of or many in the gene of the coding delta-9 desaturases of design plant expression Plant outside gene regulatory elements) it is reengineeringization transformation heterologous gene protein coding region for optimum expression.It is specific to implement Scheme includes the gene of redesign, and it passes through optimization to improve in transgenosis canola plant cell or arabidopsis thaliana cell In, rather than the table in the canola plant cell or arabidopsis thaliana cell converted with naturally occurring heterologous gene sequence Up to level (that is, generating greater protein matter).

(that is, some amino acid are by more than a kind of password for the plasticity provided due to the redundancy by genetic code/degeneracy Cuckoo is determined), the genome evolution in different organism or organism classes already leads to the difference selection of synonym.This is " close Numeral is had a preference for " reflected in the average base composition of protein coding region.For example, with containing quite relatively low G+C contents The organism of genome is using more the 3rd codons with A or T in synonym, and those have higher G+ The organism of C content is using more in the 3rd codon with G or C.Additionally it is believed that " secondary " codon in mRNA In the presence of the absolute translation rate of the mRNA can be reduced, especially in the relative of electrically charged tRNA corresponding with secondary codon When abundance is relatively low.The extension of this reasoning is to reduce translation rates for a variety of secondary codons at least by indivedual secondary codons It can be superposition.Therefore, in particular expression host the mRNA with of a relatively high secondary codon content can have it is corresponding compared with Low translation rate.This speed can be reflected by the corresponding low-level of encoding proteins matter.

In the optimization gene of engineered coding delta-9 desaturases (or other to be planted in Canola or arabidopsis Thing, such as rice, tobacco, corn, cotton or soybean) in express, if the codon-bias of expected host plant after measured, It is helpful.There are a variety of publicly available DNA sequence data storehouses, wherein the password on Plant Genome can be found The information of the protein coding region of son distribution or various plant genes.

Codon-bias is expressive host (for example, plant, such as Canola or arabidopsis) using to encode its protein Amino acid codon statistical distribution.Codon-bias can be with the codon of relatively all amino acid of single password Frequency of use calculate.Or, codon-bias can be used for encoding particular amino acid with single password, relative to the ammonia The frequency of all other codon (synonym) of base acid is calculated.

In the code area optimized for the plant design of expression of delta-9 delta 8 desaturase genes, it should be determined that plant is preferred Main (" first choice ") codon, and preferably codon second, third, the 4th selection etc. (there are multiple choices When).It is then possible to the DNA sequence dna of new coding delta-9 delta 8 desaturase genes amino acid sequences be designed, wherein new DNA sequences Row are to replace that to encode host preferable (the first preferable, second excellent with natural DNA sequence (coding desaturase) difference The property selected, the 3rd preferable or the 4th preferable, etc.) codon to be to provide the amino acid in amino acid sequence at each position. Then, to new sequence analysis restriction enzyme sites, it can be created by modifying.By by these codons with next excellent The property selected codon is replaced further to modify the presumption restriction site of identification to remove restriction site.It can be influenceed in sequence Other sites of transcription or the translation of heterologous sequence are extrons:Introne engagement (5 ' or 3 '), how poly- A addition signal and/ Or RNA polymerase termination signal.Can further analytical sequence, and modified to reduce the frequency of TA or CG doublets Rate.External in these duplexs, the sequence district's groups with identical more than about 6 G or C nucleotide can also negatively affect sequence Transcription or translation.Hence it is advantageous to, by next preferable that codon of the first or second selection, etc. is used to selection Codon is replaced to modify these district's groups.

Method as described above enables those skilled in the art to modify external gene for specified plant, makes Gene is obtained most preferably to express in plant.Methods described is further illustrated in PCT application WO 97/13402.In this way, can make The optimized synthetic gene that is functionally equal with desaturase/gene with some embodiments converts host, including Plant.More guidances on generating synthetic gene may refer to such as United States Patent (USP) 5,380,831.

Once designing the DNA sequence dna optimized through plant on paper or in computer chip, it can close in the lab Into actual DNA molecular to correspond precisely to the sequence of design in sequence.Can by the cloned dna molecule of such synthesis, and Otherwise operate, completely just as they are derived from nature or natural origin.

D. it is used for the method for genetic transformation plant material

Some embodiments are related to the method for inverted cell of the generation comprising one or more nucleic acid molecules, the core Acid molecule includes the sequence at least 60% identical nucleotide sequence with being selected from the group：SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24 and SEQ ID NO:25.Such nucleic acid molecules can also include for example non- Encode regulating element, such as promoter.Can also be by other sequences and non-coding regulatory element and transcribed sequence of nucleic acid molecules Import together in cell.These other sequences can include 3 ' transcription terminators, 3 ' polyadenylation signals, other untranslateds Sequence, transhipment or targeting sequence, selection marker, enhancer and operator.

Usually, method for transformation comprises the following steps：Suitable host cell is selected, it is thin to convert host with recombinant vector Born of the same parents, and obtain inverted host cell.For by DNA import cell in technology be well known to a person skilled in the art.One As, these methods are segmented into 5 classes：(1) chemical method (Graham and Van der Eb (1973) Virology 54 (2): 536-9；Zatloukal etc. (1992) Ann.N.Y.Acad.Sci.660:136-53)；(2) physical method, such as microinjection (Capechi(1980)Cell 22(2):479-88), electroporation (Wong and Neumann (1982) Biochim.Biophys.Res.Commun.107(2):584-7；Fromm etc. (1985) Proc.Natl.Acad.Sci.USA 82(17):5824-8；United States Patent (USP) 5,384,253) and Particle Acceleration (Johnston and Tang (1994) Methods Cell Biol.43(A):353-65；Fynan etc. (1993) Proc.Natl.Acad.Sci.USA 90 (24):11478-82；(3) it is viral Carrier (Clapp (1993) Clin.Perinatol.20 (1):155-68；Lu etc. (1993) J.Exp.Med.178 (6):2089- 96；Eglitis and Anderson (1988) Biotechniques 6 (7):608-14)；(4) receptor-mediated mechanism (Curiel Deng (1992) Hum.Gen.Ther.3 (2):147-54；Wagner etc. (1992) Proc.Natl.Acad.Sci.USA89 (13): 6099-103)；(5) mechanism of bacteria mediated, such as uses agrobacterium.Or, can be by the reproduction of direct injection plant Nucleic acid is introduced directly into pollen by organ.Zhou etc. (1983) Methods in Enzymology 101:433；Hess (1987)Intern.Rev.Cytol.107:367；Luo etc. (1988) Plant Mol.Biol.Reporter 6:165；Pena Deng (1987) Nature 325:274.Other method for transformation include such as protoplast transformation, such as United States Patent (USP) 5, in 508,184 Illustrate.Nucleic acid molecules can also be injected into immature embryo.Neuhaus etc. (1987) Theor.Appl.Genet.75: 30。

It is most-often used be used to convert plant cell method be：Agrobacterium-mediated DNA transfer methods (Fraley etc. (1983)Proc.Natl.Acad.Sci.USA 80:4803) (such as United States Patent (USP) 5,824,877；United States Patent (USP) 5,591,616； United States Patent (USP) 5,981,840；With illustrate in United States Patent (USP) 6,384,301) and Biolistic or the method for microparticle bombardment mediation (that is, particle gun) (is such as recorded in United States Patent (USP) 5,550,318；United States Patent (USP) 5,538,880；United States Patent (USP) 6,160,208； United States Patent (USP) 6,399,861；With United States Patent (USP) 6,403,865).Generally, Nuclear transformation is desired, but is expecting specificity turn Change plastid, in the case of such as chloroplaset or amyloplast, can to certain plants species such as arabidopsis, tobacco, potato and The delivering that Canola species are mediated using the particulate of desired nucleic acid molecules is come transforming plant plastides.

Agrobacterium-mediated conversion is realized via using the genetically engineered soil bacteria for belonging to Agrobacterium.It is several Agrobacterium species mediate transfer is referred to as the specific DNA of " T-DNA ", and it can will be any desired with genetically engineered transform as DNA parts are carried in many plant species.Marking the main matter of the pathogenesis process of T-DNA mediations is：Induce virulence Gene and process and shift TDNA.This process is the theme of many summaries.See such as 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) inTransgenic Plants, Kung and Wu compile, Academic Press, San Diego, CA, page 49 page-the 87；Binns and Howitz (1994) inBacterical Pathogenesis of Plants and Animals, Dang volumes, Berlin:Springer Verlag., the 119-38 pages； Hooykaas and Beijersbergen (1994) Ann.Rev.Phytopathol.32:157-79；Lessl and Lanka (1994) Cell77:321-4；And Zupan and Zambryski (1995) Annual Rev.Phytopathol.27:583-618.

In order to select inverted plant cell or score (regardless of method for transformation), the DNA imported in cell can So that containing function is to generate the gene of compound in reproducible plant tissue, the compound is assigned to plant tissue To the resistance of otherwise virose compound.Alternatively, the gene interested that screening or standards of grading thing are used include but It is not limited to β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase and antibiotic or herbicide tolerant Gene.The example of antibiotics resistance gene includes assigning to penicillin, kanamycins (and neomycin, G418, bleomycin)；First Aminopterin (and TMP)；Chloramphenicol；With the gene of the resistance of tetracycline.For example, glyphosate (glyphosate) resistance can To be assigned by herbicide resistance gene.Della-Cioppa etc. (1987) Bio/Technology 5:579-84.It can also perform Other selection devices, are included, but not limited to, e.g. to phosphinothricin (phosphinothricin), double third ammonia phosphorus and favorable selection Tolerance (Joersbro etc. (1998) Mol.Breed.4 of mechanism:111-7), and think in embodiment of the present invention In the range of.

It is then possible to allow by selection or Screening and Identification and support regeneration suitable culture medium in cultivate it is inverted Cell maturation into plant.

Presently disclosed method can be used together with any transformable plant cell or tissue.As used herein , transformable cell and tissue include but is not limited to further breed to produce those cell or tissues of plant.This Art personnel approve that many plant cells or tissue are transformable, wherein in insertion exogenous DNA and appropriate culture bar Plant cell or tissue can form the plant of differentiation after part.Be suitable for these purposes tissue can include but is not limited to not into Ripe embryo, pelta (scutellar) tissue, suspended cell culture, immature inflorescence, branch separate living tissue, section explant, more Injured tissue, Hypocotyl Tissues, cotyledon, root and leaf.

It is as known in the art from inverted plant protoplast or explant regeneration, development and culture plant. Weissbach and Weissbach (1988)Methods for Plant Molecular Biology, (eds.) Academic Press,Inc.,San Diego,CA；Horsch etc. (1985) Science 227:1229-31.This regeneration and growing method are led to Often comprise the following steps：The inverted cell of selection, and the usual stage of those cell culture to embryonic development is small to what is taken root The plant stage.Similarly, the embryo and seed of regeneration of transgenic.In this method, typically be there is into Selective agar medium in transformant In the case of cultivate, the cell of the Selective agar medium conversion chosen successfully and the regeneration for inducing plant plumule (shoot). Fraley etc. (1993) Proc.Natl.Acad.Sci.USA 80:4803.These young shoots were generally obtained in 2 to 4 months.This Afterwards, the transgenosis of gained young shoot of taking root is planted in suitable plant growth culture medium such as soil.It will can survive sudden and violent The cell of selective agent is exposed to, or is scored at positive cell in the screening test method and is trained in the culture medium for supporting plant regeneration Support.It is then possible to which young shoot is transferred in suitable root induction culture medium, the root induction culture medium contains selective agent and prevented The antibiotic of bacterial growth.Many young shoots can form root.Then, these are migrated to soil or other culture mediums to allow root Continue to develop.Usually, method as outlined above can be different according to the specified plant strain of use, and therefore, method Details in the judgement of those skilled in the art.

The genetically modified plants of regeneration can provide homozygous transgenic plants with self-pollination.Or, can be by regeneration certainly Pollen and non-transgenic plant that genetically modified plants obtain, it is preferable that the inbred line cross of the important species of agronomy.On the contrary, The pollen from non-transgenic plant can be used to carry out the genetically modified plants pollination to regeneration.

The nucleotide sequence of conversion can be passed to its offspring by genetically modified plants.Preferably, genetically modified plants are in conversion Nucleotide sequence in terms of be homozygosis, and after generative propagation and due to generative propagation by the sequence entail it is all thereafter Generation.Can the generation of free genetically modified plants seed culture offspring.It is then possible to by these other plant self-pollinations to generate Real plant breeding strain.

Can be to progenies gene expression from these plants, etc..Can be by several frequently seen method such as Western blot, Northern traces, immunoprecipitation and ELISA (enzyme-linked immunosorbent assay) detection gene expressions. The presence for the DNA that inverted plant analysis can be imported and the table assigned by the nucleic acid molecules and amino acid molecular of the present invention Up to level and/or fatty acid profile.Those skilled in the art will know that available for many methods for analyzing inverted plant.For example, Method for plant analysis includes but is not limited to Southern traces or Northern traces, the method for PCR-based, bioid Learn determination method, phenotype screening, field evaluations and immunodiagnosis determination method.

For specificity conversion dicotyledon method be well known to a person skilled in the art.To many crops, The including but not limited to member of Arabidopsis, cotton (cotton (Gossypium hirsutum)), soybean (soybean (Glycine Max)), the member of peanut (peanut (Arachis hypogaea)) and Btassica describe using these methods conversion and Plant regeneration.To cotton (United States Patent (USP) 5,004,863；United States Patent (USP) 5,159,135；United States Patent (USP) 5,518,908)；Soybean (United States Patent (USP) 5,569,834；United States Patent (USP) 5,416,011；McCabe etc. (1988) Biotechnology 6:923； Christou etc. (1988) Plant Physiol.87:671-4)；(United States Patent (USP) 5,463,174)；Peanut (Cheng etc. (1996)Plant Cell Rep.15:653-7；McKently etc. (1995) Plant Cell Rep.14:699-703)；Luxuriant wood Melon；With pea (Grant etc. (1995) Plant Cell Rep.15:254-8) deliver main by using Agrobacterium tumefaciens Conversion dicotyledon and the method for obtaining genetically modified plants.

Method for transforming monocots is also as known in the art.To many crops, including but do not limit In barley (barley (Hordeum vulgarae))；Maize (maize (Zea mays))；Oat (oat (Avena sativa))；Orchard grass (orchard grass (Dactylis glomerata))；(rice (Oryza sativa), including India and Japan become rice Kind)；Sorghum (sorghum (Sorghum bicolor))；Sugarcane (sugarcane (Saccharum sp))；Alta fascue (alta fascue (Festuca arundinacea))；Meadow grass (turfgrass) species are (for example, agrostis stolonifera (Agrostis Stolonifera), English grass (Poa pratensis), cone fringe Herba Stenotaphri helferi (Stenotaphrum secundatum))； Wheat (wheat (Triticum aestivum))；Described with clover (clover (Medicago sativa)) and use these methods Conversion and plant regeneration.It will be apparent to one skilled in the art that can use and change many method for transformation with The genetically modified plants stable to many target crop generations interested.

