CN108949807A - A method of improving plant polyunsaturated fatty acid yield - Google Patents

Abstract

The present invention relates to a kind of methods for improving plant polyunsaturated fatty acid yield.Inventor's discovery takes part in FAD2 positive-sense strand and is overexpressed the gene for causing gene silencing, is based on the discovery, and the present invention improves the polyunsaturated fatty acid in plant by gene found in knockout or silenced plant.The present invention is being cultivated rich in having significant application value in polyunsaturated fatty acid plant and crops, is used especially for cultivating production high added value oil crops.

Description

A method of improving plant polyunsaturated fatty acid yield

Technical field

The present invention relates in the multiple genic identifications and utilization more particularly to plant that participate in FAD2 transgene silencing The improvement method of polyunsaturated fatty acid.

Background technique

Grease is important one of the reserve substance of plant, and main component is triacylglycerol.Fatty acyl on glycerol backbone, There is different physicochemical properties due to carbochain length and the difference of unsaturated bond position.Typically simply use carbon atom number: How much double key number (X:Y) indicates fatty acid chain length and unsaturated bond, and such as most common fatty acid has palmitinic acid (hexadecanoic acid, be abbreviated as 16:0), stearic acid (octadecanoic acid, be abbreviated as 18:0), oleic acid (9- Octadecenoic acid, (9Z)-, cis- Δ 9octadecenoic acid, cis- 18 carbon, one alkene [9] acid are abbreviated as 18:1), linoleic acid (9,12-Octadecadienoic acid, (9Z, 12Z)-, cis, cis- Δ⁹,Δ¹²Octadecadienoic acid, cis--ten eight carbon diene [9,12] acid, is abbreviated as 18:2) and linolenic acid (9,12,15- Octadecatrienoic acid, (9Z, 12Z, 15Z)-, Δ⁹,Δ¹²,Δ¹⁵Octadecatrienoic acids, cis- ten Eight carbon triolefins [9,12,15] acid, be abbreviated as 18:3) etc. types.

Polyunsaturated fatty acid is the key component for maintaining cell membrane system mobility.Due in people and other mammals Polyunsaturated fatty acid cannot be synthesized, it is necessary to absorb from food.Therefore, polyunsaturated fatty acid to the mankind and domestic animal also by for Necessary fatty acid, intake are rich in the food of more saturated fatty acids, have extremely important meaning for the mankind and domestic animal.In plant In grease storage organ, polyunsaturated fatty acid, which mainly passes through eukaryon approach, to be completed.During oil and fat accumulation, FAD2 Oleic acid acyl group on (Fatty acid desaturase 2) major catalytic lecithin (phosphatidylchline) forms Asia Oleic acid acyl group.Linoleic acid again can be raw in 3 formation double bonds of ω under the action of FAD3 (Fatty acid desaturase 3) At alpha-linolenic acid (Ohlrogge and Browse, 1995).Since the function of arabidopsis FAD2 and FAD3 are identified, very Also carried out the research to them on more plants, identification including gene function and by genetic engineering improved seed fatty acid at Point.Being overexpressed BnFAD3 can successfully promote plant to generate more linolenic acids in Seed development.In seed specific Under promoter driving, the overexpression in arabidopsis is that linolenic acid content can be increased to 28.3- from 19.7% by BnFAD3 35.3% (Cartea et al.1998)；Since linoleic acid is the substrate that linolenic acid is formed, only just may be used by improving linoleic acid It can be further improved linolenic content.However, vegetable seeds Linoleic acid cannot but be effectively improved by being overexpressed AtFAD2 Content.In the research of above-mentioned expression BnFAD3, AtFAD2 is also overexpressed using identical promoter, in 41 transgenic lines In, only 4 T2 seed linoleic acid contents have obvious raising, and increase rate is less than 4%.The about table of half overexpression strain Type is similar to antisense strand strain, shows linoleic acid reduction and oleic acid increases, oleic acid highest runs up to 1.8 times of wild type (Cartea et al.,1998).In all documents that inventor collected, maximum increase rate is in arabidopsis seed Specifically expressing manioca JcFAD2, transgenosis system Linoleic acid content only increase to 34.7-36.3% from the 31.6% of wild type (Wu et al., 2013).

Summary of the invention

SEQ ID NO.8 encodes FAD2 enzyme.Inventor the study found that be overexpressed AtFAD2 be not only difficult to improve it is linoleic Content usually will cause the accumulation of FAD2 product linoleic reduction and its substrate oleic acid in most transgenic lines. In extreme strain, mutant of the fatty acid phenotype similar to FAD2 gene.In only a few strain, linoleic acid also only has very little amplitude Raising (Fig. 1).

Further study show that being overexpressed the positive-sense strand of the code area AtFAD2, cause in most of strain endogenous and outer Source is transferred to the gene silencing (hereinafter referred FAD2 transgene silencing) of FAD2, the total expression of FAD2 and its product how unsaturated rouge The ratio of fat acid and substrate oleic acid is closely related (attached drawing 2).In the root of constitutive expression AtFAD2, it has been found that FAD2 turns Gene silencing (attached drawing 3).

These results indicate that transgene silencing can all be caused in the different tissues of plant by being overexpressed FAD2, and this The ratio that gene silencing occurs is high, amplitude is big.

Further, inventor, which studies, finds that following gene takes part in gene silencing caused by FAD2 is overexpressed, and utilizes These mutant, the content that FAD2 co-suppression can be released, improve polyunsaturated fatty acid:

6 (RDR6) albumen of sequential coding RNA-Dependent RNA Polymerase shown in SEQ ID NO.1, at this It is overexpressed AtFAD2 in the mutant of albumen, in whole strains, FAD2 transgene silencing phenomenon is released from, it is determined that RDR6 ginseng With FAD2 transgene silencing；It is overexpressed FAD2 in rdr6, the content of linoleic acid plus linolenic acid can be improved 10% or more, surpassed The amplitude (attached drawing 4) of all correlative studys before crossing.Further analysis is overexpressed the strain of FAD2 under rdr6 mutant background, Polyunsaturated fatty acid improve strain in, the total expression of FAD2 can increase substantially, amplitude can be up to ten times with Upper (attached drawing 5).

3 (SGS3) albumen of sequential coding SUPPRESSOR OF GENE SILENCING shown in SEQ ID NO.2, at this It is overexpressed AtFAD2 in the mutant of albumen, in whole strains, polyunsaturated fatty acid is significantly accumulated, and FAD2 transgenosis is heavy Silent phenomenon is released from (attached drawing 6), it is determined that SGS3 participates in FAD2 transgene silencing；It can use sgs3 mutant and cross table in FAD2 Up to the middle content for improving polyunsaturated fatty acid.

Sequential coding Dicer Like 2 (DCL2) albumen shown in SEQ ID NO.3 crosses table in the mutant of the albumen Up to AtFAD2, in the strain of part, polyunsaturated fatty acid rises, and FAD2 transgene silencing phenomenon is released from, it is determined that DCL2 It participates in FAD2 transgene silencing (attached drawing 7).

Sequential coding Dicer Like 4 (DCL4) albumen shown in SEQ ID NO.4 crosses table in the mutant of the albumen Up to AtFAD2, in small part strain, polyunsaturated fatty acid rises, and FAD2 transgene silencing phenomenon is released from, it is determined that DCL4 participates in FAD2 transgene silencing (attached drawing 8).