Any plant can be selected to be used in presently disclosed method.The preferred plant modified according to the present invention includes Such as, but not limited to containing seed plant, arabidopsis (Arabidopsis thaliana), Common Borage, (Common Borage belongs to (Borago) Species), Canola (Canola) (Btassica (Brassica) species), castor-oil plant (castor-oil plant (Ricinus communis)), cocoa Beans (cocoa (Theobroma cacao)), corn (maize), cotton (Gossypium (Gossypium) species), two section shepherd's purse category (Crambe) species, sepal distance flower spp (Cuphea) species, flax (flax species), small shore Chrysanthemum (Lesquerella) and whale Oily grass (Limnanthes) species, Linola, nasturtium (Nasturtium (Tropaeolum) species), Oenothera (Oenothera) species, olive (olive) (wooden slippers olive belongs to (Olea) species), palm (oil palm belongs to (Elaeis) species), peanut (Arachis (Arachis) species), rapeseed (rapeseed), safflower (safflower belongs to (Carthamus) species), soybean (soybean Belong to (Glycine) and wild soybean (Soja) species), sunflower (Helianthus (Helianthus) species), tobacco (Nicotiana (Nicotiana) species), Vernonia (Vernonia) species, wheat (Triticum (Triticum) species), barley (barley Belong to (Hordeum) species), rice (Oryza (Oryza) species), oat (Avena (Avena) species), sorghum (sorghum (Sorghum) species) and rye (Secale (Secale) species) or grass family (Gramineae) other members.

It will be apparent to one skilled in the art that can use and change many method for transformation with emerging to many senses The genetically modified plants of the target crop generation stabilization of interest.

E. transgenic seed

In some embodiments, transgenic seed can include delta-9 desaturase polypeptides, its include with The sequence at least 80% identical amino acid sequence of group：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.At this In a little and other embodiments, transgenic seed can include the sequence at least 60% identical nucleotide sequence with being selected from the group： SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24 and SEQ ID NO:25. In some embodiments, transgenic seed can show the saturated fatty acid of reduction (for example, palmitic acid aliphatic acid and/or hard Resin acid aliphatic acid) level.Genetically modified plants harvest seed can be educated with self energy, it is possible to offspring's generation for cultivating inverted plant Generation, including comprising at least one nucleotide sequence such as listed above, and optionally at least a kind of other gene or core interested The hybrid plant strain of acid con-struct.

Following examples are provided to illustrate some specific features and/or embodiment.These embodiments should not be construed as Specific features or embodiment described by limiting the invention to.

Embodiment

Embodiment 1：PDAB7305 construct designs

The desaturase of aspergillus nidulans Δ -9 (AnD9DS) enzyme is previously in U.S. Patent Application Serial Number 2008/0260933 It is open, it is SEQ ID NO herein:1.Synthesis includes polynucleotide sequence (the SEQ ID NO of AnD9DS v3 coded sequences: 2), and be introduced into Plasmid Constructs pDAB7305 be used for agrobacterium-mediated Plant Transformation (Fig. 1).The construct of gained Contain three plant transcription units (being also been described as expression casette, the two is used interchangeably).First plant transcription unit (PTU)(SEQ ID NO:3) by RB7 matrix attachment regions (RB7MAR；International patent application no WO9727207), Kidney bean Kidney bean egg White promoter (Pv Phas promoters；U.S. Patent number 5,504,200), AnD9DS coded sequences (the desaturase v3 of An Δs 9), Bean phaseolin 3' non-translational regions (Pv Phas 3'UTR；U.S. Patent number 5,504,200) and bean phaseolin 3' matrix Attachment region (Pv Phas 3'MAL；U.S. Patent number 5,504,200) constitute.Second PTU (SEQ ID NO:4) by Lesquerella fendleri KCS3 promoter (LfKCS3 promoters；U.S. Patent number 7,253,337), AnD9DS coding Sequence (the desaturase v3 of An Δs 9) and Agrobacterium tumefaciens ORF 233' non-translational regions (AtuORF23 3'UTR；U.S. Patent number 5,428,147) constitute.3rd PTU (SEQ ID NO:5) by Agrobacterium tumefaciens mannopine synthase promoter (AtuMas Promoter；Barker,R.F.,Idler,K.B.,Thompson,D.V.,Kemp,J.D.,(1983),a polynucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti Plasmid pTi15955, Plant Molecular Biology, 2 (6), 335-50), phosphinothricin acetyl transferase gene (PAT；Wohlleben etc., (1988) Gene, 70：25-37) and the 3' non-translational regions (AtuORF1 of Agrobacterium tumefaciens ORF 1 3′UTR；Huang et al.,(1990)J.Bacteriol.,172:1814-1822) constitute.By limiting enzymic digestion and sequencing Confirm the construct.Finally, the construct is transformed into Agrobacterium tumefaciens and stored up as glycerine and stored.

Embodiment 2：The agrobacterium-mediated conversion to Canola (Brassica napus) hypocotyl

It is prepared by agrobacterium

Containing streptomysin (100mg/mL) and grand mould using the agrobacterium bacterial strain containing pDAB7305 binary plasmids YEP culture mediums (the Bacto Peptone of plain (50mg/mL)^TM(20.0g/L) and yeast extract (10.0g/L)) lining out, And incubated 2 days in 28 DEG C.Scraped using aseptic inoculation ring from 2 days on streak plate containing pDAB7305 binary plasmids through propagation Agrobacterium bacterial strain.Then the agrobacterium inoculation containing pDAB7305 binary plasmids scraped is entered into 150mL into nothing The improvement YEP liquid with streptomysin (100mg/mL) and spectinomycin (50mg/mL) in the baffled flasks of bacterium 500mL In, and with 200rpm in 28 DEG C of shakes.M- culture mediums (LS salt is resuspended in after culture is centrifuged；3% glucose；The B5 of improvement Vitamin；1 μM of basic element of cell division；1μM 2,4-D；PH 5.8) in, and it is diluted to suitable concentration (50 Klett units, use Spectrophotometer is measured), Canola hypocotyl is converted afterwards.

Canola is converted：

Seed is sprouted：By canola seeds (kind Nexera 710^TM) surface sterilizing 10 minutes in 10%Clorox, and Three times (seed is contained in steel filter screen in this process) is rinsed with sterile distilled water.Seed is planted in Phytatray^TMIn 1/2MS Canolas culture medium (1/2MS, 2% sucrose, 0.8% agar) (each Phytatray contained^TMFor 25 seeds) on To be sprouted, and it is placed on the growth protocols for being set to 25 DEG C and 16 hours illumination/8 hour dark photoperiods Percival^TMIn growth room 5 days for sprout.

Pretreatment：At the 5th day, cut-out length about 3mm hypocotyl section, discarded remaining and bud under aseptic condition Partly (by the way that hypocotyl section is immersed into the sterile milliQ of 10mL during process is cut out^TMPrevent them from drying in water). Percival with the growth protocols for being set to 22-23 DEG C and 16 hours illumination/8 hour dark photoperiods^TMIt is pre- in growth room Before processing, by hypocotyl section on aseptic filter paper in callus inducing medium MSK1D1 (MS；The 1mg/L basic elements of cell division； 1mg/L 2,4-D；3% sucrose；0.7% plant agar (Phytagar)) on horizontal positioned 3 days.

With the co-cultivation of agrobacterium：The previous day is co-cultured in agrobacterium, with the soil containing pDAB7305 binary plasmids Earth bacillus strain is inoculated with the flask of the YEP culture mediums containing suitable antibiotic.By hypocotyl section from filter paper callus induction Culture medium MSK1D1 is transferred to the empty 100x25mm Petri containing 10mL liquid M culture mediums^TMCulture dish, to prevent hypocotyl from saving Section is dried.Section is scooped out using scraper in this stage, and is transferred to new culture medium.Liquid M culture mediums are removed with pipette, and 40mL Agrobacterium cell suspensions are added to Petri^TMCulture dish (500 sections and 40mL Agrobacterium solutions).By each hypocotyl Section is handled 30 minutes under conditions of the culture dish of periodicity vortex oscillation hypocotyl section so that hypocotyl is molten in agrobacterium Submergence is kept in liquid.At the end of process phase, Agrobacterium solution liquid relief is entered in waste beaker, handled with autoclave, And discard and (remove Agrobacterium solution completely excessive to place Agrobacterium growth).Hypocotyl through processing is returned with tweezers Revolution moves to the initial flat panel of the MSK1D1 culture mediums covered containing useful filter paper, notes ensuring that each section is moist.Will be inverted Hypocotyl section be put back into together with unconverted control hypocotyl section under reduction light intensity (covering flat board by using aluminium foil) Percival^TMGrowth room, and the hypocotyl section through processing and agrobacterium are co-cultured 3 days.

Callus induction on Selective agar medium：After co-culturing 3 days, hypocotyl section tweezers are individually turned Move to callus inducing medium MSK1D1H1 (MS；The 1mg/L basic elements of cell division；1mg/L 2,4-D；0.5g/L MES；5mg/ L AgNO₃；300mg/L Ticarcillin/Clavulanate Acids (Timentin)；200mg/L Carbenicillins (Carbenicillin)；1mg/L Herbiace^TM；3% sucrose；0.7% plant agar) on, growth protocols are set as 22-26 DEG C.By hypocotyl section in culture medium Upper grappling, but it is not buried into culture medium.

Selection and shoot regeneration：On callus inducing medium after 7 days, it will generate (callusing) of callus Hypocotyl section is transferred to the selectable shoot regeneration culture medium 1MSB3Z1H1 (MS of tool；3mg/L BAP；1mg/L zeatin；0.5gm/ L MES；5mg/L AgNO₃；300mg/L Ticarcillin/Clavulanate Acids^TM；200mg/L Carbenicillins；1mg/L Herbiace；3% sucrose；0.7% Plant agar) on.After 14 days, the hypocotyl section for having sent bud is transferred to the regeneration culture medium with enhanced selection 2MSB3Z1H3(MS；3mg/L BAP；1mg/L zeatin；0.5gm/L MES；5mg/L AgNO₃；300mg/l Ticarcillin/Clavulanate Acids^TM； 200mg/L Carbenicillins；3mg/L Herbiace^TM；3% sucrose；0.7% plant agar), growth protocols are set as 22-26 DEG C.

Bud extends：After 14 days, the hypocotyl section with hair tonic bud is transferred to bud elongation training from regeneration culture medium 2 Support base MSMESH5 (MS；300mg/L Ticarcillin/Clavulanate Acids^TM；5mg/L Herbiace^TM；2% sucrose；0.7%TC agar), growth protocols It is set as 22-26 DEG C.The bud extended is separated from hypocotyl section, and is transferred to MSMESH5., will be in elongation training after 14 days The remaining bud not extended in the first round culture for supporting base is transferred to fresh bud Extending culture base MSMESH5.In this stage, lose Abandon all remaining hypocotyl fragments for not producing bud.

Root induction：After being cultivated 14 days in bud elongation medium, the bud of separation is transferred to MSMEST culture mediums (MS； 0.5g/L MES；300mg/L Ticarcillin/Clavulanate Acids^TM；2% sucrose；0.7%TC agar) with 22-26 DEG C of progress root induction.By it is any For the first time during transfer MSMEST culture mediums the bud do not taken root after incubation be transferred on MSMEST culture mediums second or Third round is incubated, until bud bears root.

PCR is analyzed：Inverted Canola hypocotyl fragment regeneration has after the bud of root, and determination method is confirmed with PCR molecules To its further analysis.Leaf texture is obtained from green bud, tests whether there is pat selectable marker genes by PCR.Abandon any Chlorisis bud, is analyzed without PCR.It is accredited as pat selectable marker genes and exists for the sample of the positive on MSMEST culture mediums Preserve and cultivate, to continue to send and extend bud and root.Abandon to be analyzed and identified according to PCR and be free of pat selection marker things for feminine gender The sample of gene.

The inverted canola plant that there is the positive buds of PCR and root comprising selectable marker gene is transplanted to greenhouse In soil in.After canola plant is colonized in soil, canola plant is further analyzed, passes through Invader^TMIt is quantitative The copy number of PCR determination methods and Southern trace standard measure pat expression casettes.Confirm to contain at least one pat gene table The transgenosis T copied up to box₀Canola plant promotes further analysis seed fatty acid profile.By FAME analyticals from These transgenosis T₀Seed (the i.e. T that canola plant is obtained₁Canola seeds), to identify total saturated fat compared with check plant Fat acid reduction event (total saturated fatty acid content by by all saturated fatty acids, including short chain and long chain fatty acids add and To determine).

Embodiment 3：The T obtained from transgenosis pDAB7305 canola plants₁The FAME analyses of canola seeds

For (segregating) T separated₁Canola seeds, by FAME analyticals to identify this The T of sample₀Canola event：Its T produced₁Total saturated fatty acid (C14 of canola seeds:0, C16：0, C18：0, C20:0, C22:0, C24:0) reduced compared with the seed that the check plant grown under the conditions of identical is obtained.To all total saturated fatty acids (TSFA) summation is quantified, and is compared with negative control plant.Using scheme described below to single T₁Kano Seed is drawn to complete FAME analyses.To from each single Canola T₀24 single T altogether of event₁Canola seeds are carried out Determine, and to being quantified from each individual TSFA results.

Single canola seeds sample is homogenized in heptane using steel ball grinder, wherein contains Heptadecanoic acide in heptane Triglyceride (triheptadecanoin) (Nu-Chek prep) is used as triacylglycerol internal standard.Before homogenizing, add The solution of the freshly prepared sodium methoxides of 0.25M (Sigma-Aldrich, St.Louis, MO) in methyl alcohol.Extraction is constant at 40 DEG C Shake lower progress.The Endogenous fatty acid rate of recovery is normalized with the rate of recovery of the substitute C17 aliphatic acid methylated.Repeat 3 Secondary FAME (fatty acid methyl ester) extraction, merges post analysis by heptane layer.The FAME of gained is analyzed by GC-FID, using next From SGE capillary column BPX 70 (15m × 0.25mm × 0.25 μm).Every kind of FAME is identified according to retention time, and passes through note Enter from Matreya LLC (Pleasant Gap, PA) rapeseed oil with reference to pre-composition as calibration standard, while adding suitably Long chain fatty acids (Nu-Chek Prep, Elysian MN), to be quantified.

Large quantities of seed analysises are made up of 50mg aliquots (10 to 15 seeds are combined), and are added according to above-mentioned same approach To implement after a few modifications.It is oily three times with heptane extraxtion first in order to promote derivative reaction completely.Then, by the oil of merging The aliquot for corresponding to 1 seed of extract derivative chemical conversion FAME as described in above-mentioned single subscheme.By at the 4th time Check that endogenous FAME presence carrys out the completeness of confirmatory reaction in extraction/derivatization.

Based on the FAME results that substantially reduce compared with compareing rapeseed plant TSFA are shown, identify and have selected three and turn base Because of Canola event (event 2182 [12] -138.001, [12] -125.001 of event 2182 and [12] -156.001 of event 2182) It is advanced to T₁Generation.Determine two other vegetable fatty acid inclusion category.These categories include monounsaturated fatty acids (MUFA：C16:1, C18:11 and C20:11) with polyunsaturated fatty acid (PUFA：C18:12 and C18:13) concentration, lists these Category is to show T₁The TSFA reduced in seed effect (table 1).

Table 1：With several Nexera 710^TMUntransformed control plants are compared, and are obtained from three transgenosis Canola events Single T₁Seed TSFA, MUFA and PUFA content are summarized.N* represents the single T analyzed each plant generations₁The quantity of seed.

With Nexera 710^TMUntransformed control plants are compared, and the average TSFA of transgenosis Canola event is significantly reduced. While TSFA reduction, it was observed that MUFA contents (C18:1 and C16:1) increase.The increase of MUFA contents is AnD9DS mistakes Express and introduce the direct result of double bond at 9th carbon (Δ 9) place of the saturated fatty acid from carboxylic acid functional.It is interesting that PUFA contents are not with phosphoglyceride desaturase FAD2 synthesis C18:2 substrate MUFA accumulation and increase.

By T₁Canola plant event grows in greenhouse and from being fertilized, the transgenosis of infiltration is fixed on into progeny plants In.To multiple T from each transgenic event₁Canola plant completes Invader^TMQuantitative PCR detection.These result tables T that is bright, being obtained from three events₁PDAB7305 T chain of the canola plant containing about 2 or 3 copies is integrated (at the top of Fig. 3 Small figure).Copy number can not be specifically determined, because pDAB7305 T chains intergrants are divided in three events with different copy numbers From.Allow T₁Canola plant event growth and maturity and fertilization certainly in greenhouse.Harvest the T produced₂Canola seeds are used to pass through FAME determination methods carry out fatty acid profile analysis.

Embodiment 4：The T obtained from transgenosis pDAB7305 canola plants₂The FAME analyses of canola seeds

Pass through the previously described large quantities of T of FAME analyticals₂Canola seeds, with identification and check plant (Nexera 710^TM) compared to the T of total saturated fatty acid reduction₁Canola plant strain (Fig. 2).The yield of each plant is made Recorded for the seed grams of each plant.By yield result and the desaturase transgenosis of aspergillus nidulans Δ -9 containing stable integration Transgenic positive control event 218-11.30 (referring to WO2006/042049) (also described herein as 218-11.30HS50 or Yield 218-11.30HL) is compared, and check plant is planted together with the genetically modified plants of the present invention.218-11.30 plant Similar transgenosis is all expressed with transgenosis canola plant disclosed in this theme.However, this theme is used to convert in disclosing The construct of 2182 [12] -125.Sx001,2182 [12] -138.Sx001 and 2182 [12] -156.Sx00 canola plants is Different, because it includes second PTU, the PTU is driven by Lesquerella fendleri KCS3 promoters and the promoter The AnD9DS coded sequences composition of dynamic expression, and by Agrobacterium tumefaciens ORF23 3' non-translational regions institute flank.

The quantitative TSFA (%) to genetically modified plants disclosed in this theme, and with turning base from positive control 218-11.30HL Because of canola plant and negative control Nexera 710^TMThe TSFA (%) that plant obtains is compared.Identify this small number of theme Disclosed genetically modified plants contain the TSFA of higher level, and its level is similar to the Nexera 710 of negative control^TMPlant.These Plant is due to that the invalid body (sibling nulls) born of the same parents for separating and producing occurs during from fertilization for transgenosis, and not Include the copy of transgenosis disclosed in any this theme actively expressed.These results demonstrate that identifying T₁Canola plant In, large quantities of T₂Total saturated fatty acid content of canola seeds is reduced to less than 3.5%.

Further analyze T₂Canola strain, to determine pDAB7305T which Canola strain contains low copy number Chain intergrant, and produce high T₂Seed production.(Fig. 3).

Canola plant individual disclosed in this such theme of selection：The TSFA contained is less than 3.5%, and the yield of generation surpasses 10g is crossed, and the T chain copy numbers contained are minimum.Based on these three standards, (TSFA levels are less than 3.5%, high seed production and low Copy number), it have selected seven canola plants and promote to further characterize TSFA spectrums.Using Invader Assay from each T₁One plant of strain determines copy number.Table 2.By these T₂Canola plant strain is transferred to greenhouse, growth and maturity and from by Essence.Further analyze T₂Canola plant strain, and T is harvested by FAME determination methods₃Seed carries out fatty acid profile analysis.

Table 2：List based on maximum output (seed weight), minimum PAT copy numbers (T1 plants) and minimum TSFA selections T₂Canola strain.

Embodiment 5：T₂The molecule of Canola strain confirms

For the T2 Canola things containing pat expression casettes (and AnD9DS expression casettes of close linkage) selected Part, molecule integration mode is characterized using quantitative PCR and Southern engram analysis.

Confirm that AnD9DS is integrated by hydrolysis probes determination method

Being determined by hydrolysis probes confirms the presence of AnD9DS expression casettes.Determine (similar by hydrolysis probes first In TAQMAN^TM) the T2 canola plants separated are screened, to confirm the presence of pat transgenosis.The data produced from these researchs For determining transgene copy number, and the transgenosis Canola event for selecting to be used to being returned and being advanced to offspring.

Tissue sample is collected in 96 orifice plates, KLECO is used^TMTissue pulverizer and stainless shot (Hoover Precision Products, Cumming, GA), in Qiagen RLT^TMTissue maceration is carried out in buffer solution.In tissue maceration Afterwards, according to manufacturer's recommendation scheme, Biosprint 96 is used^TMBotanical agents box (Qiagen, Germantown, MD) is with height Flux version isolated genes group DNA.Genomic DNA Quant-IT Pico Green DNA assay kit^TM(Molecular Probes, Invitrogen, Carlsbad, CA) it is quantitative.Use BIOROBOT3000^TMAutomated fluid processor (Qiagen, Germantown, MD), the genomic DNA after quantifying is adjusted to about 2ng/ μ L to be determined for hydrolysis probes.By usingThe real-time PCR of 480 systems (Roche Applied Science, Indianapolis, IN) is carried out It is similar toThe transgene copy number that the hydrolysis probes of measure are determined is determined.Use Probe Design Software 2.0 are designed for pat and internal reference gene HMG1 (Weng etc. (2005) .J.AOAC Int.88(2):Measure 577-84).In order to expand, 1 × final concentration is prepared in 10 μ L volume multiple reactions480 probe main mixtures (contain 0.4 μM of every kind of primer for AnD9DS and pat, and 0.2 μ M every kind of probe) (table 3).Two step amplified reactions are carried out, are extended 40 seconds at 60 DEG C, fluorescence is captured.All samples are run, and Averaging loop threshold value (Ct) value is used for the analysis of each sample.The analysis of real-time PCR data is used using Relative quantification moduleSoftware version 1.5 is carried out, and based on Δ Δ Ct methods.Control includes：From single copy calibration The genome DNA sample of product, and the known two copies inspection product that each run includes.Table 4 lists hydrolysis probes survey Fixed result.Determined using qPCR from each T₁The N strains (and average, to provide the value in table 4) of strain determine copy number.

Table 3：The primer and probe sequence determined for pat and the hydrolysis probes of internal reference (HMG1).

Table 4:AnD9DS events (the T determined using hydrolysis probes determination method₂Plant) copy number result

The result that hydrolysis probes are determined determine two strains (2182 [12] -138.Sx001.Sx094 and 2182 [12] - 138.SX001.Sx090), it has relative standard deviation (such as table 4 suitable with positive control plant (218-11.30 (HL)) Show SD) and the coefficient of variation (being as shown in table 4 CV%) combination.(218-11.30 HL) check plant was previously accredited as AnD9DS genes containing two fixed copies insert (WO 2006042049).Pass through the Canola strain (2182 that will be selected [12] -138.Sx001.Sx094 and 2182 [12] -138.SX001.Sx090) compared with 218-11.30 (HL) check plant Compared with identifying will the specific Canola strain containing the pDAB7305T chain intergrants of fixed copy.

Confirm AnD9DS genome conformities by Southern engram analysis.

Being set up using Southern engram analysis proves the integration mode of T chain DNA fragments, and identification contains total length AnD9DS The Canola strain of expression casette.Generate the integration of the provable transgenic insertions in Canola genome and complete The data of property.Detailed Southern engram analysis are carried out using the PCR amplification probes for being specific to AnD9DS expression casettes.Should The hybridization of genomic DNA of the probe with being digested by specific restriction enzyme identifies the genomic DNA piece with specified molecular weight Section, is characterized with the pattern of these fragments for being advanced to follow-on transgenic event.

Tissue sample is collected in 2mL conical pipes and lyophilized 2 days.Use KLECKO^TMTissue pulverizer and tungsten pearl carry out group Knit dipping.After tissue maceration, CTAB separable programming isolated genes groups DNA is used.Use Qiagen Genomic Tips^TMExamination Genomic DNA is further purified in agent box.Kit (Molecular is determined with Quant-IT Pico Green DNA TM Probes, Invitrogen, Carlsbad, CA) quantitative genomic DNA.It will be adjusted through quantitative genomic DNA to consistent dense Degree.

For each sample, with restriction enzyme BamHI (New England Biolabs, Beverley, MA) fully 4 μ of digestion G genomic DNAs.Use Quick Precipitation Solution^TM(Edge Biosystems, Gaithersburg, MD), according to manufacturer's recommendation scheme by precipitating the DNA come concentrating, assimilating.Then by genomic DNA at 65 DEG C resuspension 1 hour in 25 μ L water.The sample of resuspension is loaded on 0.8% Ago-Gel prepared in 1X TAE, and with 1.1V/ Cm electrophoresis in 1X TAE buffer solutions is stayed overnight.It is denatured (0.2M NaOH/0.6M NaCl) successively to gel 30 minutes, and in (0.5M Tris-HCl (pH7.5)/1.5M NaCl) 30 minutes.

Using spill and paper handkerchief is chromatographed, 20X SSC solution is passively penetrated gel and be attracted to through processing IMMOBILON^TMOn NY+ transfer membranes (Millipore, Billerica, MA), DNA fragmentation is transferred on nylon membrane.Transfer Afterwards, by the film of short duration washings of 2X SSC, with STRATALINKER^TM1800 (Stratagene, LaJolla, CA) are crosslinked, and 80 Vacuum bakeout 3 hours at DEG C.

It is using 400 types hybridization incubator (Robbins Scientific, Sunnyvale, CA), trace is molten with prehybridization Liquid (Perfect Hyb plus, Sigma, St.Louis, MO) is incubated 1 hour at 65 DEG C in glass roller bottles.From containing whole The PCR fragment of individual coded sequence prepares probe.Use QIAEX II gel extraction kit^TMPCR amplicons are purified, and With DIG DNA Labeling Kit^TM(Roche Applied BioSciencse, Indianapolis, IN) is marked.Trace exists Hybridized overnight at 65 DEG C, denatured probe is added directly into hybridization buffer.After hybridization, at 65 DEG C with 0.1 × SSC/0.1% SDS washs trace 40 minutes successively.Finally, by trace exposed to storage phosphorus imaging screen, and Molecular Dynamics are used Storm 860^TMImaging system images.

The Southern engram analysis completed in this research are used to determine copy number, and confirm selected event in Kano Draw and contain AnD9DS expression casettes in genome.Table 5 is provided from based on the selected strain of the standard defined in above-mentioned table 2 Multiple T₂The band distribution of plant.Control strain does not contain selection marker thing, this confirms that PCR data.From three events Selection most of strains show homogeneous band pattern (number and size), except from event 2182 [12]- Outside 125.Sx001.Sx014 strain.Event 2182 [12] -138.Sx001 all three T₂System's display has consistent band The T of pattern₂Colony.

Table 5：To multiple T from four transgenic events₂Canola strain and NEXERA 710^TMCanola control is planted The general introduction for the Southern analyses that thing is completed.Compared by the known standard items with being run on Ago-Gel beside sample Relatively come determine each sample observe band size.

Embodiment 6：The T of selected Canola strain₃Seed production

From T₂The selected plant of Canola strain growth and maturity in greenhouse.From plant harvest seed.Seed is cleaned, and It is determined that each T₂The seed production (table 6) of Canola strain.By the yield of the seed from each strain with carrying out comfortable identical bar Unconverted check plant (the Nexera grown under part^TM710GS) yield of the seed obtained is compared.Table 6 give from Each T₂The yield result for the different plants that Canola strain is obtained.These results indicate that each plant and the strain of test Yield is variable.But T₂Ka Nala strains average product (2182 [12] -125.Sx001.Sx014,2182 [12] - 138.Sx001.Sx085,2182 [12] -138.Sx001.Sx090,2182 [12] -138.Sx001.Sx094 and 2182 [12] - It is 156.Sx001.Sx049) relatively approximate, and do not deviate significantly from check plant (Nexera^TM710G5 and 218-11.30 (HL))。

Table 6：From Canola transgenosis product strain and unconverted Nexera^TM710GS check plants seed production (with Total grams meter) ANNOVA analyses.The plant products result that same letter is connected in bracket is not significantly different (p<0.05).

Event/strain	Plant number	Ratio	Yield (g)
				218-11.30(HL)	12	0.06186	5.7233333(A,B)
2182[12]-125.Sx001.Sx014	35	0.18041	5.9874286(A,B)
				2182[12]-138.Sx001.Sx085	34	0.17526	6.8394118(A)
2182[12]-138.Sx001.Sx090	33	0.17010	6.4887879(A,B)
				2182[12]-138.Sx001.Sx094	35	0.18041	5.0302857(B)
2182[12]-156.Sx001.Sx049	33	0.17010	6.3715152(A,B)
				NexeraTM710G5	12	0.06186	7.1666667(A,B)
Amount to	194	1.00000	---

Embodiment 7：The T obtained from transgenosis pDAB7305 canola plants₃The FAME analyses of canola seeds

Pass through the single and large quantities of T of previously described FAME analyticals₃Canola seeds, to characterize each strain Fatty acid profile, so as to identify the specific strain for causing total saturated fatty acid to reduce compared with check plant.Quantitative total saturated fat The summation of acid, and be compared with positive control and negative control plant.As a result, it was confirmed that in selected canola plant strain In, single and large quantities of T₃The total saturated fatty acid and saturated fatty acid of canola seeds are (by palmitic acid and stearic acid content sum It is determined that) content is reduced to less than 3.5%.

, it is surprising that two strains, 2182 [12] -138.SX001.SX085 and 2182 [12] - 138.Sx001.Sx094, the TSFA levels of accumulation are average below 3.0%.Describe first by comprising less than 3.0% saturated fat The Canola strain that large quantities of seeds of fat acid are constituted.Table 7 and table 8.

In addition, the Canola strain of a subgroup is analyzed for seed FAME, to determine in single canola seeds The total saturated fatty acid and the floor level of saturated fatty acid level resulted in.From minimum total saturated fat based on large quantities of seeds The Canola strain that fat is sour and high-caliber plant products are selected obtains seed, for the sub- FAME analyses of single.Use FAME methods, each strain analyzes 288 individual seeds altogether.The summary of analysis is shown in Table 9.All single from selected plant The average TSFA of son is less than 2.8%, hence it is evident that the TSFA levels less than 3.5%.It is minimum in the level of single subcard Nola TSFA levels are 2.25%.This is substantially less than Nexera^TMThe TSFA levels 5.11% that 710G5 check plants are obtained.Finally, base is turned Because the largest percentage of TSFA in Canola strain is no more than 3.5%, one-seeded average saturated level is 2.52%, is far below 3.5%.Table 9 and Fig. 4.