Sequential coding DCL2 and DCL4 albumen shown in SEQ ID NO.3 and SEQ ID NO.4, in the double prominent of two kinds of albumen AtFAD2 is overexpressed in variant, almost in all strains, polyunsaturated fatty acid is obviously increased, FAD2 transgene silencing phenomenon It is released from, it is determined that DCL2 and DCL4 funtion part overlapping in participating in FAD2 transgene silencing, while the double-mutant knocked out, Very effective it can release above-mentioned transgene silencing (attached drawing 9)；It is overexpressed FAD2 under dcl2dcl4 double-mutant background, ten The ratio highest of eight carbon polyunsaturated fatty acids and saturation monounsaturated fatty acids can be improved about 2 times.

2 (MOS2) albumen of sequential coding MODIFIER OF SNC1 shown in SEQ ID NO.5, in the mutant of the albumen Middle overexpression AtFAD2, in whole strains, polyunsaturated fatty acid is obviously increased, and FAD2 transgene silencing phenomenon is released from, It has been determined that MOS2 participates in FAD2 transgene silencing (attached drawing 10).

Sequential coding Dicer Like 1 (DCL1) albumen shown in SEQ ID NO.6 crosses table in the mutant of the albumen Up to AtFAD2, in most of strain, polyunsaturated fatty acid is obviously increased, and FAD2 transgene silencing phenomenon is released from, and is determined DCL1 participates in FAD2 transgene silencing (attached drawing 11).FAD2,18 carbon how unsaturateds are overexpressed under dcl1 mutant background Content of fatty acid highest can be increased to 60% from 40%.

Sequential coding ARGONAUTE 1 (AGO1) albumen, is overexpressed in the mutant of the albumen shown in SEQ ID NO.7 AtFAD2, in most of strain, polyunsaturated fatty acid is obviously increased, and FAD2 transgene silencing phenomenon is released from, it is determined that AGO1 participates in FAD2 transgene silencing (attached drawing 12).FAD2,18 carbon how unsaturated rouge are overexpressed under ago1 mutant background Fat acid can be improved 5 from 3 highests compareed with the ratio of saturation monounsaturated fatty acids.

SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6 and SEQ ID NO.7 is separately encoded RDR6, SGS3, DCL2, DCL4, MOS2, DCL1 and AGO1 albumen, in these mutant In, FAD2 transgene silencing is released from, and being overexpressed AtFAD2 can be improved the expression quantity of FAD2, improves linoleic acid content and total The content of polyunsaturated fatty acid.In genetic engineering, the mutant that can use these albumen is overexpressed FAD2, improves mostly not The content of saturated fatty acid.

Compared with existing FAD2 is in plant genetic engineering using status, the invention has the characteristics that and effect:

(1) it has further clarified FAD2 and has been overexpressed positive-sense strand, strong gene silencing can be caused in most strains.

(2) to have found that the genes such as RDR6, SGS3, DCL2, DCL4, MOS2, DCL1 and AGO1 take part in for the first time above-mentioned FAD2 transgene silencing process.

(3) in RDR6 mutant, being overexpressed FAD2 can be improved ten times of transcriptional level of FAD2 or more.RDR6, It is overexpressed in the isogenic single mutant of SGS3, DCL2, DCL4, MOS2, DCL1 and AGO1 or the multimutation body that they are combined FAD2, polyunsaturated fatty acid increase rate can be more than 10% or 18 carbon polyunsaturated fatty acid and single unsaturated lipid The ratio of fat acid improves 2 times or more.Polyunsaturated fatty acid especially in vegetable seeds and root significantly improves.

Detailed description of the invention

Fig. 1 is the fatty acid variation that FAD2 is overexpressed T2 for strain in arabidopsis wild type seeds.Fat in legend Sour type: red pillar is oleic acid (18:1), represents the substrate of FAD2；Green pillar be linoleic acid (18:2) and linolenic acid (18: 3) sum represents the product of FAD2.Abscissa P_phaseolin: FAD2/Col-0 indicates that FAD2 is opened in seed specific phaseolin Mover drives lower arabidopsis thaliana transformation Col-0 wild type separate transgenic system obtained, and fad2-1 is a mutant of FAD2. Ordinate refers to that fatty acid shown in transgenosis system is corresponding to arabidopsis wild type Col fatty relative to the net change amount of control The difference of acid content is net increase on abscissa, is only reduces under horizontal seat.AtFAD2 is overexpressed in most of transgenic line The linoleic reduction of its product is shown in system.In FAD2 transgenosis system, the accumulation of oleic acid and the reduction of polyunsaturated fatty acid Close to the phenotype for being even more than fad2 mutant.

Fig. 2 is that arabidopsis FAD2 transgenic line fatty acid component and FAD2 expression are analyzed.Abscissa is wild type And FAD2 transgenosis T2 strain.The ratio of A figure ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.B Figure ordinate is the expression of FAD2, is indicated with the ratio of the expression quantity with wild type.Expression is fixed using Q-PCR technology Amount, using 5 ' the endogenous FAD2 of UTR primer detection, using the total FAD2 of code area primer detection.Abscissa represents wild type in figure Transgenic line of the Col-0 and AtFAD2 in Col-0.It is overexpressed in AtFAD2 strain in seed, FAD2 gene expression It is horizontal consistent with levels of polyunsaturated fatty acids.In the strain that linoleic acid reduces, endogenous and external source FAD2 expression is reduced, a In the strain that other linoleic acid improves, total FAD2 is largely accumulated.

Fig. 3 is that FAD2 transgene silencing occurs for the root of 35S promoter constitutive expression AtFAD2 strain.A figure abscissa For fatty acid species, including oleic acid, linoleic acid plus linolenic acid.Ordinate is the content of related fatty acids.The pillar of different colours Represent wild type Col-0 and Col-0 transgenic line 1,2,3,4,5 and 7.B figure abscissa is wild type Col-0 and transgenic line System, transgenic line are that the AtFAD2 of 35S driving is overexpressed system.Ordinate is FAD2 expression, with the expression with wild type The ratio of amount indicates.Different colours pillar indicates the expression of total FAD2 and endogenous FAD2.Expression uses Q-PCR technology It is quantitative, using 5 ' the endogenous FAD2 of UTR primer detection, using the total FAD2 of code area primer detection.The raising of FAD2 substrate oleic acid, product More saturated fatty acids reduce, and endogenous and external source FAD2 expression reduces.

Fig. 4 is to be overexpressed FAD2 in rdr6 mutant to increase substantially content of polyunsaturated fatty acid.Abscissa P_phaseolin: FAD2/rdr6-11 indicates that rdr6-11 and FAD2 FAD2 under the driving of seed specific phaseolin promoter exists Transgenosis system (Pphaseolin:FAD2/rdr6-11) in arabidopsis rdr6-11 mutant.Rdr6-11 is one of RDR6 Afunction mutant.The ratio of ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.In legend Black is rdr6-11 mutant, brown Pphaseolin:FAD2/rdr6-11.It is expressed in wild type with FAD2 most of Strain product reduces difference, and all there is no silencings when expression FAD2 in rdr6 mutant, and product is sub- in a large amount of strains The ratio of the sum of oleic acid and linolenic acid and substrate oleic acid can be improved one times or more.

Fig. 5 is that FAD2/rdr6 transgenosis system fatty acid phenotype and FAD2 expression are analyzed.Abscissa is rdr6 and at it The transgenosis system of FAD2 is overexpressed under background.A figure ordinate indicates the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid Ratio.B figure ordinate is the expression of FAD2, is indicated with the ratio of the expression quantity with wild type.Expression uses Q- Round pcr is quantitative, using 5 ' the endogenous FAD2 of UTR primer detection, using the total FAD2 of code area primer detection.FAD2 is mutated in rdr6 Polyunsaturated fatty acid can be improved in whole strains by being overexpressed in body, be overexpressed FAD2 transgene silencing in rdr6 mutant In be released from completely, the total expression of FAD2 can be improved more than 10 times or more.