Table 7 is shown and unconverted Nexera^TM218-11.30 (HL) positive control plant that 710G5 is compareed and converted Compare, the T of homogeneous Canola strain colony in 5 heredity₂The distribution of the large quantities of FAME analyses of mature seed.Obtain all individuals The average value of measured value (N), to represent the TSFA and saturation percentage of canola plant colony.2182 are designated with boldface letter [12] -138.Sx001.Sx085 and 2182 [12] -138.Sx001.Sx094, because the average TSFA percentages of the two strains Less than 3.00%.

Table 7

Table 8 is shown and Nexera^TM710G5 controls are compared, the single T obtained from event 2182 [12] -138.Sx001₂ The minimum T of progeny plants₃The large quantities of FAME of mature seed and plant products.The result of display is the percentage of oil, TSFA percentage Than the percentage (being determined by summarizing palmitic acid and stearic acid content) and seed production of, saturated fatty acid.

Table 8

Table 9 is shown from selected T₂The T of strain₃The distribution of the sub- FAME analysis results of single.With being shown in the mutually table Average value (average value), minimum value (minimum) and the maximum (maximum) of TSFA and saturated fatty acid percentage, under the conditions of same The Nexera of growth^TM710G5 control Canola seed plants are compared.Compared to Nexera^TM710G5 compares canola plant, selected The T of event₃Total saturated fatty acid (TSFA) and saturated fatty acid level in seed have reduction.

Table 9

Sequence table

<110>The Dow Agrosciences, LLC.

DJ Jia Qiaodi

PAO writes from memory Lip river

MA thompsons

TA Walsh

BR Wilson's

M Wei Erte

<120>The generation of transgenosis Canola having less than 3.5% total saturated fatty acid

<130> 71671

<160> 33

<170> PatentIn version 3.5

<210> 1

<211> 455

<212> PRT

<213>Aspergillus nidulans

<400> 1

Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys

1 5 10 15

Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys

20 25 30

His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr

35 40 45

Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile

50 55 60

Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly

65 70 75 80

Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu

85 90 95

Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile

100 105 110

Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr

115 120 125

Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu

130 135 140

Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp

145 150 155 160

Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn

165 170 175

Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val

180 185 190

Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly

195 200 205

Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser

210 215 220

Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro

225 230 235 240

Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His

245 250 255

Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp

260 265 270

His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu

275 280 285

Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys

290 295 300

Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr

305 310 315 320

Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp

325 330 335

Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala

340 345 350

Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly

355 360 365

Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe

370 375 380

Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser

385 390 395 400

Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp

405 410 415

Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly

420 425 430

Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro

435 440 445

Ile Pro Thr Ala Asp Ala Ala

450 455

<210> 2

<211> 1368

<212> DNA

<213>Artificial sequence

<220>

<223>Encode the desaturase v3 of aspergillus nidulans Delta 9 synthesizing ribonucleotide

<400> 2

atgtctgctc caaccgctga catcagggct agggctccag aggctaagaa ggttcacatc 60

gctgataccg ctatcaacag gcacaattgg tacaagcacg tgaactggct caacgtcttc 120

ctcatcatcg gaatcccact ctacggatgc atccaagctt tctgggttcc acttcaactc 180

aagaccgcta tctgggctgt gatctactac ttcttcaccg gacttggaat caccgctgga 240

taccacaggc tttgggctca ctgctcatac tctgctactc ttccacttag gatctggctt 300

gctgctgttg gaggaggagc tgttgaggga tctatcagat ggtgggctag ggatcacagg 360

gctcatcata ggtacaccga taccgacaag gacccatact ctgttaggaa gggacttctc 420

tactctcacc ttggatggat ggtgatgaag cagaacccaa agaggatcgg aaggaccgac 480

atctctgatc tcaacgagga cccagttgtt gtttggcaac acaggaacta cctcaaggtt 540

gtgttcacca tgggacttgc tgttccaatg cttgttgctg gacttggatg gggagattgg 600

cttggaggat tcgtgtacgc tggaatcctt aggatcttct tcgttcaaca agctaccttc 660

tgcgtgaact ctcttgctca ctggcttgga gatcaaccat tcgatgatag gaactctcct 720

agggatcacg tgatcaccgc tcttgttacc cttggagagg gataccacaa cttccaccac 780

gagttcccat ctgactacag gaacgctatc gagtggcacc agtacgatcc taccaagtgg 840

tctatctggg cttggaagca acttggattg gcttacgatc tcaagaagtt cagggctaac 900

gagatcgaga agggaagggt tcaacaactt cagaagaagc ttgataggaa gagggctact 960

cttgattggg gaaccccact tgatcaactt ccagtgatgg aatgggatga ctacgttgag 1020

caagctaaga acggaagggg acttgttgct atcgctggag ttgttcacga tgttaccgac 1080

ttcatcaagg atcacccagg aggaaaggct atgatctctt ctggaatcgg aaaggatgct 1140

accgctatgt tcaacggagg agtgtactac cactctaacg cagctcacaa ccttcttagc 1200

accatgaggg tgggagtgat caggggagga tgcgaggttg agatctggaa gagggctcag 1260

aaggagaacg ttgagtacgt tagggatgga tctggacaaa gggtgatcag ggctggagag 1320

caaccaacca agatcccaga gccaatccca accgctgatg ctgcttga 1368

<210> 3

<211> 2912

<212> DNA

<213>Artificial sequence

<220>

<223>Include the desaturase v3 of aspergillus nidulans delta 9 the first plant transcription unit

<400> 3

ctcccagtat cattatagtg aaagttttgg ctctctcgcc ggtggttttt tacctctatt 60

taaaggggtt ttccacctaa aaattctggt atcattctca ctttacttgt tactttaatt 120

tctcataatc tttggttgaa attatcacgc ttccgcacac gatatcccta caaatttatt 180

atttgttaaa cattttcaaa ccgcataaaa ttttatgaag tcccgtctat ctttaatgta 240

gtctaacatt ttcatattga aatatataat ttacttaatt ttagcgttgg tagaaagcat 300

aatgatttat tcttattctt cttcatataa atgtttaata tacaatataa acaaattctt 360

taccttaaga aggatttccc attttatatt ttaaaaatat atttatcaaa tatttttcaa 420

ccacgtaaat ctcataataa taagttgttt caaaagtaat aaaatttaac tccataattt 480

ttttattcga ctgatcttaa agcaacaccc agtgacacaa ctagccattt ttttctttga 540

ataaaaaaat ccaattatca ttgtattttt tttatacaat gaaaatttca ccaaacaatg 600

atttgtggta tttctgaagc aagtcatgtt atgcaaaatt ctataattcc catttgacac 660

tacggaagta actgaagatc tgcttttaca tgcgagacac atcttctaaa gtaattttaa 720

taatagttac tatattcaag atttcatata tcaaatactc aatattactt ctaaaaaatt 780

aattagatat aattaaaata ttactttttt aattttaagt ttaattgttg aatttgtgac 840

tattgattta ttattctact atgtttaaat tgttttatag atagtttaaa gtaaatataa 900

gtaatgtagt agagtgttag agtgttaccc taaaccataa actataagat ttatggtgga 960

ctaattttca tatatttctt attgctttta ccttttcttg gtatgtaagt ccgtaactgg 1020

aattactgtg ggttgccatg acactctgtg gtcttttggt tcatgcatgg atgcttgcgc 1080

aagaaaaaga caaagaacaa agaaaaaaga caaaacagag agacaaaacg caatcacaca 1140

accaactcaa attagtcact ggctgatcaa gatcgccgcg tccatgtatg tctaaatgcc 1200

atgcaaagca acacgtgctt aacatgcact ttaaatggct cacccatctc aacccacaca 1260

caaacacatt gcctttttct tcatcatcac cacaaccacc tgtatatatt cattctcttc 1320

cgccacctca atttcttcac ttcaacacac gtcaacctgc atatgcgtgt catcccatgc 1380

ccaaatctcc atgcatgttc caaccacctt ctctcttata taatacctat aaatacctct 1440

aatatcactc acttctttca tcatccatcc atccagagta ctactactct actactataa 1500

taccccaacc caactcatat tcaatactac tctaggatcc aacaatgtct gctccaaccg 1560

ctgacatcag ggctagggct ccagaggcta agaaggttca catcgctgat accgctatca 1620

acaggcacaa ttggtacaag cacgtgaact ggctcaacgt cttcctcatc atcggaatcc 1680

cactctacgg atgcatccaa gctttctggg ttccacttca actcaagacc gctatctggg 1740

ctgtgatcta ctacttcttc accggacttg gaatcaccgc tggataccac aggctttggg 1800

ctcactgctc atactctgct actcttccac ttaggatctg gcttgctgct gttggaggag 1860

gagctgttga gggatctatc agatggtggg ctagggatca cagggctcat cataggtaca 1920

ccgataccga caaggaccca tactctgtta ggaagggact tctctactct caccttggat 1980

ggatggtgat gaagcagaac ccaaagagga tcggaaggac cgacatctct gatctcaacg 2040

aggacccagt tgttgtttgg caacacagga actacctcaa ggttgtgttc accatgggac 2100

ttgctgttcc aatgcttgtt gctggacttg gatggggaga ttggcttgga ggattcgtgt 2160

acgctggaat ccttaggatc ttcttcgttc aacaagctac cttctgcgtg aactctcttg 2220

ctcactggct tggagatcaa ccattcgatg ataggaactc tcctagggat cacgtgatca 2280

ccgctcttgt tacccttgga gagggatacc acaacttcca ccacgagttc ccatctgact 2340

acaggaacgc tatcgagtgg caccagtacg atcctaccaa gtggtctatc tgggcttgga 2400

agcaacttgg attggcttac gatctcaaga agttcagggc taacgagatc gagaagggaa 2460

gggttcaaca acttcagaag aagcttgata ggaagagggc tactcttgat tggggaaccc 2520

cacttgatca acttccagtg atggaatggg atgactacgt tgagcaagct aagaacggaa 2580

ggggacttgt tgctatcgct ggagttgttc acgatgttac cgacttcatc aaggatcacc 2640

caggaggaaa ggctatgatc tcttctggaa tcggaaagga tgctaccgct atgttcaacg 2700

gaggagtgta ctaccactct aacgcagctc acaaccttct tagcaccatg agggtgggag 2760

tgatcagggg aggatgcgag gttgagatct ggaagagggc tcagaaggag aacgttgagt 2820

acgttaggga tggatctgga caaagggtga tcagggctgg agagcaacca accaagatcc 2880

cagagccaat cccaaccgct gatgctgctt ga 2912

<210> 4

<211> 2517

<212> DNA

<213>Artificial sequence

<220>

<223>Include the desaturase v3 of aspergillus nidulans delta 9 the second plant transcription unit

<400> 4

gaattcggaa atgggccaag tgaaatggaa atagagcttc aatccattta gtcccactca 60

aaatggtgct cgaattatat ttagttacgt tcgaatcaga caaccaagta tttggttaat 120

aaaaaccact cgcaacaaag gaaaaacacc aagcgcgtgc gtccaacatc cgacggaagg 180

ggggtaatgt ggtccgaaaa ccttacaaaa atctgacgtc atctaccccc gaaaacgttg 240

aatcgtcaac gggggtagtt ttcgaattat ctttttttta ggggcagttt tattaatttg 300

ctctagaaat tttatgattt taattaaaaa aagaaaaaga atatttgtat atttattttt 360

tatactcttt ttttgtccaa ctatttctct tattttggca actttaacta gactagtaac 420

ttatgtcaat gtgtatggat gcatgagagt gagtatacac atgtctaaat gcatgcctta 480

tgaaagcaac gcaccacaaa acgaagaccc ctttacaaat acatctcatc ccttagtacc 540

ctcttactac tgtcccgaca caaactcaaa acaaggtacc ctgcagggat ccaacaatgt 600

ctgctccaac cgctgacatc agggctaggg ctccagaggc taagaaggtt cacatcgctg 660

ataccgctat caacaggcac aattggtaca agcacgtgaa ctggctcaac gtcttcctca 720

tcatcggaat cccactctac ggatgcatcc aagctttctg ggttccactt caactcaaga 780

ccgctatctg ggctgtgatc tactacttct tcaccggact tggaatcacc gctggatacc 840

acaggctttg ggctcactgc tcatactctg ctactcttcc acttaggatc tggcttgctg 900

ctgttggagg aggagctgtt gagggatcta tcagatggtg ggctagggat cacagggctc 960

atcataggta caccgatacc gacaaggacc catactctgt taggaaggga cttctctact 1020

ctcaccttgg atggatggtg atgaagcaga acccaaagag gatcggaagg accgacatct 1080

ctgatctcaa cgaggaccca gttgttgttt ggcaacacag gaactacctc aaggttgtgt 1140

tcaccatggg acttgctgtt ccaatgcttg ttgctggact tggatgggga gattggcttg 1200

gaggattcgt gtacgctgga atccttagga tcttcttcgt tcaacaagct accttctgcg 1260

tgaactctct tgctcactgg cttggagatc aaccattcga tgataggaac tctcctaggg 1320

atcacgtgat caccgctctt gttacccttg gagagggata ccacaacttc caccacgagt 1380

tcccatctga ctacaggaac gctatcgagt ggcaccagta cgatcctacc aagtggtcta 1440

tctgggcttg gaagcaactt ggattggctt acgatctcaa gaagttcagg gctaacgaga 1500

tcgagaaggg aagggttcaa caacttcaga agaagcttga taggaagagg gctactcttg 1560

attggggaac cccacttgat caacttccag tgatggaatg ggatgactac gttgagcaag 1620

ctaagaacgg aaggggactt gttgctatcg ctggagttgt tcacgatgtt accgacttca 1680

tcaaggatca cccaggagga aaggctatga tctcttctgg aatcggaaag gatgctaccg 1740

ctatgttcaa cggaggagtg tactaccact ctaacgcagc tcacaacctt cttagcacca 1800

tgagggtggg agtgatcagg ggaggatgcg aggttgagat ctggaagagg gctcagaagg 1860

agaacgttga gtacgttagg gatggatctg gacaaagggt gatcagggct ggagagcaac 1920

caaccaagat cccagagcca atcccaaccg ctgatgctgc ttgagtagtt agcttaatca 1980

cctaggtcac cagcataatt tttattaatg tactaaatta ctgttttgtt aaatgcaatt 2040

ttgctttctc gggattttaa tatcaaaatc tatttagaaa tacacaatat tttgttgcag 2100

gcttgctgga gaatcgatct gctatcataa aaattacaaa aaaattttat ttgcctcaat 2160

tattttagga ttggtattaa ggacgcttaa attatttgtc gggtcactac gcatcattgt 2220

gattgagaag atcagcgata cgaaatattc gtagtactat cgataattta tttgaaaatt 2280

cataagaaaa gcaaacgtta catgaattga tgaaacaata caaagacaga taaagccacg 2340

cacatttagg atattggccg agattactga atattgagta agatcacgga atttctgaca 2400

ggagcatgtc ttcaattcag cccaaatggc agttgaaata ctcaaaccgc cccatatgca 2460

ggagcggatc attcattgtt tgtttggttg cctttgccaa catgggagtc caaggtt 2517

<210> 5

<211> 1828

<212> DNA

<213>Artificial sequence

<220>

<223>Plant transcription unit comprising phosphinothricin acetyl transferase

<400> 5

cctgcaggga gatttttcaa atcagtgcgc tagacgtgac gtaagtatcc gagtcagttt 60

ttatttttct actaatttgg tcgtttattt cggcgtgtag gacatggcaa ccgggcctga 120

atttcgcggg tattctgttt ctattccaac tttttcttga tccgcagcca ttaacgactt 180

ttgaatagat acgtctaggg tcgagggggg atccgtcgag ggggtccacc aaaaacgtaa 240

gcgcttacgt acatggtcga gggggtccac caaaaacgta agcgcttacg tacatggtcg 300

agggggtcca ccaaaaacgt aagcgcttac gtacatggtc gagggggtcc accaaaaacg 360

taagcgctta cgtacatggt cgactagagc gtgacgctcg cggtgacgcc atttcgcctt 420

ttcagaaatg gataaatagc cttgcttcct attatatctt cccaaattac caatacatta 480

cactagcatc tgaatttcat aaccaatctc gatacaccaa atcgcagatc tggatcccaa 540

accatgtctc cggagaggag accagttgag attaggccag ctacagcagc tgatatggcc 600

gcggtttgtg atatcgttaa ccattacatt gagacgtcta cagtgaactt taggacagag 660

ccacaaacac cacaagagtg gattgatgat ctagagaggt tgcaagatag atacccttgg 720

ttggttgctg aggttgaggg tgttgtggct ggtattgctt acgctgggcc ctggaaggct 780

aggaacgctt acgattggac agttgagagt actgtttacg tgtcacatag gcatcaaagg 840

ttgggcctag gatctacatt gtacacacat ttgcttaagt ctatggaggc gcaaggtttt 900

aagtctgtgg ttgctgttat aggccttcca aacgatccat ctgttaggtt gcatgaggct 960

ttgggataca cagcccgggg tacattgcgc gcagctggat acaagcatgg tggatggcat 1020

gatgttggtt tttggcaaag ggattttgag ttgccagctc ctccaaggcc agttaggcca 1080

gttacccaaa tctgagtagt tagcttaatc acctagagct cgatcggcgg caatagcttc 1140

ttagcgccat cccgggttga tcctatctgt gttgaaatag ttgcggtggg caaggctctc 1200

tttcagaaag acaggcggcc aaaggaaccc aaggtgaggt gggctatggc tctcagttcc 1260

ttgtggaagc gcttggtcta aggtgcagag gtgttagcgg gatgaagcaa aagtgtccga 1320

ttgtaacaag atatgttgat cctacgtaag gatattaaag tatgtattca tcactaatat 1380

aatcagtgta ttccaatatg tactacgatt tccaatgtct ttattgtcgc cgtatgtaat 1440

cggcgtcaca aaataatccc cggtgacttt cttttaatcc aggatgaaat aatatgttat 1500

tataattttt gcgatttggt ccgttatagg aattgaagtg tgcttgaggt cggtcgccac 1560

cactcccatt tcataatttt acatgtattt gaaaaataaa aatttatggt attcaattta 1620

aacacgtata cttgtaaaga atgatatctt gaaagaaata tagtttaaat atttattgat 1680

aaaataacaa gtcaggtatt atagtccaag caaaaacata aatttattga tgcaagttta 1740

aattcagaaa tatttcaata actgattata tcagctggta cattgccgta gatgaaagac 1800

tgagtgcgat attatggtgt aatacata 1828

<210> 6

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223>Patv5 forward primers

<400> 6

acaagagtgg attgatgatc tagagaggt 29

<210> 7

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223>Patv5 reverse primers

<400> 7

ctttgatgcc tatgtgacac gtaaacagt 29

<210> 8

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223>Pat v5 probes

<400> 8

ccagcgtaag caataccagc cacaacacc 29

<210> 9

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>HMG1 forward primers

<400> 9

cctctctacc accgtctcac atg 23

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>HMG1 reverse primers

<400> 10

gatctggccg gactgtttca 20

<210> 11

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223>HMG1 probes

<400> 11

cgctcctcag ctaccacctc aacca 25

<210> 12

<211> 1523

<212> DNA

<213>Artificial sequence

<220>

<223>MgD9Ds pcr amplified fragment

<400> 12

gaattcatgg cttcgtcatc ttcctccgtg ccggagttgg ctgccgcctt ccctgatggc 60

actaccgact tcaagcccat gaggaacacc aagggctacg acgtcagcaa gccgcacatt 120

tccgagacac ctatgacact caagaactgg cataagcacg tcaactggct caacaccacc 180

ttcatcttgt ttgtgcccct ggctggtctc atatccactt actgggtccc tctgcagtgg 240

aagacggctg tatgggctgt cgtctactac ttcaacaccg gcctgggaat tactgccggt 300

aagtggctct tgaacaaacg agctaggccg ccgccctgta tccaatcatc tgtatccatc 360

cctagatgct aactagaaaa cttgcgggtt accaccgact ttgggctcac agctcgtaca 420

aggcctcgct tccgctcaaa atctaccttg ccgccgttgg cgctggtgcc gtcgagggct 480

ccatcagatg gtggtccaac ggtcaccgcg cacaccaccg atacaccgat accgagaagg 540

acccctactc agtccgcaag ggtctcctgt actcacacat gggatggatg cttctgaagc 600

agaaccccaa gaagcagggc cgcaccgaca tcaccgacct gaacgaggac cccgttgtcg 660

tttggcagca ccgcaacttc ctcaagtgtg ttatcttcat ggccctcgtc ttccccacac 720

ttgtggctgg ccttggctgg ggtgactact ggggaggttt catctacgga ggtattctgc 780

gtgtcttctt cgtccagcag gccaccttct gcgtcaactc gcttgcccac tggctcggtg 840

accagccttt cgacgatcgc aactcgccgc gtgatcacgt catcacagcc ctggtcaccc 900

ttggagaggg ataccacaac ttccaccacg agttcccttc ggactaccgc aacgctattg 960

agtggtacca gtatgacccc accaagtggt caatctggat ctggaagcag cttggtcttg 1020

cccacaacct gaagcagttc cgccaaaacg agattgagaa gggacgcgtc cagcagctgc 1080

agaagaagct cgaccagaag cgcgccaagc ttgattgggg tattcccttg gagcagcttc 1140

ccgttgttag ctgggatgac tttgttgagc agtccaagaa cggaaaggct tggattgcag 1200

ttgccggtgt catccacgat gttggtgact tcatcaagga ccaccctggt ggcagagctc 1260

tcatcaactc ggccattggc aaggacgcaa ccgcaatctt caacggcggt gtttacaacc 1320

actccaacgc cgctcacaac ctgctctcga ctatgcgtgt gggtgttttg cgtggcggct 1380

gcgaggttga gatctggaag cgcgcccagt ccgaaaacaa ggacgtctca accgtcgttg 1440

attcttcggg taaccgcatc gtccgcgcgg gtgggcaagc gaccaaggtc gtccagcctg 1500

ttccgggtgc tcaggccgcg tga 1523

<210> 13

<211> 1428

<212> DNA

<213>Artificial sequence

<220>

<223>The MgD9Ds clones of intronless

<400> 13

atggcttcgt catcttcctc cgtgccggag ttggctgccg ccttccctga tggcactacc 60

gacttcaagc ccatgaggaa caccaagggc tacgacgtca gcaagccgca catttccgag 120

acacctatga cactcaagaa ctggcataag cacgtcaact ggctcaacac caccttcatc 180

ttgtttgtgc ccctggctgg tctcatatcc acttactggg tccctctgca gtggaagacg 240

gctgtatggg ctgtcgtcta ctacttcaac accggcctgg gaattactgc cggttaccac 300

cgactttggg ctcacagctc gtacaaggcc tcgcttccgc tcaaaatcta ccttgccgcc 360

gttggcgctg gtgccgtcga gggctccatc agatggtggt ccaacggtca ccgcgcacac 420

caccgataca ccgataccga gaaggacccc tactcagtcc gcaagggtct cctgtactca 480

cacatgggat ggatgcttct gaagcagaac cccaagaagc agggccgcac cgacatcacc 540

gacctgaacg aggaccccgt tgtcgtttgg cagcaccgca acttcctcaa gtgtgttatc 600

ttcatggccc tcgtcttccc cacacttgtg gctggccttg gctggggtga ctactgggga 660

ggtttcatct acggaggtat tctgcgtgtc ttcttcgtcc agcaggccac cttctgcgtc 720

aactcgcttg cccactggct cggtgaccag cctttcgacg atcgcaactc gccgcgtgat 780

cacgtcatca cagccctggt cacccttgga gagggatacc acaacttcca ccacgagttc 840

ccttcggact accgcaacgc tattgagtgg taccagtatg accccaccaa gtggtcaatc 900

tggatctgga agcagcttgg tcttgcccac aacctgaagc agttccgcca aaacgagatt 960

gagaagggac gcgtccagca gctgcagaag aagctcgacc agaagcgcgc caagcttgat 1020

tggggtattc ccttggagca gcttcccgtt gttagctggg atgactttgt tgagcagtcc 1080

aagaacggaa aggcttggat tgcagttgcc ggtgtcatcc acgatgttgg tgacttcatc 1140

aaggaccacc ctggtggcag agctctcatc aactcggcca ttggcaagga cgcaaccgca 1200

atcttcaacg gcggtgttta caaccactcc aacgccgctc acaacctgct ctcgactatg 1260

cgtgtgggtg ttttgcgtgg cggctgcgag gttgagatct ggaagcgcgc ccagtccgaa 1320

aacaaggacg tctcaaccgt cgttgattct tcgggtaacc gcatcgtccg cgcgggtggg 1380

caagcgacca aggtcgtcca gcctgttccg ggtgctcagg ccgcgtga 1428

<210> 14

<211> 1997

<212> DNA

<213> Leptosphaeria nodorum

<400> 14

cccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 60

caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 120

tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 180

tgaccatgat tacgccaagc tcgaaattaa ccctcactaa agggaacaaa agctggagct 240

ccaccgcggt ggcggccgct ctagaactag tggatccccc gggctgcagg aattcggcac 300

gagtatgcct tcccaccagg ctgttgctgg catgcaggcc atcgaccccg agtttgtcaa 360

gcagccgtct cctatggcga gcacctcgga gcccaaccgc aactccaagt acgatcctaa 420

gaagccgcac attacagaca tgcccatcac gcggtcaaac tggtaccagc atgtcaactg 480

gctcaacgtc atcttcatca tcggcgtgcc tctcgctggc tgcgtcgccg ccttctggac 540

ccctctgcag tggaagaccg ctgcgtgggc tgtcatctac tatttctgga ctggcctcgg 600

tatcaccgcc ggataccatc gtctctgggc acacaagtca tacaacgccg gtcttcctct 660

gaggatctgg ctcgccgccg tcggcgctgg tgctgttgag ggttccatcc gctggtggag 720

ccgtgaccac cgcgcccacc accgctacac cgacaccaac aaggacccct acagtgtccg 780

caagggcctt ctctacagcc atctcggatg gatggtcatg aagcagaacc ccaagcgtat 840

cggccgcacc gacatcaccg acttgaacga ggaccccgtt gtcgtctggc agcacaagaa 900

ctacatcaag gccgtcgtca ccatgggctt gatctttccc tctgccgtcg ccggtctcat 960

gtggggcgat tggatgggtg gcttcatcta cgctggtatc ctccgtatct tcttcgtcca 1020

gcaggccacc ttctgcgtca actcgcttgc tcactggctc ggtgaccagc ccttcgacga 1080

ccgcaactct cctcgtgacc acgtcattac cgctcttgtc actctcggag agggctacca 1140

caacttccac cacgagttcc cctccgacta ccgcaacgcc atcgagtggc accagtacga 1200

ccctaccaag tggtccatct ggctgtggag caagctcggc ctcgcctcca acctcaagca 1260

gttccgctcc aacgaaatcg agaagggtcg tgtccagcag ctccagaaga agattgacca 1320

gaagcgcgcc aagctcgact ggggtgtccc tctcgaccag ctgcctgtca tagaatggga 1380

cgactatgtc gagcaggcca agaacggccg tggtctcatc gctgtcgctg gtgtcgttca 1440

tgacgttacc gacttcatca acgagcaccc cggtggcaag acgcttatca agagcggcgt 1500

tggcaaggat gccaccgcca tgttcaacgg cggtgtctac ttccactcca acggagccca 1560

caacctcctt tctaccatga gggttggtgt catccgcggt ggctgtgaag ttgagatctg 1620

gaagcgcgct cagcgtgaga acaaggatgt cggtctggtc ctggacgacg caggcaaccc 1680

aatcatcagg gctggtaacc agattaccaa ggttgcgcaa cccattcaga gtgctagtgc 1740

agcatagatt ggatcttcat cttcacgagc gatgtatggc gtttggttgt ctctcttcct 1800

tggcggacag agtaatattc aatttcttag cgatcgttag aaagcatcat ggttacgatg 1860

ctcagtcatg ttagatggcg tatgtttgta gccttcctcg agtgattggs tatgaaaagt 1920

agcctcacgg cctagaccaa gaatgaaaac attcacgatt tcagaaaaaa aaaaaaaaaa 1980

aaactcgagg gggggcc 1997

<210> 15

<211> 1370

<212> DNA

<213> Leptosphaeria nodorum

<400> 15

ggatccatgg cggccttgga cagcattcca gaggataagg ctacctcgtc gaaatcgact 60

catattcaat atcaagaagt aacttttcgg aactggtata agaagataaa ttggctcaac 120

acgacgctgg tggtgctcat acccgctctt ggactctacc taacacgcac cacgccactt 180

acacgaccta cgctcatctg gtccgtcctg tactacttct gcacagcttt cggcatcaca 240

ggcggatatc atcgactatg gagtcatcgc agctactccg ctcgtctacc gctacgctta 300