Fig. 6 is to be overexpressed FAD2 in sgs3 mutant to increase substantially content of polyunsaturated fatty acid.Abscissa is Sgs3 mutant and transgenic line, sgs3 are an afunction mutant of SGS3, and FAD2/sgs3 indicates FAD2 in sgs3 The strain expressed in mutant.The ratio of ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.FAD2 Transgene silencing is released from completely in mutant.It is different that most of strain product reduction is expressed in wild type from FAD2, There is no silencings for whole strain when expressing FAD2 in sgs3 mutant, and in a large amount of strains product linoleic acid and linolenic acid it It can be improved one times or more with the ratio with substrate oleic acid.

Fig. 7 is the ratio reduction for being overexpressed FAD2 in dcl2 mutant and transgene silencing occurring.Abscissa is prominent for dcl2 Variant and transgenic line, dcl2 are an afunction mutant of DCL2, and FAD2/dcl2 indicates FAD2 in dcl2 mutant The strain of middle expression.The ratio of ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.FAD2 transgenosis Silencing a part in mutant is released from.It is different that most of strain product reduction is expressed in wild type from FAD2, in dcl2 The ratio that transgene silencing occurs when expressing FAD2 in mutant reduces, and in some strains product linoleic acid and linolenic acid it It can be improved one times or more with the ratio with substrate oleic acid.

Fig. 8 is the ratio reduction for being overexpressed FAD2 in dcl4 mutant and transgene silencing occurring.Abscissa is prominent for dcl4 Variant and transgenic line, dcl4 are an afunction mutant of DCL4, and FAD2/dcl4 indicates FAD2 in dcl4 mutant The strain of middle expression.The ratio of ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.FAD2 transgenosis Silencing a part in mutant is released from.It is different that most of strain product reduction is expressed in wild type from FAD2, in dcl4 The ratio that transgene silencing occurs when expressing FAD2 in mutant reduces, and in some strains product linoleic acid and linolenic acid it It can be improved one times or more with the ratio highest with substrate oleic acid.

Fig. 9 is that FAD2 transgene silencing is almost released from dcl2dcl4 mutant.Abscissa is dcl2dcl4 Mutant and transgenic line, dcl2dcl4 are the afunction double-mutant of DCL2 and DCL4, and FAD2/dcl2dcl4 is indicated The strain that FAD2 is expressed in dcl2dcl4 mutant.Ordinate indicates the sum of FAD2 product linoleic acid and linolenic acid and substrate oil The ratio of acid.Expressing most of strain product in wild type with FAD2 reduces completely different, the table in dcl2dcl4 mutant There is a situation where transgene silencings almost to be released completely when up to FAD2, and in a large amount of strains product linoleic acid and linolenic acid it It can be improved one times or so with the ratio highest with substrate oleic acid.

Figure 10 is that FAD2 transgene silencing is released from completely in mos2 mutant.Abscissa is mos2 mutant and turns base Because of strain, mos2 is an afunction mutant of MOS2, and FAD2/mos2 indicates the strain that FAD2 is expressed in mos2 mutant System.The ratio of ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.It is expressed in wild type with FAD2 Most of strain product reduction is completely different, and it is heavy that transgenosis occurs when expressing FAD2 in mos2 mutant, in detected strain Silent situation is all released from, and the sum of product linoleic acid and linolenic acid and the ratio of substrate oleic acid can mention in a large amount of strains Double left and right.

Figure 11 is the fatty acid variation that FAD2 is overexpressed T2 for strain in arabidopsis dcl1-9 mutant seeds.dcl1-9 For an afunction mutant of DCL1.Ordinate is fatty acid percentage in figure.Orange pillar is mutant, other pillars For different transgenic lines.Transgenic line is that FAD2 converts quasi- south under the driving of seed specific phaseolin promoter The transgenosis system of mustard dcl1-9 mutant.Abscissa is fatty acid component: oleic acid (18:1) and linoleic acid plus linolenic acid it is total It measures (18:2+18:3).Most of strain polyunsaturated fatty acid increases substantially.

Figure 12 is that FAD2 transgene silencing is released from completely in ago1 mutant.Abscissa is ago1 mutant and turns base Because of strain, ago1-25 is an afunction mutant of AGO1, and FAD2/ago1-25 indicates FAD2 in ago1-25 mutant The strain of middle expression.The ratio of ordinate expression the sum of FAD2 product linoleic acid and linolenic acid and substrate oleic acid.It is out of office with FAD2 It is completely different that most of strain product reduction is expressed in raw type, when expressing FAD2 in ago1-25 mutant, in detected strain There is a situation where transgene silencings to be all released from, and the sum of product linoleic acid and linolenic acid and substrate oleic acid in a large amount of strains Ratio can be improved one times or so.

Specific embodiment

The present invention encodes the arabidopsis single mutation of the gene of RDR6, SGS3, DCL2, DCL4, MOS2, DCL1 or AGO1 albumen Body refers to that DNA sequence dna mutates, causes coded sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 protein part or completely lose the vegetable material of function. These materials are overexpressed in transgenosis system for FAD2, for improving content of polyunsaturated fatty acid in plant.Coding RDR6, The Arabidopsis Mutants of the gene of SGS3, DCL2, DCL4, MOS2, DCL1 or AGO1 albumen be combined with each other to be formed it is more than one The material of gene mutation is overexpressed in transgenosis system, for improving content of polyunsaturated fatty acid in plant for FAD2.

In following embodiment, only relatively special material and research method are given to introduce.Conventional test method and Related conventional biochemical reagent is not described in detail, and research method refers to " Molecular Cloning:A Laboratory guide " (J. Sha Mubulu It is gram equal to write) and " Lipid Analysis " (William W.Christie and Xianlin Han work) etc..

Embodiment 1: the building of plant expression vector

1. vegetable material prepares

Arabidopsis used in this test is wild type Col-0.Arabidopsis seed is placed 2-4 days in the environment of 4 DEG C and is carried out together After stepization processing, in the culture of manual control culturing room, growth conditions are as follows: temperature condition is 22 DEG C, and the photoperiod was 16 small time According to/8 hours dark, light intensity was 100-130 μ E m^-2s^-1。

2. RNA is extracted in arabidopsis seed

Arabidopsis seed total serum IgE is extracted using conventional Trizon method.

3.RNA reverse transcription synthesizes the first chain cDNA

Reverse transcription is carried out using Takara kit (Code NO.RR047A).

4. design of primers and gene cloning

According to SEQ ID No.8 sequence design overall length primer, primer is as follows:

FAD2-F:5 '-ATGGGTGCAGGTGGAAGA-3 '

FAD2-R:5’-GGTCATAACTTATTGTTGTACCAG-3’

Using cDNA obtained by reverse transcription as template, using FAD2-F and FAD2-R as primer, PCR obtains FAD2 full length gene.

5. expression vector establishment

The AtFAD2 gene that clone obtains is connected in cloning vector p-EASY (Quan Shi King Company), sun is screened after conversion Property clone, sequencing identification sequence.It is connected using restriction enzyme ECOR I with I double digestion of Xba, AtFAD2 gene is connected to It is pGW-MCS+FAD2 before Gateway on entry vector pGW-MCS.