ttcctagcct tcacaggcgc cggagccatc caaggtagtg ctcgatggtg gagcgcaaat 360

caccgcgccc accaccgatg gaccgacaca atgaaggacc cctactccgt tatgcgcggc 420

ctattattct cgcacatcgg atggatggta ttgaacagcg accccaaagt caaaggccga 480

acagacgtca gtgatctcga cagcgacccc gtcgtagtct ggcagcacaa gcactacggc 540

aagtgcctgc tgttcgccgc gtggatattc cccatgatcg tagccggcct cggatgggga 600

gattggtggg gaggccttgt ctacgccggc atcattcgag cgtgtttcgt ccagcaggcg 660

acattttgcg tgaactctct cgcgcattgg atcggcgagc agccgttcga cgacagacgc 720

acgcctcgag accacgtttt gacagcgttg gtaacgatgg gagaaggata tcataacttc 780

caccacgaat tcccaagcga ttatcgcaac gcgatcatct ggtaccaata cgaccctacc 840

aaatggctca tttacctctt ctccctcggc cccttccccc tcgcatactc gctcaaaacc 900

ttccggtcca atgagattga aaaagggcgg ttgcaacaac aacaaaaagc cctggacaag 960

aagcgctcag gacttgattg gggcctaccc ctcttccaac tccctgtcat atcgtgggac 1020

gacttccaag cgcgttgcaa agagtccggc gagatgctgg ttgctgtcgc aggtgtgatt 1080

cacgacgtca gccagtttat tgaagatcac cctggaggca ggagtttgat tcggagtgcg 1140

gtgggcaaag atgggacagg gatgtttaat ggaggcgtat atgagcacag taatgcggcg 1200

cataatctgt tgtcgacaat gagggtggga gtgcttagag gtgggcagga ggtggaggtg 1260

tggaagaagc agagagtgga tgttttaggg aagagcgaca ttttgagaca ggttacgcgg 1320

gtggagaggt tggttgaggg ggctgtggct gcgtagctaa ctgaccatgg 1370

<210> 16

<211> 1428

<212> DNA

<213> Magnaporthe grisea

<400> 16

atggcttcgt catcttcctc cgtgccggag ttggctgccg ccttccctga tggcactacc 60

gacttcaagc ccatgaggaa caccaagggc tacgacgtca gcaagccgca catttccgag 120

acacctatga cactcaagaa ctggcataag cacgtcaact ggctcaacac caccttcatc 180

ttgtttgtgc ccctggctgg tctcatatcc acttactggg tccctctgca gtggaagacg 240

gctgtatggg ctgtcgtcta ctacttcaac accggcctgg gaattactgc cggttaccac 300

cgactttggg ctcacagctc gtacaaggcc tcgcttccgc tcaaaatcta ccttgccgcc 360

gttggcgctg gtgccgtcga gggctccatc agatggtggt ccaacggtca ccgcgcacac 420

caccgataca ccgataccga gaaggacccc tactcagtcc gcaagggtct cctgtactca 480

cacatgggat ggatgcttct gaagcagaac cccaagaagc agggccgcac cgacatcacc 540

gacctgaacg aggaccccgt tgtcgtttgg cagcaccgca acttcctcaa gtgtgttatc 600

ttcatggccc tcgtcttccc cacacttgtg gctggccttg gctggggtga ctactgggga 660

ggtttcatct acggaggtat tctgcgtgtc ttcttcgtcc agcaggccac cttctgcgtc 720

aactcgcttg cccactggct cggtgaccag cctttcgacg atcgcaactc gccgcgtgat 780

cacgtcatca cagccctggt cacccttgga gagggatacc acaacttcca ccacgagttc 840

ccttcggact accgcaacgc tattgagtgg taccagtatg accccaccaa gtggtcaatc 900

tggatctgga agcagcttgg tcttgcccac aacctgaagc agttccgcca aaacgagatt 960

gagaagggac gcgtccagca gctgcagaag aagctcgacc agaagcgcgc caagcttgat 1020

tggggtattc ccttggagca gcttcccgtt gttagctggg atgactttgt tgagcagtcc 1080

aagaacggaa aggcttggat tgcagttgcc ggtgtcatcc acgatgttgg tgacttcatc 1140

aaggaccacc ctggtggcag agctctcatc aactcggcca ttggcaagga cgcaaccgca 1200

atcttcaacg gcggtgttta caaccactcc aacgccgctc acaacctgct ctcgactatg 1260

cgtgtgggtg ttttgcgtgg cggctgcgag gttgagatct ggaagcgcgc ccagtccgaa 1320

aacaaggacg tctcaaccgt cgttgattct tcgggtaacc gcatcgtccg cgcgggtggg 1380

caagcgacca aggtcgtcca gcctgttccg ggtgctcagg ccgcgtga 1428

<210> 17

<211> 1062

<212> DNA

<213> Helicoverpa zea

<400> 17

atggctccaa atatatcgga ggatgtgaac ggggtgctct tcgagagtga tgcagcgacg 60

ccggacctgg cgctgtccac gccgcctgtg cagaaggctg acaacaggcc caagcaactg 120

gtgtggagga acatactact gttcgcgtat cttcacttag cggctcttta cggaggttat 180

ctgttcctct tctcagctaa atggcagaca gacatatttg cctacatcct gtatgtgatc 240

tccgggcttg gtatcacggc tggagcacat cgcctgtggg cccacaagtc ctacaaagct 300

aaatggcctc tccgagttat cctggtcatc tttaacacag tggcattcca ggatgccgct 360

atggactggg cgcgcgacca ccgcatgcat cacaagtact cggaaaccga tgctgatcct 420

cataatgcga cccgaggatt cttcttctct cacattggct ggctgcttgt caggaaacat 480

cccgacctta aggagaaggg caagggactc gacatgagcg acttacttgc tgaccccatt 540

ctcaggttcc agaaaaaata ctacctgatc ctgatgccct tggcttgctt cgtgatgcct 600

accgtgattc ctgtgtactt ctggggtgaa acctggacca acgcattctt tgtggcggcc 660

atgttccgct acgcgttcat cctaaatgtg acgtggctcg tcaactctgc cgctcacaag 720

tggggagaca agccctacga caaaagcatt aagccttccg aaaacttgtc ggtcgccatg 780

ttcgctctcg gagaaggatt ccacaactac caccacactt tcccttggga ctacaaaact 840

gctgagctgg gcaacaacaa actcaacttc actaccacct ttattaactt cttcgctaaa 900

attggctggg cttacgacct gaagacagtg tctgatgata tcgtcaagaa cagggtgaag 960

cgcactggtg acggctccca ccacctgtgg ggctggggag acgaaaatca atccaaagaa 1020

gaaattgatg ccgctatcag aatcaatcct aaggacgatt aa 1062

<210> 18

<211> 1350

<212> DNA

<213> Leptosphaeria nodorum

<400> 18

atggcggcct tggacagcat tccagaggat aaggctacct cgtcgaaatc gactcatatt 60

caatatcaag aagtaacttt tcggaactgg tataagaaga taaattggct caacacgacg 120

ctggtggtgc tcatacccgc tcttggactc tacctaacac gcaccacgcc acttacacga 180

cctacgctca tctggtccgt cctgtactac ttctgcacag ctttcggcat cacaggcgga 240

tatcatcgac tatggagtca tcgcagctac tccgctcgtc taccgctacg cttattccta 300

gccttcacag gcgccggagc catccaaggt agtgctcgat ggtggagcgc aaatcaccgc 360

gcccaccacc gatggaccga cacaatgaag gacccctact ccgttatgcg cggcctatta 420

ttctcgcaca tcggatggat ggtattgaac agcgacccca aagtcaaagg ccgaacagac 480

gtcagtgatc tcgacagcga ccccgtcgta gtctggcagc acaagcacta cggcaagtgc 540

ctgctgttcg ccgcgtggat attccccatg atcgtagccg gcctcggatg gggagattgg 600

tggggaggcc ttgtctacgc cggcatcatt cgagcgtgtt tcgtccagca ggcgacattt 660

tgcgtgaact ctctcgcgca ttggatcggc gagcagccgt tcgacgacag acgcacgcct 720

cgagaccacg ttttgacagc gttggtaacg atgggagaag gatatcataa cttccaccac 780

gaattcccaa gcgattatcg caacgcgatc atctggtacc aatacgaccc taccaaatgg 840

ctcatttacc tcttctccct cggccccttc cccctcgcat actcgctcaa aaccttccgg 900

tccaatgaga ttgaaaaagg gcggttgcaa caacaacaaa aagccctgga caagaagcgc 960

tcaggacttg attggggcct acccctcttc caactccctg tcatatcgtg ggacgacttc 1020

caagcgcgtt gcaaagagtc cggcgagatg ctggttgctg tcgcaggtgt gattcacgac 1080

gtcagccagt ttattgaaga tcaccctgga ggcaggagtt tgattcggag tgcggtgggc 1140

aaagatggga cagggatgtt taatggaggc gtatatgagc acagtaatgc ggcgcataat 1200

ctgttgtcga caatgagggt gggagtgctt agaggtgggc aggaggtgga ggtgtggaag 1260

aagcagagag tggatgtttt agggaagagc gacattttga gacaggttac gcgggtggag 1320

aggttggttg agggggctgt ggctgcgtag 1350

<210> 19

<211> 1428

<212> DNA

<213>Artificial sequence

<220>

<223>The desaturase sequence of Canola optimization

<400> 19

atggccagca gttcttcaag tgtgccagaa cttgccgcag ctttccctga tgggacaacg 60

gacttcaaac ccatgaggaa caccaaaggc tatgatgtct ccaaacctca catctctgaa 120

acaccgatga ctttgaagaa ctggcacaaa catgtgaact ggctcaacac cacattcatt 180

ctctttgttc cactggctgg gttgatctca acctattggg ttcctcttca atggaaaact 240

gcagtgtggg cagttgtgta ctacttcaac actggacttg ggatcactgc tggctaccat 300

agattgtggg cacattcctc ttacaaggcc agcttgcctc tcaaaatcta ccttgccgca 360

gttggtgctg gagccgttga aggttccata agatggtgga gcaacggaca cagagcacat 420

cacagataca cagacacaga gaaagatcct tactcagtga ggaagggatt gctctacagc 480

cacatgggtt ggatgctctt gaagcagaat ccaaagaagc aagggaggac ggacattact 540

gatctgaatg aggacccagt tgtggtctgg caacatagga actttctcaa gtgtgtgatc 600

ttcatggctt tggtctttcc cacccttgtt gctggcctgg gatggggaga ctactgggga 660

ggtttcatct atggagggat cttgagagtg ttctttgttc agcaagccac cttctgtgtc 720

aactcacttg cacattggct tggtgatcaa ccgtttgatg acagaaactc tccacgtgac 780

catgtcataa ctgctcttgt cacgctgggt gaaggctatc acaactttca ccatgagttt 840

ccgtcagact atagaaatgc gattgagtgg tatcagtatg accccacgaa gtggagcatt 900

tggatttgga agcaacttgg acttgctcac aatctcaagc agttcagaca gaatgagata 960

gagaagggaa gggttcaaca gttgcagaag aaactggatc agaagagagc gaaacttgat 1020

tggggaatac cgttggaaca actccctgtt gtgtcttggg atgactttgt tgaacagtca 1080

aagaatggca aggcatggat tgctgttgct ggtgtcattc acgatgttgg tgacttcatc 1140

aaggatcatc ctggtggacg tgctctcatc aactctgcga ttggcaaaga tgccacagcg 1200

atcttcaatg gaggtgtcta caatcattca aatgccgcac acaaccttct ctccaccatg 1260

agggttggtg tcctccgtgg agggtgcgaa gtggagatat ggaaacgtgc tcaaagtgag 1320

aacaaagatg tctctactgt ggttgatagt tctggcaacc gtattgtgag agctggtgga 1380

caagctacca aagtggttca gccagtccct ggtgctcaag cagcttga 1428

<210> 20

<211> 1062

<212> DNA

<213>Artificial sequence

<220>

<223>The desaturase sequence of Canola optimization

<400> 20

atggctccca acatttctga ggatgtcaat ggtgttcttt ttgagtcaga tgcggcaacc 60

cctgatttgg ctctttccac accacctgtg caaaaagctg acaacagacc caagcaactt 120

gtgtggagga acattttgct tttcgcttac ttgcacctcg cagctctcta cggaggctat 180

ttgtttctct tcagtgcaaa atggcagacc gacattttcg cttacattct ttatgtcatc 240

tctggactgg ggataactgc tggggcacat agactctggg ctcacaagtc atacaaagcc 300

aagtggccac tcagagttat actggtcatc ttcaacacgg ttgcctttca agacgctgct 360

atggattggg ctcgtgacca tagaatgcat cacaagtaca gcgagaccga cgcggaccca 420

cacaatgcaa cgagaggttt cttcttctct cacattggct ggcttcttgt taggaaacat 480

cctgatctga aagaaaaagg gaagggactc gacatgagtg atctccttgc tgatccaata 540

ctccgttttc agaagaagta ctatctgatc ctcatgcctc tggcctgttt tgtgatgcca 600

accgttatcc cggtttactt ttggggagaa acttggacaa atgctttctt cgtggcagcc 660

atgttccgtt atgctttcat cctgaatgtt acctggttgg tgaactctgc cgcacacaag 720

tggggagaca aaccctatga caagtccatc aagccttccg aaaacctttc agttgcgatg 780

tttgctttgg gagaaggatt tcacaattac catcacactt ttccgtggga ctacaagaca 840

gcagagcttg gaaacaacaa gttgaacttc acaacaacgt tcatcaattt ctttgcgaaa 900

atcggttggg cctatgattt gaagactgtg agtgatgaca ttgtcaagaa cagggtcaag 960

agaactggcg atggaagcca tcatctctgg ggctggggtg atgagaatca gagcaaagaa 1020

gagatagatg cagccattag gatcaaccct aaagacgatt ga 1062

<210> 21

<211> 1350

<212> DNA

<213>Artificial sequence

<220>

<223>The desaturase sequence of Canola optimization

<400> 21

atggctgcac ttgatagcat ccctgaggac aaagcaacta gctccaagtc aacccacata 60

cagtaccaag aggtcacgtt taggaactgg tacaagaaaa tcaactggct caacacgacc 120

cttgttgtcc tcattcctgc tcttgggttg tacttgacga gaaccacacc tctcaccaga 180

cctaccctca tttggtctgt tctctactat ttctgtacag cgtttggcat cactggtggc 240

taccacagac tttggtccca taggtcttac agtgcgaggt tgccattgag actcttcctg 300

gctttcactg gagctggtgc gatccaaggt tctgcaagat ggtggtcagc caatcatagg 360

gcacatcacc gttggacgga caccatgaag gacccctact ctgtgatgag aggactgctg 420

ttctcccaca taggttggat ggttctcaac tctgatccaa aggtcaaagg cagaacagat 480

gtttctgatc ttgactctga tcccgtcgtt gtgtggcaac acaaacacta tggcaagtgt 540

ttgctctttg ccgcttggat ctttccgatg atagtggctg ggctgggttg gggagattgg 600

tggggtggac ttgtctatgc tggcatcata cgtgcctgct ttgttcagca agccactttc 660

tgtgtcaact cattggcaca ttggataggt gaacaaccgt ttgatgacag acgtactcca 720

agggatcatg ttctgactgc gttggtcaca atgggagaag gataccacaa cttccaccat 780

gagtttccga gtgactacag aaatgccatc atttggtatc agtatgaccc tacaaagtgg 840

ctcatctatc tcttcagctt gggtcccttc ccattggcct actctctcaa gaccttccgt 900

tccaatgaga ttgagaaagg aaggcttcag caacagcaaa aggctcttga caagaaaaga 960

agtggtcttg attggggact tcctctcttc cagcttccag tgatctcatg ggatgacttt 1020

caagctcgtt gcaaagaaag tggagagatg cttgttgctg ttgctggagt gatccatgat 1080

gtctcccagt tcattgaaga tcatcctggt gggaggagcc tcattagaag tgctgttggg 1140

aaagatggga ctggcatgtt caatggtgga gtgtatgaac attcaaacgc cgcacacaac 1200

ttgctgagca caatgagagt tggagtcttg agaggtggac aagaagtgga ggtttggaag 1260

aaacagaggg tggatgttct tgggaagtca gacattcttc gtcaagtgac aagggtggag 1320

cgtctggtgg aaggagctgt tgcagcgtga 1350

<210> 22

<211> 1350

<212> DNA

<213>Artificial sequence

<220>

<223>The desaturase of Canola optimization

<400> 22

atggctgctc ttgattctat cccagaggat aaggctacct cttctaagtc tacccacatc 60

caataccaag aagttacctt caggaactgg tacaagaaga tcaactggct taacaccacc 120

cttgttgttc ttatcccagc tcttggactt taccttacca ggaccacccc acttaccagg 180

ccaaccctta tctggtctgt tctttactac ttctgcaccg ctttcggaat aaccggagga 240

taccacaggc tttggtctca caggtcttac tctgctaggc ttccacttag gcttttcctt 300

gctttcaccg gagctggagc tatccaagga tctgctagat ggtggtctgc taaccacagg 360

gctcaccaca ggtggaccga taccatgaag gacccatact ctgttatgag gggacttctt 420

ttctctcaca tcggatggat ggttcttaac tctgatccaa aggttaaggg aaggaccgat 480

gtttctgatc ttgattctga tccagttgtt gtttggcaac acaagcacta cggaaagtgc 540

cttcttttcg ctgcttggat cttcccaatg atcgttgctg gacttggatg gggagattgg 600

tggggaggac ttgtttacgc tggaatcatc agggcttgct tcgttcaaca agctaccttc 660

tgcgttaact ctcttgctca ctggatcgga gagcaaccat tcgacgatag gaggacccca 720

agggatcacg ttcttaccgc tcttgttacc atgggagagg gataccacaa cttccaccac 780

gagttcccat ctgattacag gaacgctatc atctggtacc aatacgatcc aaccaagtgg 840

cttatctacc ttttctctct tggaccattc ccacttgctt actctcttaa gaccttcagg 900

tctaacgaga tcgagaaggg aaggcttcaa caacaacaaa aggctcttga taagaagagg 960

tctggacttg attggggact tccacttttc caacttccag ttatctcttg ggatgatttc 1020

caagctaggt gcaaggagtc tggagagatg cttgttgctg ttgctggagt tatccacgat 1080

gtttctcaat tcatcgagga tcacccagga ggaaggtctc ttatcaggtc tgctgttgga 1140

aaggatggaa ccggaatgtt caacggagga gtttacgagc actctaacgc tgctcacaac 1200

cttctttcta ccatgagggt tggagttctt aggggaggac aagaggttga ggtttggaag 1260

aagcaaaggg ttgatgttct tggaaagtca gatatcctta ggcaagttac cagggttgag 1320