GFP expression cassette on pK7WG2D is cut off with restriction enzyme Hind III, and spends phosphorylase processing, is utilized DsRED expression cassette on T4 ligase reaction forming, constructed carrier are named as pK7WG2D-DsRED.

GFP expression cassette on pK7WG2D and 35S promoter are cut off with restriction enzyme Hind III and Spe I, utilized Phaseolin promoter on T4 ligase reaction forming is pK7WG2D-Pha；Use restriction enzyme Hind III will again PK7WG2D-Pha is cut, and spends phosphorylase processing, constructed using DsRED expression cassette on T4 ligase reaction forming Carrier is named as pK7WG2D-Pha-DsRED.

PGW-MCS+FAD2 is carried out with above-mentioned pK7WG2D-DsRED and pK7WG2D-Pha-DsRED respectively using LR enzyme LR reaction, by Spec resistance screening, obtained plant expression vector is named as pK7WG2D-DsRED+FAD2 and pK7WG2D- Pha-DsRED+FAD2。

Embodiment 2: the arabidopsis AtFAD2 of mediated by agriculture bacillus is converted in wild type and various mutations body

1, vegetable material prepares

Arabidopsis used in this test is wild type Col-0, rdr6-11, sgs3-14, mos2-2, dcl2-1, dcl4-2T, Dcl1-9 and ago1-25 single mutant, dcl2-1/dcl4-2T double-mutant.Arabidopsis seed places 2-4 in the environment of 4 DEG C After the processing of its synchronizing, in the culture of manual control culturing room, growth conditions are as follows: temperature condition is 22 DEG C, and the photoperiod is 16 hours illumination/8 hour dark, light intensity are 100-130 μ E m^-2s^-1。

2, above-mentioned pK7WG2D-DsRED+FAD2 and pK7WG2D-Pha-DsRED+FAD2 is expressed using very fast freeze-thaw method Carrier is transformed into Agrobacterium (GV3101 bacterial strain).

3, in arabidopsis full-bloom stage, genetic transformation is carried out to arabidopsis and other mutant using inflorescence dip method, it will PK7WG2D-DsRED+FAD2 carrier is transformed into Col-0 plant, and pK7WG2D-Pha-DsRED+FAD2 is transformed into wild type Col-0 and rdr6-11, sgs3-14, mos2-2, dcl2-1, dcl4-2T, dcl2-1/dcl4-2T, dcl1-9 and ago1-25 In single double-mutant.It collects FAD2 genetic transformation and obtains arabidopsis seed in 1.5ml centrifuge tube, it can be in green light (excitation wavelength Under conditions of 543nm), red fluorescence is inspired.Through red optical filter, relative to unconverted WT lines Col- For 0, the red fluorescence of positive transformants seed sending can be observed by the naked eye, T1 can be filtered out for transgenosis according to this Seed.T1 is sprouted for seed, is T1 for transgenic plant, moves it to using vermiculite, perlite, peat as the basin alms bowl of matrix In, it allows it to continue to grow to harvest in culturing room, is T2 for seed.The T2 obtained under variant genetic background divides for seed Col-0 strain 1, strain 2 Bian Hao be named as until marked all strains, all strains be obtain at random independently turn base Because being；If the T2 of Col-0 background is Col-0 strain 1, Col-0 strain 2 ... Col-0 strain n for seed tag.Mutant background The T2 of lower transgenosis is similar to Col-0 for seed number.According to the difference of promoter used, the transformant of 35S promoter carrier System is named as 35S:FAD2, and the strain of Phaseolin promoter vector conversion is named as Pphaseolin:FAD2.Without special note It is bright outer, expression Phaseolin promoter vector conversion.After the background material of conversion is labeled in oblique line, such as in rdr6-11 background Conversion, is labeled as/rdr6-11.

Embodiment 3: Fatty Acids in Seeds constituent analysis

1, plant and seed material prepare

T1 generation and T2 are selected for the seed for having fluorescence in seed, is used for fatty acid analysis.

2, seed fat acid analysis

By T1 or T2 for positive seeds under natural light aeration-drying until weight do not change.To (Poirier et Al., Plant Physiol, 1999,121 (4): 1359-1366.) method improves, arabidopsis Fatty Acids in Seeds carried out It extracts and analyzes.Method is as follows after improvement:

For T1 band seed, takes a seed and 100 μ L 1M concentrated sulfuric acid methanol solution extracting solutions are added, be then put in Water-bath carries out fatty acid extraction and carries out esterification, and after it is cooled to room temperature, 100 μ l 0.9%NaCl (w/v) are added eventually It only reacts, is subsequently added into 50 μ l n-hexanes, 2300rpm centrifugation 3min extracts fatty acid methyl ester after oscillation mixes, and uses pipettor The careful organic phase for drawing upper layer, is placed in the gas phase in the HPLC sample injection bottle of n-hexane rinse, for fatty acid component Chromatography, method are the same.

For T2 for seed, takes 10 seeds and 200 μ L 1M concentrated sulfuric acid methanol solution extracting solutions are added, be then put in Water-bath carries out fatty acid extraction and carries out esterification, and after it is cooled to room temperature, 200 μ l 0.9%NaCl (w/v) are added eventually It only reacts, is subsequently added into 100 μ l n-hexanes, later step is same as above.

Root sample is derived from the entire root of 40 days plant, and 200 μ L 1M concentrated sulfuric acid methanol solution extracting solutions are added, then put In water-bath carry out fatty acid extraction and carrying out esterification, after it is cooled to room temperature, 200 μ l 0.9%NaCl (w/ are added V) reaction is terminated, is subsequently added into 100 μ l n-hexanes, later step is same as above.Using 35S promoter in root under Col-0 background The result for being overexpressed FAD2 is shown in Figure of description 3A.

Col-0 wild type, rdr6-11, sgs3-14, mos2-2, dcl2-1, dcl4-2T, dcl2-1/dcl4-2T, The result for being overexpressed FAD2 under dcl1-9 and ago1-25 mutant background is shown in Figure of description 1,4,6,7,8,9,10,11 respectively With 12.The ratio of content of polyunsaturated fatty acid (18:2+18:3) either polyunsaturated fatty acid and monounsaturated fatty acids ((18:2+18:3)/18:1) is improved compared with the control, illustrates that these genes take part in FAD2 co-suppression.It also turns out simultaneously, Using the overexpression of these mutant or their silencing strain combination FAD2, can be used to improve polyunsaturated fat in seed The content of acid.

Embodiment 4: gene expression analysis

1, vegetable material prepares

T1 is sprouted for seed, is T1 for transgenic plant, moves it to using vermiculite, perlite, peat as the basin of matrix In alms bowl, it is allowed to continue to grow in culturing room to bloom.Arabidopsis flowering time is marked at the florescence, is harvested within Post flowering 12-14 days Seed, as developmental arabidopsis seed；The seed for not having markd flower to bear harvests after maturation.

2, RNA is extracted in arabidopsis seed and reverse transcription method is the same as method in expression vector establishment.