aggcttgttg agggagctgt tgctgcttga 1350

<210> 23

<211> 1062

<212> DNA

<213>Artificial sequence

<220>

<223>The desaturase of Canola optimization

<400> 23

atggctccaa acatctctga ggatgttaac ggagttcttt tcgagtctga tgctgctacc 60

ccagatcttg ctctttctac cccaccagtt caaaaggctg ataacaggcc aaagcaactt 120

gtttggagga acatccttct tttcgcttac cttcaccttg ctgctcttta cggaggatac 180

cttttccttt tctctgctaa gtggcaaacc gatatcttcg cttacatcct ttacgttatc 240

tctggacttg gaataaccgc tggagcacac aggctttggg ctcacaagtc ttacaaggct 300

aagtggccac ttagggttat ccttgttatc ttcaacaccg ttgctttcca agacgctgct 360

atggattggg ctagggatca caggatgcac cacaagtact ctgagaccga cgctgatcca 420

cacaacgcta ccaggggatt cttcttctct cacatcggat ggcttcttgt taggaagcac 480

ccagatctta aggagaaggg aaagggactt gatatgtctg atcttcttgc tgatccaatc 540

cttaggttcc aaaagaagta ctaccttatc cttatgccac ttgcttgctt cgttatgcca 600

accgttatcc cagtttactt ctggggagag acctggacca acgctttctt cgttgctgct 660

atgttcaggt acgctttcat ccttaacgtt acctggcttg ttaactctgc tgctcacaag 720

tggggagata agccatacga taagtctatc aagccatctg agaacctttc tgttgctatg 780

ttcgctcttg gagagggatt ccacaactac caccacacct tcccatggga ttacaagacc 840

gctgagcttg gaaacaacaa gcttaacttc accaccacct tcatcaactt cttcgctaag 900

atcggatggg cttacgatct taagaccgtt tctgatgata tcgttaagaa cagggttaag 960

aggaccggag atggatcaca ccacctttgg ggatggggag atgagaacca atctaaggag 1020

gagatcgatg ctgctatcag gatcaaccca aaggatgatt ga 1062

<210> 24

<211> 1368

<212> DNA

<213>Aspergillus nidulans

<400> 24

atgtctgctc caaccgctga catcagggct agggctccag aggctaagaa ggttcacatc 60

gctgataccg ctatcaacag gcacaattgg tacaagcacg tgaactggct caacgtcttc 120

ctcatcatcg gaatcccact ctacggatgc atccaagctt tctgggttcc acttcaactc 180

aagaccgcta tctgggctgt gatctactac ttcttcaccg gacttggaat caccgctgga 240

taccacaggc tttgggctca ctgctcttac tctgctactc ttccacttag gatctggctt 300

gctgctgttg gaggaggagc tgttgaggga tctatcagat ggtgggctag ggatcacagg 360

gctcatcata ggtacaccga taccgacaag gacccatact ctgttaggaa gggacttctc 420

tactctcacc ttggatggat ggtgatgaag cagaacccaa agaggatcgg aaggaccgac 480

atctctgatc tcaacgagga cccagttgtt gtttggcaac acaggaacta cctcaaggtt 540

gtgttcacca tgggacttgc tgttccaatg cttgttgctg gacttggatg gggagattgg 600

cttggaggat tcgtgtacgc tggaatcctt aggatcttct tcgttcaaca agctaccttc 660

tgcgtgaact ctcttgctca ctggcttgga gatcaaccat tcgatgatag gaactctcct 720

agggatcacg tgatcaccgc tcttgttacc cttggagagg gataccacaa cttccaccac 780

gagttcccat ctgactacag gaacgctatc gagtggcacc agtacgatcc taccaagtgg 840

tctatctggg cttggaagca acttggattg gcttacgatc tcaagaagtt cagggctaac 900

gagatcgaga agggaagggt tcaacaactt cagaagaagc ttgataggaa gagggctact 960

cttgattggg gaaccccact tgatcaactt ccagtgatgg aatgggatga ctacgttgag 1020

caagctaaga acggaagggg acttgttgct atcgctggag ttgttcacga tgttaccgac 1080

ttcatcaagg atcacccagg aggaaaggct atgatctctt ctggaatcgg aaaggatgct 1140

accgctatgt tcaacggagg agtgtactac cactctaacg cagctcacaa ccttcttagc 1200

accatgaggg tgggagtgat caggggagga tgcgaggttg agatctggaa gagggctcag 1260

aaggagaacg ttgagtacgt tagggatgga tctggacaaa gggtgatcag ggctggagag 1320

caaccaacca agatcccaga gccaatccca accgctgatg ctgcttga 1368

<210> 25

<211> 1368

<212> DNA

<213>Artificial sequence

<220>

<223>AnD9DS v3 silent mutants

<400> 25

atgtctgctc caaccgctga catcagggct agggctccag aggctaagaa ggttcacatc 60

gctgataccg ctatcaacag gcacaattgg tacaagcacg tgaactggct caacgtcttc 120

ctcatcatcg gaatcccact ctacggatgc atccaagctt tctgggttcc acttcaactc 180

aagaccgcta tctgggctgt gatctactac ttcttcaccg gacttggaat caccgctgga 240

taccacaggc tttgggctca ctgctcatac tctgctactc ttccacttag gatctggctt 300

gctgctgttg gaggaggagc tgttgaggga tctatcagat ggtgggctag ggatcacagg 360

gctcatcata ggtacaccga taccgacaag gacccatact ctgttaggaa gggacttctc 420

tactctcacc ttggatggat ggtgatgaag cagaacccaa agaggatcgg aaggaccgac 480

atctctgatc tcaacgagga cccagttgtt gtttggcaac acaggaacta cctcaaggtt 540

gtgttcacca tgggacttgc tgttccaatg cttgttgctg gacttggatg gggagattgg 600

cttggaggat tcgtgtacgc tggaatcctt aggatcttct tcgttcaaca agctaccttc 660

tgcgtgaact ctcttgctca ctggcttgga gatcaaccat tcgatgatag gaactctcct 720

agggatcacg tgatcaccgc tcttgttacc cttggagagg gataccacaa cttccaccac 780

gagttcccat ctgactacag gaacgctatc gagtggcacc agtacgatcc taccaagtgg 840

tctatctggg cttggaagca acttggattg gcttacgatc tcaagaagtt cagggctaac 900

gagatcgaga agggaagggt tcaacaactt cagaagaagc ttgataggaa gagggctact 960

cttgattggg gaaccccact tgatcaactt ccagtgatgg aatgggatga ctacgttgag 1020

caagctaaga acggaagggg acttgttgct atcgctggag ttgttcacga tgttaccgac 1080

ttcatcaagg atcacccagg aggaaaggct atgatctctt ctggaatcgg aaaggatgct 1140

accgctatgt tcaacggagg agtgtactac cactctaacg cagctcacaa ccttcttagc 1200

accatgaggg tgggagtgat caggggagga tgcgaggttg agatctggaa gagggctcag 1260

aaggagaacg ttgagtacgt tagggatgga tctggacaaa gggtgatcag ggctggagag 1320

caaccaacca agatcccaga gccaatccca accgctgatg ctgcttga 1368

<210> 26

<211> 475

<212> PRT

<213> Magnaporthe grisea

<400> 26

Met Ala Ser Ser Ser Ser Ser Val Pro Glu Leu Ala Ala Ala Phe Pro

1 5 10 15

Asp Gly Thr Thr Asp Phe Lys Pro Met Arg Asn Thr Lys Gly Tyr Asp

20 25 30

Val Ser Lys Pro His Ile Ser Glu Thr Pro Met Thr Leu Lys Asn Trp

35 40 45

His Lys His Val Asn Trp Leu Asn Thr Thr Phe Ile Leu Phe Val Pro

50 55 60

Leu Ala Gly Leu Ile Ser Thr Tyr Trp Val Pro Leu Gln Trp Lys Thr

65 70 75 80

Ala Val Trp Ala Val Val Tyr Tyr Phe Asn Thr Gly Leu Gly Ile Thr

85 90 95

Ala Gly Tyr His Arg Leu Trp Ala His Ser Ser Tyr Lys Ala Ser Leu

100 105 110

Pro Leu Lys Ile Tyr Leu Ala Ala Val Gly Ala Gly Ala Val Glu Gly

115 120 125

Ser Ile Arg Trp Trp Ser Asn Gly His Arg Ala His His Arg Tyr Thr

130 135 140

Asp Thr Glu Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser

145 150 155 160

His Met Gly Trp Met Leu Leu Lys Gln Asn Pro Lys Lys Gln Gly Arg

165 170 175

Thr Asp Ile Thr Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His

180 185 190

Arg Asn Phe Leu Lys Cys Val Ile Phe Met Ala Leu Val Phe Pro Thr

195 200 205

Leu Val Ala Gly Leu Gly Trp Gly Asp Tyr Trp Gly Gly Phe Ile Tyr

210 215 220

Gly Gly Ile Leu Arg Val Phe Phe Val Gln Gln Ala Thr Phe Cys Val

225 230 235 240

Asn Ser Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn

245 250 255

Ser Pro Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly

260 265 270

Tyr His Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile

275 280 285

Glu Trp Tyr Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ile Trp Lys

290 295 300

Gln Leu Gly Leu Ala His Asn Leu Lys Gln Phe Arg Gln Asn Glu Ile

305 310 315 320

Glu Lys Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Gln Lys Arg

325 330 335

Ala Lys Leu Asp Trp Gly Ile Pro Leu Glu Gln Leu Pro Val Val Ser

340 345 350

Trp Asp Asp Phe Val Glu Gln Ser Lys Asn Gly Lys Ala Trp Ile Ala

355 360 365

Val Ala Gly Val Ile His Asp Val Gly Asp Phe Ile Lys Asp His Pro

370 375 380

Gly Gly Arg Ala Leu Ile Asn Ser Ala Ile Gly Lys Asp Ala Thr Ala

385 390 395 400

Ile Phe Asn Gly Gly Val Tyr Asn His Ser Asn Ala Ala His Asn Leu

405 410 415

Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Cys Glu Val Glu

420 425 430

Ile Trp Lys Arg Ala Gln Ser Glu Asn Lys Asp Val Ser Thr Val Val

435 440 445

Asp Ser Ser Gly Asn Arg Ile Val Arg Ala Gly Gly Gln Ala Thr Lys

450 455 460

Val Val Gln Pro Val Pro Gly Ala Gln Ala Ala

465 470 475

<210> 27

<211> 353

<212> PRT

<213> Helicoverpa zea

<400> 27

Met Ala Pro Asn Ile Ser Glu Asp Val Asn Gly Val Leu Phe Glu Ser

1 5 10 15

Asp Ala Ala Thr Pro Asp Leu Ala Leu Ser Thr Pro Pro Val Gln Lys

20 25 30

Ala Asp Asn Arg Pro Lys Gln Leu Val Trp Arg Asn Ile Leu Leu Phe

35 40 45

Ala Tyr Leu His Leu Ala Ala Leu Tyr Gly Gly Tyr Leu Phe Leu Phe

50 55 60

Ser Ala Lys Trp Gln Thr Asp Ile Phe Ala Tyr Ile Leu Tyr Val Ile

65 70 75 80

Ser Gly Leu Gly Ile Thr Ala Gly Ala His Arg Leu Trp Ala His Lys

85 90 95

Ser Tyr Lys Ala Lys Trp Pro Leu Arg Val Ile Leu Val Ile Phe Asn

100 105 110

Thr Val Ala Phe Gln Asp Ala Ala Met Asp Trp Ala Arg Asp His Arg

115 120 125

Met His His Lys Tyr Ser Glu Thr Asp Ala Asp Pro His Asn Ala Thr

130 135 140

Arg Gly Phe Phe Phe Ser His Ile Gly Trp Leu Leu Val Arg Lys His

145 150 155 160

Pro Asp Leu Lys Glu Lys Gly Lys Gly Leu Asp Met Ser Asp Leu Leu

165 170 175

Ala Asp Pro Ile Leu Arg Phe Gln Lys Lys Tyr Tyr Leu Ile Leu Met

180 185 190

Pro Leu Ala Cys Phe Val Met Pro Thr Val Ile Pro Val Tyr Phe Trp

195 200 205

Gly Glu Thr Trp Thr Asn Ala Phe Phe Val Ala Ala Met Phe Arg Tyr

210 215 220

Ala Phe Ile Leu Asn Val Thr Trp Leu Val Asn Ser Ala Ala His Lys

225 230 235 240

Trp Gly Asp Lys Pro Tyr Asp Lys Ser Ile Lys Pro Ser Glu Asn Leu

245 250 255

Ser Val Ala Met Phe Ala Leu Gly Glu Gly Phe His Asn Tyr His His

260 265 270

Thr Phe Pro Trp Asp Tyr Lys Thr Ala Glu Leu Gly Asn Asn Lys Leu

275 280 285

Asn Phe Thr Thr Thr Phe Ile Asn Phe Phe Ala Lys Ile Gly Trp Ala

290 295 300

Tyr Asp Leu Lys Thr Val Ser Asp Asp Ile Val Lys Asn Arg Val Lys

305 310 315 320

Arg Thr Gly Asp Gly Ser His His Leu Trp Gly Trp Gly Asp Glu Asn

325 330 335

Gln Ser Lys Glu Glu Ile Asp Ala Ala Ile Arg Ile Asn Pro Lys Asp

340 345 350

Asp

<210> 28

<211> 449

<212> PRT

<213> Leptosphaeria nodorum

<400> 28

Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys

1 5 10 15

Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys

20 25 30

Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu

35 40 45

Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile

50 55 60

Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly

65 70 75 80

Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu

85 90 95

Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala

100 105 110

Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr

115 120 125

Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile

130 135 140

Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp

145 150 155 160

Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His

165 170 175

Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val

180 185 190

Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly

195 200 205

Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser

210 215 220

Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro

225 230 235 240

Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His

245 250 255

Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp

260 265 270

Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly

275 280 285

Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile

290 295 300

Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg

305 310 315 320

Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser

325 330 335

Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val

340 345 350

Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His

355 360 365

Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr

370 375 380

Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn

385 390 395 400

Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val

405 410 415

Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile

420 425 430

Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala

435 440 445

Ala

<210> 29

<211> 455

<212> PRT

<213>Aspergillus nidulans

<400> 29

Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys

1 5 10 15

Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys

20 25 30

His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr

35 40 45

Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile

50 55 60

Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly

65 70 75 80

Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu

85 90 95

Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile

100 105 110

Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr

115 120 125

Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu

130 135 140

Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp

145 150 155 160

Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn

165 170 175

Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val

180 185 190

Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly

195 200 205

Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser

210 215 220

Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro

225 230 235 240

Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His

245 250 255

Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp

260 265 270

His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu

275 280 285

Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys

290 295 300

Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr

305 310 315 320

Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp

325 330 335

Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala

340 345 350

Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly

355 360 365

Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe

370 375 380

Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser

385 390 395 400

Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp

405 410 415

Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly

420 425 430

Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro

435 440 445

Ile Pro Thr Ala Asp Ala Ala

450 455

<210> 30

<211> 455

<212> PRT

<213>Aspergillus nidulans

<400> 30

Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys

1 5 10 15

Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys

20 25 30

His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr

35 40 45

Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile

50 55 60

Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly

65 70 75 80

Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu

85 90 95

Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile

100 105 110

Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr

115 120 125

Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu

130 135 140

Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp

145 150 155 160

Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn

165 170 175

Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val

180 185 190

Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly

195 200 205

Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser

210 215 220

Leu Ala Leu Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro

225 230 235 240

Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His

245 250 255

Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp

260 265 270

His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu

275 280 285

Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys

290 295 300

Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr

305 310 315 320

Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp

325 330 335

Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala

340 345 350

Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly

355 360 365

Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe

370 375 380

Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser

385 390 395 400

Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp

405 410 415

Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly

420 425 430

Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro

435 440 445

Ile Pro Thr Ala Asp Ala Ala

450 455

<210> 31

<211> 510

<212> PRT

<213> Saccharomyces cerevisiae

<400> 31

Met Pro Thr Ser Gly Thr Thr Ile Glu Leu Ile Asp Asp Gln Phe Pro

1 5 10 15

Lys Asp Asp Ser Ala Ser Ser Gly Ile Val Asp Glu Val Asp Leu Thr

20 25 30

Glu Ala Asn Ile Leu Ala Thr Gly Leu Asn Lys Lys Ala Pro Arg Ile

35 40 45

Val Asn Gly Phe Gly Ser Leu Met Gly Ser Lys Glu Met Val Ser Val

50 55 60

Glu Phe Asp Lys Lys Gly Asn Glu Lys Lys Ser Asn Leu Asp Arg Leu

65 70 75 80

Leu Glu Lys Asp Asn Gln Glu Lys Glu Glu Ala Lys Thr Lys Ile His

85 90 95

Ile Ser Glu Gln Pro Trp Thr Leu Asn Asn Trp His Gln His Leu Asn

100 105 110

Trp Leu Asn Met Val Leu Val Cys Gly Met Pro Met Ile Gly Trp Tyr

115 120 125

Phe Ala Leu Ser Gly Lys Val Pro Leu His Leu Asn Val Phe Leu Phe

130 135 140

Ser Val Phe Tyr Tyr Ala Val Gly Gly Val Ser Ile Thr Ala Gly Tyr

145 150 155 160

His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala His Trp Pro Leu Arg

165 170 175

Leu Phe Tyr Ala Ile Phe Gly Cys Ala Ser Val Glu Gly Ser Ala Lys

180 185 190

Trp Trp Gly His Ser His Arg Ile His His Arg Tyr Thr Asp Thr Leu

195 200 205

Arg Asp Pro Tyr Asp Ala Arg Arg Gly Leu Trp Tyr Ser His Met Gly

210 215 220

Trp Met Leu Leu Lys Pro Asn Pro Lys Tyr Lys Ala Arg Ala Asp Ile

225 230 235 240

Thr Asp Met Thr Asp Asp Trp Thr Ile Arg Phe Gln His Arg His Tyr

245 250 255

Ile Leu Leu Met Leu Leu Thr Ala Phe Val Ile Pro Thr Leu Ile Cys

260 265 270

Gly Tyr Phe Phe Asn Asp Tyr Met Gly Gly Leu Ile Tyr Ala Gly Phe

275 280 285

Ile Arg Val Phe Val Ile Gln Gln Ala Thr Phe Cys Ile Asn Ser Met

290 295 300

Ala His Tyr Ile Gly Thr Gln Pro Phe Asp Asp Arg Arg Thr Pro Arg

305 310 315 320

Asp Asn Trp Ile Thr Ala Ile Val Thr Phe Gly Glu Gly Tyr His Asn

325 330 335

Phe His His Glu Phe Pro Thr Asp Tyr Arg Asn Ala Ile Lys Trp Tyr

340 345 350

Gln Tyr Asp Pro Thr Lys Val Ile Ile Tyr Leu Thr Ser Leu Val Gly

355 360 365

Leu Ala Tyr Asp Leu Lys Lys Phe Ser Gln Asn Ala Ile Glu Glu Ala

370 375 380

Leu Ile Gln Gln Glu Gln Lys Lys Ile Asn Lys Lys Lys Ala Lys Ile

385 390 395 400

Asn Trp Gly Pro Val Leu Thr Asp Leu Pro Met Trp Asp Lys Gln Thr

405 410 415

Phe Leu Ala Lys Ser Lys Glu Asn Lys Gly Leu Val Ile Ile Ser Gly

420 425 430

Ile Val His Asp Val Ser Gly Tyr Ile Ser Glu His Pro Gly Gly Glu

435 440 445

Thr Leu Ile Lys Thr Ala Leu Gly Lys Asp Ala Thr Lys Ala Phe Ser

450 455 460

Gly Gly Val Tyr Arg His Ser Asn Ala Ala Gln Asn Val Leu Ala Asp

465 470 475 480

Met Arg Val Ala Val Ile Lys Glu Ser Lys Asn Ser Ala Ile Arg Met

485 490 495

Ala Ser Lys Arg Gly Glu Ile Tyr Glu Thr Gly Lys Phe Phe

500 505 510

<210> 32

<211> 68

<212> PRT

<213>Artificial sequence

<220>

<223>AnD9DS N-terminal residue 1-68

<400> 32

Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys

1 5 10 15

Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys

20 25 30

His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr

35 40 45

Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile

50 55 60

Trp Ala Val Ile

65

<210> 33

<211> 175

<212> PRT

<213>Artificial sequence

<220>

<223> C-terminal residues 281-455 of AnD9DS

<400> 33

Ser Ile Trp Ala Trp Lys Gln Leu Gly Leu Ala Tyr Asp Leu Lys Lys

1 5 10 15

Phe Arg Ala Asn Glu Ile Glu Lys Gly Arg Val Gln Gln Leu Gln Lys

20 25 30

Lys Leu Asp Arg Lys Arg Ala Thr Leu Asp Trp Gly Thr Pro Leu Asp

35 40 45

Gln Leu Pro Val Met Glu Trp Asp Asp Tyr Val Glu Gln Ala Lys Asn

50 55 60

Gly Arg Gly Leu Val Ala Ile Ala Gly Val Val His Asp Val Thr Asp

65 70 75 80

Phe Ile Lys Asp His Pro Gly Gly Lys Ala Met Ile Ser Ser Gly Ile

85 90 95

Gly Lys Asp Ala Thr Ala Met Phe Asn Gly Gly Val Tyr Tyr His Ser

100 105 110

Asn Ala Ala His Asn Leu Leu Ser Thr Met Arg Val Gly Val Ile Arg

115 120 125

Gly Gly Cys Glu Val Glu Ile Trp Lys Arg Ala Gln Lys Glu Asn Val

130 135 140

Glu Tyr Val Arg Asp Gly Ser Gly Gln Arg Val Ile Arg Ala Gly Glu

145 150 155 160

Gln Pro Thr Lys Ile Pro Glu Pro Ile Pro Thr Ala Asp Ala Ala

165 170 175

Claims (28)

1. a kind of nucleic acid molecules of the separation of the desaturase of coded delta -9, it is included and SEQ ID NO:2 at least 80% identicals Amino acid sequence.

2. the nucleic acid molecules of claim 1, also comprising at least one gene regulatory elements.

3. the nucleic acid molecules of claim 2, wherein the gene regulatory elements are selected from the group：The desaturase of saccharomyces cerevisiae Δ -9 is opened Mover, the desaturase 3'UTR/ of Δ -9 terminators, ole1 gene promoters, Kidney bean (Phaseolus vulgaris) Phaseolin Promoter, bean phaseolin 5' non-translational regions, bean phaseolin 3' non-translational regions, bean phaseolin matrix attachment regions, Lesquerella fendleri KCS3 promoters, Agrobacterium tumefaciens mannopine synthase promoter, Agrobacterium tumefaciens ORF23 3' non-translational regions, cassava vein mosaic virus promoters, Agrobacterium tumefaciens ORF1 3' non-translational regions, tobacco RB7 bases Matter attachment region, overdrives (Overdrive), T- chains border sequence, LfKCS3 promoters, the promoters of FAE 1, Myc labels and blood Solidifying element label.

4. a kind of construct, includes the first nucleic acid molecules as claimed in claim 3 and the second nucleic acid as claimed in claim 3 Molecule, wherein first nucleic acid molecules also include the first gene regulatory elements, second nucleic acid molecules include the second gene Controlling element.

5. the construct of claim 4, wherein the described first or second nucleic acid molecules contain at least two gene regulatory elements.

6. a kind of method for reducing the amount of saturated fatty acid in cell, methods described includes：

With the nucleic acid molecules transformed cells of claim 1 so that the amount of the saturated fatty acid in cell is reduced.

7. a kind of method for reducing the amount of saturated fatty acid in cell, methods described includes：

With the construct transformed cells of claim 5 so that the amount of the saturated fatty acid in cell is reduced.

8. method according to claim 7, wherein the cell is yeast cells.

9. the method according to claim 7 or 8, wherein the cell is plant cell.

10. method according to claim 9, including convert the plant cell with the nucleic acid molecules of more than one claim 1.

11. method according to claim 9, wherein the conversion to plant cell will be used to reduce by 16 in plant cell:0- The means introduced plant cell of CoA levels.

12. method according to claim 11, wherein described be used to reduce by 16 in plant cell:The means of 0-CoA levels It is the outer desaturase of plastid.

13. the method for claim 12, wherein the outer desaturase of the plastid is the desaturase being selected from the group：LnD9DS goes to satisfy And enzyme, AnD9DS desaturases, HzD9DS desaturases, and MgD9DS desaturases.

14. the outer desaturase of the method for claim 12, wherein plastid is AnD9DS desaturases.

15. method according to claim 9, wherein the plant cell is obtained from the plant being selected from the group：Arabidopsis (Arabidopsis), Common Borage category (Borago), Canola, Ricinus (Ricinus), Theobroma (Theobroma), corn Belong to (Zea), Gossypium (Gossypium), two section shepherd's purse category (Crambe), sepal distance flower spp (Cuphea), linum (Linum), small shore Chrysanthemum (Lesquerella), whale oil grass category (Limnanthes), Linola, Nasturtium (Tropaeolum), Oenothera (Oenothera), wooden slippers olive category (Olea), oil palm category (Elaeis), Arachis (Arachis), rapeseed, safflower category (Carthamus), Glycine (Glycine), wild soybean category (Soja), Helianthus (Helianthus), Nicotiana (Nicotiana), Vernonia (Vernonia), Triticum (Triticum), Hordeum (Hordeum), Oryza (Oryza), Other members of Avena (Avena), sorghum (Sorghum), Secale (Secale), and grass family (Gramineae).

16. a kind of oily seed plant, it includes the nucleotide sequence of claim 1.

17. a kind of oily seed plant, it includes the construct described in claim 5 or 6.

18. a kind of vegetable seeds, it expresses the outer desaturase of the plastid being selected from the group：SEQ ID NO:2,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,SEQ ID NO:25,SEQ ID NO:26,SEQ ID NO:27, or SEQ ID NO:28.

19. the seed of transgenosis colea system, the seed relative to the transgenosis colea system etc. genotype version have There is the saturated fatty acid level of reduction.

20. a kind of be used to create the genetic engineering plant compared with wild-type plant with the amount of saturated fatty acid in reduced plant The method of thing, methods described includes：

Vegetable material is converted with the nucleic acid molecules of claim 1；With

Inverted vegetable material is cultivated to obtain plant.

21. a kind of be used to create the genetic engineering plant compared with wild-type plant with the amount of saturated fatty acid in reduced plant The method of thing, methods described includes：

Vegetable material is converted with the construct of claim 4；With

Inverted vegetable material is cultivated to obtain plant.

22. the method described in claim 21, wherein the plant is selected from the group：Arabidopsis, Common Borage category, Canola, castor Fiber crops category, Theobroma, Zea, Gossypium, two section shepherd's purse category, sepal distance flower spp, linum, small shore Chrysanthemum, whale oil grass category, Linola, drought Lily feet belongs to, Oenothera, wooden slippers olive category, oil palm category, Arachis, rapeseed, safflower category, Glycine, wild soybean category, sunflower Category, Nicotiana, Vernonia, Triticum, Hordeum, Oryza, Avena, sorghum, Secale, and it is gramineous it is other into Member.

23. the plant that a kind of method by described in claim 21 is obtained.

24. a kind of vegetable material obtained from the plant of claim 23.

25. the vegetable material of claim 23, wherein the vegetable material is seed.

26. the oil of the plant from claim 23.

27. the oil of the plant from claim 23, wherein the oil contains the saturated fatty acid less than 3.5%.

28. the oil of the plant from claim 23, wherein the oil contains the saturated fatty acid less than 3.0%.