3, gene expression analysis

Using takara kit (Code DRR820A) in ABI QuantStudio 7Flex real-time fluorescence quantitative PCR Gene expression analysis is carried out in system.The primer is as follows:

Endogenous FAD2 analyzes primer:

QEndo-F:CTTCTTCTTCGTAGGGTG

QEndo-R:TGTTTCTGGAGATGGAGC

Total FAD2 analyzes primer:

Qtotal-F:CGGAAACCGACACCACAAA

Qtotal-R:TTCAGATCTCCCACCGAGAAA

Internal reference Tubulin primer:

Qtub-F:TTTGTGCTCATCTTGCCACGGAAC

Qtub-R:CTCAAGAGGTTCTCAGCAGTACC

FAD2 turn wild type Col development seed in, in root system and FAD2 turn rdr6 development seed in endogenous FAD2 Figure of description 2B, 3B and 5B are seen with the expression analysis result of the total FAD2 of interior external source.Reduction (the attached drawing of endogenous and total FAD2 expression 2B and 3B) illustrate that FAD2 transgenosis produces co-suppression phenomenon；Endogenous and total FAD2 is expressed in rdr6 mutant significantly It improves (Fig. 5 B), illustrates that rdr6 takes part in the RNA silencing of FAD2, while also illustrating, utilize rdr6 mutant or its silencing strain It can be with effective expression FAD2.

Quantitative parameter setting: then 95 DEG C of initial denaturation 2min carry out following circulation；94 DEG C of denaturation 15sec, 60 DEG C of annealing and Extend 1min, carries out 40 circulations altogether, last 65-95 DEG C prepares solubility curve.

The preferred embodiment of the disclosure is described in detail in conjunction with attached drawing above, still, the disclosure is not limited to above-mentioned reality The detail in mode is applied, in the range of the technology design of the disclosure, a variety of letters can be carried out to the technical solution of the disclosure Monotropic type, these simple variants belong to the protection scope of the disclosure.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the disclosure to it is various can No further explanation will be given for the combination of energy.

In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally Disclosed thought equally should be considered as disclosure disclosure of that.

Nucleotides sequence list electronic document

<110>Xibei Univ. of Agricultural & Forest Science & Technology

<120>a kind of method for improving plant polyunsaturated fatty acid

<141>

<160>

<210>1

<211>1196

<212>amino acid

<213>RDR6

<220>

<223>

<400>1

1 MGSEGNMKKS VVTQVSIGGF GESTTAKQLT DYLEDEVGIV WRCRLKTSWT

51 PPGSYPNFEI ADTSNIPSID EYKKVEPHAF VHFAVFESAG RAMDAAGQCN

101 LILDGQPLKV SLGPKNPYSL NQRRRTTVPY KLAGITLEIG TLVSRDDFFV

151 SWRAEGVDFL VDPFDNTCKF CFRKSTAFSF KDAVMHAVIN CDYKLELLVR

201 DIQTVRQYKT LHGFVLILQL ASSPRVWYRT ADDDIYDTVP GDLLDDDDPW

251 IRTTDFTQVG AIGRCHSYRV LISPRYENKL RTALDYFRMR RVQEERVRWP

301 PRIRNEPCFG EPVSDHFFCI HHKEGISFEI MFLVNSVLHR GVFNQFQLTE

351 RFFDLLRNQP KDVNIASLKH LCTYKRPVFD AYKRLKLVQE WIQKNPKLLG

401 SHEQSEDISE IRRLVITPTR AYCLPPEVEL SNRVLRRYKA VAERFLRVTF

451 MDESMQTINS NVLSYFVAPI VKDLTSSSFS QKTYVFKRVK SILTDGFKLC

501 GRKYSFLAFS ANQLRDRSAW FFAEDGKTRV SDIKTWMGKF KDKNVAKCAA

551 RMGLCFSSTY ATVDVMPHEV DTEVPDIERN GYVFSDGIGT ITPDLADEVM

601 EKLKLDVHYS PCAYQIRYAG FKGVVARWPS KSDGIRLALR DSMKKFFSKH

651 TILEICSWTR FQPGFLNRQI ITLLSVLGVP DEIFWDMQES MLYKLNRILD

701 DTDVAFEVLT ASCAEQGNTA AIMLSAGFKP KTEPHLRGML SSVRIAQLWG

751 LREKSRIFVT SGRWLMGCLD EAGILEHGQC FIQVSKPSIE NCFSKHGSRF

801 KETKTDLEVV KGYVAIAKNP CLHPGDVRIL EAVDVPQLHH MYDCLIFPQK

851 GDRPHTNEAS GSDLDGDLYF VAWDQKLIPP NRKSYPAMHY DAAEEKSLGR

901 AVNHQDIIDF FARNLANEQL GTICNAHVVH ADRSEYGAMD EECLLLAELA

951 ATAVDFPKTG KIVSMPFHLK PKLYPDFMGK EDYQTYKSNK ILGRLYRRVK

1001 EVYDEDAEAS SEESTDPSAI PYDAVLEIPG FEDLIPEAWG HKCLYDGQLI

1051 GLLGQYKVQK EEEIVTGHIW SMPKYTSKKQ GELKERLKHS YNSLKKEFRK

1101 VFEETIPDHE NLSEEEKNIL YEKKASAWYH VTYHPEWVKK SLELQDPDES

1151 SHAAMLSFAW IAADYLARIK IRSREMGSID SAKPVDSLAK FLAQRL

<210>2

<211>625

<212>amino acid

<213>SGS3

<220>

<223>

<400>2

1 MSSRAGPMSK EKNVQGGYRP EVEQLVQGLA GTRLASSQDD GGEWEVISKK

51 NKNKPGNTSG KTWVSQNSNP PRAWGGQQQG RGSNVSGRGN NVSGRGNGNG

101 RGIQANISGR GRALSRKYDN NFVAPPPVSR PPLEGGWNWQ ARGGSAQHTA

151 VQEFPDVEDD VDNASEEEND SDALDDSDDD LASDDYDSDV SQKSHGSRKQ

201 NKWFKKFFGS LDSLSIEQIN EPQRQWHCPA CQNGPGAIDW YNLHPLLAHA

251 RTKGARRVKL HRELAEVLEK DLQMRGASVI PCGEIYGQWK GLGEDEKDYE

301 IVWPPMVIIM NTRLDKDDND KWLGMGNQEL LEYFDKYEAL RARHSYGPQG

351 HRGMSVLMFE SSATGYLEAE RLHRELAEMG LDRIAWGQKR SMFSGGVRQL

401 YGFLATKQDL DIFNQHSQGK TRLKFELKSY QEMVVKELRQ ISEDNQQLNY

451 FKNKLSKQNK HAKVLEESLE IMSEKLRRTA EDNRIVRQRT KMQHEQNREE

501 MDAHDRFFMD SIKQIHERRD AKEENFEMLQ QQERAKVVGQ QQQNINPSSN

551 DDCRKRAEEV SSFIEFQEKE MEEFVEEREM LIKDQEKKME DMKKRHHEEI

601 FDLEKEFDEA LEQLMYKHGL HNEDD

<210>3

<211>1388

<212>amino acid

<213>DCL2

<220>

<223>

<400>3

1 MTMDADAMET ETTDQVSASP LHFARSYQVE ALEKAIKQNT IVFLETGSGK

51 TLIAIMLLRS YAYLFRKPSP CFCVFLVPQV VLVTQQAEAL KMHTDLKVGM

101 YWGDMGVDFW DSSTWKQEVD KYEVLVMTPA ILLDALRHSF LSLSMIKVLI

151 VDECHHAGGK HPYACIMREF YHKELNSGTS NVPRIFGMTA SLVKTKGENL

201 DSYWKKIHEL ETLMNSKVYT CENESVLAGF VPFSTPSFKY YQHIKIPSPK

251 RASLVEKLER LTIKHRLSLG TLDLNSSTVD SVEKRLLRIS STLTYCLDDL

301 GILLAQKAAQ SLSASQNDSF LWGELNMFSV ALVKKFCSDA SQEFLAEIPQ

351 GLNWSVANIN GNAEAGLLTL KTVCLIETLL GYSSLENIRC IIFVDRVITA

401 IVLESLLAEI LPNCNNWKTK YVAGNNSGLQ NQTRKKQNEI VEDFRRGLVN

451 IIVATSILEE GLDVQSCNLV IRFDPASNIC SFIQSRGRAR MQNSDYLMMV

501 ESGDLLTQSR LMKYLSGGKR MREESLDHSL VPCPPLPDDS DEPLFRVEST

551 GATVTLSSSV SLIYHYCSRL PSDEYFKPAP RFDVNKDQGS CTLYLPKSCP

601 VKEVKAEANN KVLKQAVCLK ACIQLHKVGA LSDHLVPDMV VAETVSQKLE

651 KIQYNTEQPC YFPPELVSQF SAQPETTYHF YLIRMKPNSP RNFHLNDVLL

701 GTRVVLEDDI GNTSFRLEDH RGTIAVTLSY VGAFHLTQEE VLFCRRFQIT

751 LFRVLLDHSV ENLMEALNGL HLRDGVALDY LLVPSTHSHE TSLIDWEVIR

801 SVNLTSHEVL EKHENCSTNG ASRILHTKDG LFCTCVVQNA LVYTPHNGYV

851 YCTKGVLNNL NGNSLLTKRN SGDQTYIEYY EERHGIQLNF VDEPLLNGRH

901 IFTLHSYLHM AKKKKEKEHD REFVELPPEL CHVILSPISV DMIYSYTFIP

951 SVMQRIESLL IAYNLKKSIP KVNIPTIKVL EAITTKKCED QFHLESLETL

1001 GDSFLKYAVC QQLFQHCHTH HEGLLSTKKD GMISNVMLCQ FGCQQKLQGF

1051 IRDECFEPKG WMVPGQSSAA YSLVNDTLPE SRNIYVASRR NLKRKSVADV

1101 VESLIGAYLS EGGELAALMF MNWVGIKVDF TTTKIQRDSP IQAEKLVNVG

1151 YMESLLNYSF EDKSLLVEAL THGSYMMPEI PRCYQRLEFL GDSVLDYLIT

1201 KHLYDKYPCL SPGLLTDMRS ASVNNECYAL VAVKANLHKH ILYASHHLHK

1251 HISRTVSEFE QSSLQSTFGW ESDISFPKVL GDVIESLAGA IFVDSGYNKE

1301 VVFASIKPLL GCMITPETVK LHPVRELTEL CQKWQFELSK AKDFDSFTVE

1351 VKAKEMSFAH TAKASDKKMA KKLAYKEVLN LLKNSLDY

<210>4

<211>1702

<212>amino acid

<213>DCL4

<220>

<223>

<400>4

1 MRDEVDLSLT IPSKLLGKRD REQKNCEEEK NKNKKAKKQQ KDPILLHTSA

51 ATHKFLPPPL TMPYSEIGDD LRSLDFDHAD VSSDLHLTSS SSVSSFSSSS

101 SSLFSAAGTD DPSPKMEKDP RKIARRYQVE LCKKATEENV IVYLGTGCGK

151 THIAVMLIYE LGHLVLSPKK SVCIFLAPTV ALVEQQAKVI ADSVNFKVAI

201 HCGGKRIVKS HSEWEREIAA NEVLVMTPQI LLHNLQHCFI KMECISLLIF

251 DECHHAQQQS NHPYAEIMKV FYKSESLQRP RIFGMTASPV VGKGSFQSEN

301 LSKSINSLEN LLNAKVYSVE SNVQLDGFVS SPLVKVYYYR SALSDASQST

351 IRYENMLEDI KQRCLASLKL LIDTHQTQTL LSMKRLLKRS HDNLIYTLLN

401 LGLWGAIQAA KIQLNSDHNV QDEPVGKNPK SKICDTYLSM AAEALSSGVA

451 KDENASDLLS LAALKEPLFS RKLVQLIKIL SVFRLEPHMK CIIFVNRIVT

501 ARTLSCILNN LELLRSWKSD FLVGLSSGLK SMSRRSMETI LKRFQSKELN

551 LLVATKVGEE GLDIQTCCLV IRYDLPETVT SFIQSRGRAR MPQSEYAFLV

601 DSGNEKEMDL IENFKVNEDR MNLEITYRSS EETCPRLDEE LYKVHETGAC

651 ISGGSSISLL YKYCSRLPHD EFFQPKPEFQ FKPVDEFGGT ICRITLPANA

701 PISEIESSLL PSTEAAKKDA CLKAVHELHN LGVLNDFLLP DSKDEIEDEL

751 SDDEFDFDNI KGEGCSRGDL YEMRVPVLFK QKWDPSTSCV NLHSYYIMFV

801 PHPADRIYKK FGFFMKSPLP VEAETMDIDL HLAHQRSVSV KIFPSGVTEF

851 DNDEIRLAEL FQEIALKVLF ERGELIPDFV PLELQDSSRT SKSTFYLLLP

901 LCLHDGESVI SVDWVTIRNC LSSPIFKTPS VLVEDIFPPS GSHLKLANGC

951 WNIDDVKNSL VFTTYSKQFY FVADICHGRN GFSPVKESST KSHVESIYKL

1001 YGVELKHPAQ PLLRVKPLCH VRNLLHNRMQ TNLEPQELDE YFIEIPPELS

1051 HLKIKGLSKD IGSSLSLLPS IMHRMENLLV AIELKHVLSA SIPEIAEVSG

1101 HRVLEALTTE KCHERLSLER LEVLGDAFLK FAVSRHLFLH HDSLDEGELT

1151 RRRSNVVNNS NLCRLAIKKN LQVYIRDQAL DPTQFFAFGH PCRVTCDEVA

1201 SKEVHSLNRD LGILESNTGE IRCSKGHHWL YKKTIADVVE ALVGAFLVDS

1251 GFKGAVKFLK WIGVNVDFES LQVQDACIAS RRYLPLTTRN NLETLENQLD

1301 YKFLHKGLLV QAFIHPSYNR HGGGCYQRLE FLGDAVLDYL MTSYFFTVFP

1351 KLKPGQLTDL RSLSVNNEAL ANVAVSFSLK RFLFCESIYL HEVIEDYTNF

1401 LASSPLASGQ SEGPRCPKVL GDLVESCLGA LFLDCGFNLN HVWTMMLSFL

1451 DPVKNLSNLQ ISPIKELIEL CQSYKWDREI SATKKDGAFT VELKVTKNGC

1501 CLTVSATGRN KREGTKKAAQ LMITNLKAHE NITTSHPLED VLKNGIRNEA

1551 KLIGYNEDPI DVVDLVGLDV ENLNILETFG GNSERSSSYV IRRGLPQAPS

1601 KTEDRLPQKA IIKAGGPSSK TAKSLLHETC VANCWKPPHF ECCEEEGPGH

1651 LKSFVYKVIL EVEDAPNMTL ECYGEARATK KGAAEHAAQA AIWCLKHSGF

1701 LC

<210>5

<211>462

<212>amino acid

<213>MOS2

<220>

<223>

<400>5

1 MKLSFSLPSK SKPKVTATTA DGNNAVDDGT SKEFVTEFDP SKTLANSIPK

51 YVIPPIENTW RPHKKMKNLD LPLQSGNAGS GLEFEPEVPL PGTEKPDNIS

101 YGLNLRQKVK DDSIGGDAVE ERKVSMGEQL MLQSLRRDLM SLADDPTLED

151 FESVPVDGFG AALMAGYGWK PGKGIGKNAK EDVEIKEYKK WTAKEGLGFD

201 PDRSKVVDVK AKVKESVKLD KKGVGINGGD VFFVGKEVRI IAGRDVGLKG

251 KIVEKPGSDF FVIKISGSEE EVKVGVNEVA DLGSKEEEKC LKKLKDLQLN

301 DREKDKKTSG RGRGAERGSR SEVRASEKQD RGQTRERKVK PSWLRSHIKV

351 RIVSKDWKGG RLYLKKGKVV DVVGPTTCDI TMDETQELVQ GVDQELLETA

401 LPRRGGPVLV LSGKHKGVYG NLVEKDLDKE TGVVRDLDNH KMLDVRLDQV

451 AEYMGDMDDI EY

<210>6

<211>1910

<212>amino acid

<213>DCL1

<220>

<223>

<400>6

1 MVMEDEPREA TIKPSYWLDA CEDISCDLID DLVSEFDPSS VAVNESTDEN

51 GVINDFFGGI DHILDSIKNG GGLPNNGVSD TNSQINEVTV TPQVIAKETV

101 KENGLQKNGG KRDEFSKEEG DKDRKRARVC SYQSERSNLS GRGHVNNSRE

151 GDRFMNRKRT RNWDEAGNNK KKRECNNYRR DGRDREVRGY WERDKVGSNE

201 LVYRSGTWEA DHERDVKKVS GGNRECDVKA EENKSKPEER KEKVVEEQAR

251 RYQLDVLEQA KAKNTIAFLE TGAGKTLIAI LLIKSVHKDL MSQNRKMLSV

301 FLVPKVPLVY QQAEVIRNQT CFQVGHYCGE MGQDFWDSRR WQREFESKQV

351 LVMTAQILLN ILRHSIIRME TIDLLILDEC HHAVKKHPYS LVMSEFYHTT

401 PKDKRPAIFG MTASPVNLKG VSSQVDCAIK IRNLETKLDS TVCTIKDRKE

451 LEKHVPMPSE IVVEYDKAAT MWSLHETIKQ MIAAVEEAAQ ASSRKSKWQF

501 MGARDAGAKD ELRQVYGVSE RTESDGAANL IHKLRAINYT LAELGQWCAY

551 KVGQSFLSAL QSDERVNFQV DVKFQESYLS EVVSLLQCEL LEGAAAEKVA

601 AEVGKPENGN AHDEMEEGEL PDDPVVSGGE HVDEVIGAAV ADGKVTPKVQ

651 SLIKLLLKYQ HTADFRAIVF VERVVAALVL PKVFAELPSL SFIRCASMIG

701 HNNSQEMKSS QMQDTISKFR DGHVTLLVAT SVAEEGLDIR QCNVVMRFDL

751 AKTVLAYIQS RGRARKPGSD YILMVERGNV SHAAFLRNAR NSEETLRKEA

801 IERTDLSHLK DTSRLISIDA VPGTVYKVEA TGAMVSLNSA VGLVHFYCSQ

851 LPGDRYAILR PEFSMEKHEK PGGHTEYSCR LQLPCNAPFE ILEGPVCSSM

901 RLAQQAVCLA ACKKLHEMGA FTDMLLPDKG SGQDAEKADQ DDEGEPVPGT

951 ARHREFYPEG VADVLKGEWV SSGKEVCESS KLFHLYMYNV RCVDFGSSKD

1001 PFLSEVSEFA ILFGNELDAE VVLSMSMDLY VARAMITKAS LAFKGSLDIT

1051 ENQLSSLKKF HVRLMSIVLD VDVEPSTTPW DPAKAYLFVP VTDNTSMEPI

1101 KGINWELVEK ITKTTAWDNP LQRARPDVYL GTNERTLGGD RREYGFGKLR

1151 HNIVFGQKSH PTYGIRGAVA SFDVVRASGL LPVRDAFEKE VEEDLSKGKL

1201 MMADGCMVAE DLIGKIVTAA HSGKRFYVDS ICYDMSAETS FPRKEGYLGP

1251 LEYNTYADYY KQKYGVDLNC KQQPLIKGRG VSYCKNLLSP RFEQSGESET

1301 VLDKTYYVFL PPELCVVHPL SGSLIRGAQR LPSIMRRVES MLLAVQLKNL

1351 ISYPIPTSKI LEALTAASCQ ETFCYERAEL LGDAYLKWVV SRFLFLKYPQ

1401 KHEGQLTRMR QQMVSNMVLY QFALVKGLQS YIQADRFAPS RWSAPGVPPV

1451 FDEDTKDGGS SFFDEEQKPV SEENSDVFED GEMEDGELEG DLSSYRVLSS

1501 KTLADVVEAL IGVYYVEGGK IAANHLMKWI GIHVEDDPDE VDGTLKNVNV

1551 PESVLKSIDF VGLERALKYE FKEKGLLVEA ITHASRPSSG VSCYQRLEFV

1601 GDAVLDHLIT RHLFFTYTSL PPGRLTDLRA AAVNNENFAR VAVKHKLHLY

1651 LRHGSSALEK QIREFVKEVQ TESSKPGFNS FGLGDCKAPK VLGDIVESIA

1701 GAIFLDSGKD TTAAWKVFQP LLQPMVTPET LPMHPVRELQ ERCQQQAEGL

1751 EYKASRSGNT ATVEVFIDGV QVGVAQNPQK KMAQKLAARN ALAALKEKEI

1801 AESKEKHINN GNAGEDQGEN ENGNKKNGHQ PFTRQTLNDI CLRKNWPMPS

1851 YRCVKEGGPA HAKRFTFGVR VNTSDRGWTD ECIGEPMPSV KKAKDSAAVL

1901 LLELLNKTFS

<210>7

<211>1050

<212>amino acid

<213>AGO1

<220>

<223>

<400>7

1 MVRKRRTDAP SEGGEGSGSR EAGPVSGGGR GSQRGGFQQG GGQHQGGRGY

51 TPQPQQGGRG GRGYGQPPQQ QQQYGGPQEY QGRGRGGPPH QGGRGGYGGG

101 RGGGPSSGPP QRQSVPELHQ ATSPTYQAVS SQPTLSEVSP TQVPEPTVLA

151 QQFEQLSVEQ GAPSQAIQPI PSSSKAFKFP MRPGKGQSGK RCIVKANHFF

201 AELPDKDLHH YDVTITPEVT SRGVNRAVMK QLVDNYRDSH LGSRLPAYDG

251 RKSLYTAGPL PFNSKEFRIN LLDEEVGAGG QRREREFKVV IKLVARADLH

301 HLGMFLEGKQ SDAPQEALQV LDIVLRELPT SRIRYIPVGR SFYSPDIGKK

351 QSLGDGLESW RGFYQSIRPT QMGLSLNIDM SSTAFIEANP VIQFVCDLLN

401 RDISSRPLSD ADRVKIKKAL RGVKVEVTHR GNMRRKYRIS GLTAVATREL

451 TFPVDERNTQ KSVVEYFHET YGFRIQHTQL PCLQVGNSNR PNYLPMEVCK

501 IVEGQRYSKR LNERQITALL KVTCQRPIDR EKDILQTVQL NDYAKDNYAQ

551 EFGIKISTSL ASVEARILPP PWLKYHESGR EGTCLPQVGQ WNMMNKKMIN

601 GGTVNNWICI NFSRQVQDNL ARTFCQELAQ MCYVSGMAFN PEPVLPPVSA

651 RPEQVEKVLK TRYHDATSKL SQGKEIDLLI VILPDNNGSL YGDLKRICET

701 ELGIVSQCCL TKHVFKMSKQ YMANVALKIN VKVGGRNTVL VDALSRRIPL

751 VSDRPTIIFG ADVTHPHPGE DSSPSIAAVV ASQDWPEITK YAGLVCAQAH

801 RQELIQDLFK EWKDPQKGVV TGGMIKELLI AFRRSTGHKP LRIIFYRDGV

851 SEGQFYQVLL YELDAIRKAC ASLEAGYQPP VTFVVVQKRH HTRLFAQNHN

901 DRHSVDRSGN ILPGTVVDSK ICHPTEFDFY LCSHAGIQGT SRPAHYHVLW

951 DENNFTADGL QSLTNNLCYT YARCTRSVSI VPPAYYAHLA AFRARFYMEP

1001 ETSDSGSMAS GSMARGGGMA GRSTRGPNVN AAVRPLPALK ENVKRVMFYC

<210>8

<211>1869

<212>DNA

<213>FAD2

<220>

<223>

<400>8

1 TTTTTTCACA AGTAAAAAAT GGGTTATTTG CGGTAAATAA AAATACCAGA

51 TATTTTGAAT TGATTAAAAA GGTTGAAATA AGAGAGGAGG GGAAAGAAAA

101 GAAGGTGGGG GCCCAGTATG AAAGGGAAAG GTGTCATCAA ATCATCTCTC

151 TCTCTCTCTC TCTACCTTCG ACCCACGGGC CGTGTCCATT TAAAGCCCTG

201 TCTCTTGCCA TTCCCCATCT GACCACCAGA AGAAGAGCCA CACACTCACA

251 AATTAAAAAG AGAGAGAGAG AGAGAGAGAC AGAGAGAGAG AGAGATTCTG

301 CGGAGGAGCT TCTTCTTCGT AGGGTGTTCA TCGTTATTAA CGTTATCGCC

351 CCTACGTCAG CTCCATCTCC AGAAACATGG GTGCAGGTGG AAGAATGCCG

401 GTTCCTACTT CTTCCAAGAA ATCGGAAACC GACACCACAA AGCGTGTGCC

451 GTGCGAGAAA CCGCCTTTCT CGGTGGGAGA TCTGAAGAAA GCAATCCCGC

501 CGCATTGTTT CAAACGCTCA ATCCCTCGCT CTTTCTCCTA CCTTATCAGT

551 GACATCATTA TAGCCTCATG CTTCTACTAC GTCGCCACCA ATTACTTCTC

601 TCTCCTCCCT CAGCCTCTCT CTTACTTGGC TTGGCCACTC TATTGGGCCT

651 GTCAAGGCTG TGTCCTAACT GGTATCTGGG TCATAGCCCA CGAATGCGGT

701 CACCACGCAT TCAGCGACTA CCAATGGCTG GATGACACAG TTGGTCTTAT

751 CTTCCATTCC TTCCTCCTCG TCCCTTACTT CTCCTGGAAG TATAGTCATC

801 GCCGTCACCA TTCCAACACT GGATCCCTCG AAAGAGATGA AGTATTTGTC

851 CCAAAGCAGA AATCAGCAAT CAAGTGGTAC GGGAAATACC TCAACAACCC

901 TCTTGGACGC ATCATGATGT TAACCGTCCA GTTTGTCCTC GGGTGGCCCT

951 TGTACTTAGC CTTTAACGTC TCTGGCAGAC CGTATGACGG GTTCGCTTGC

1001 CATTTCTTCC CCAACGCTCC CATCTACAAT GACCGAGAAC GCCTCCAGAT

1051 ATACCTCTCT GATGCGGGTA TTCTAGCCGT CTGTTTTGGT CTTTACCGTT

1101 ACGCTGCTGC ACAAGGGATG GCCTCGATGA TCTGCCTCTA CGGAGTACCG

1151 CTTCTGATAG TGAATGCGTT CCTCGTCTTG ATCACTTACT TGCAGCACAC

1201 TCATCCCTCG TTGCCTCACT ACGATTCATC AGAGTGGGAC TGGCTCAGGG

1251 GAGCTTTGGC TACCGTAGAC AGAGACTACG GAATCTTGAA CAAGGTGTTC

1301 CACAACATTA CAGACACACA CGTGGCTCAT CACCTGTTCT CGACAATGCC

1351 GCATTATAAC GCAATGGAAG CTACAAAGGC GATAAAGCCA ATTCTGGGAG

1401 ACTATTACCA GTTCGATGGA ACACCGTGGT ATGTAGCGAT GTATAGGGAG

1451 GCAAAGGAGT GTATCTATGT AGAACCGGAC AGGGAAGGTG ACAAGAAAGG

1501 TGTGTACTGG TACAACAATA AGTTATGAGG ATGATGGTGA AGAAATTGTC

1551 GACCTTTCTC TTGTCTGTTT GTCTTTTGTT AAAGAAGCTA TGCTTCGTTT

1601 TAATAATCTT ATTGTCCATT TTGTTGTGTT ATGACATTTT GGCTGCTCAT

1651 TATGTTATGT GGGAAGTTAG TGTTCAAATG TTTTGTGTCG GTATTGTTCT

1701 TCTCATCGCT GTTTTGTTGG GATCGTAGAA ATGTGACCTT CGGACAGTAA

1751 AACTCTTGTA CTAAAACTAT CTCCCTATTG GCATTTCTTA AACTTTTAAT

1801 AGTTACGTGC TCGTAGTGAA TCTTGACTTG AGTCAACTTC TTGTTTAAGA

1851 CCTGCCAAGT GTATAAGAG

<210>9

<211>383

<212>amino acid

<213>FAD2

<220>

<223>

<400>9

1 MGAGGRMPVP TSSKKSETDT TKRVPCEKPP FSVGDLKKAI PPHCFKRSIP

51 RSFSYLISDI IIASCFYYVA TNYFSLLPQP LSYLAWPLYW ACQGCVLTGI

101 WVIAHECGHH AFSDYQWLDD TVGLIFHSFL LVPYFSWKYS HRRHHSNTGS

151 LERDEVFVPK QKSAIKWYGK YLNNPLGRIM MLTVQFVLGW PLYLAFNVSG

201 RPYDGFACHF FPNAPIYNDR ERLQIYLSDA GILAVCFGLY RYAAAQGMAS

251 MICLYGVPLL IVNAFLVLIT YLQHTHPSLP HYDSSEWDWL RGALATVDRD

301 YGILNKVFHN ITDTHVAHHL FSTMPHYNAM EATKAIKPIL GDYYQFDGTP

351 WYVAMYREAK ECIYVEPDRE GDKKGVYWYN NKL

Claims (5)

1. a kind of method for releasing FAD2 transgene silencing, which is characterized in that knock out or silencing causes FAD2 transgene silencing Gene, the gene are to encode functional SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, any albumen shown in SEQ ID NO.6 and SEQ ID NO.7 gene or homologous gene；Alternatively, institute State the gene or homologous gene that gene is albumen shown in functional SEQ ID NO.3 and SEQ the ID NO.4 sequence of coding.

2. a kind of expression of FAD2 gene, which is characterized in that method, which is included under suitable genetic background gene, to be expressed FAD2 gene, the suitable genetic background gene do not include encoding functional SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, any albumen shown in SEQ ID NO.6 and SEQ ID NO.7 gene or Homologous gene；Alternatively, the suitable genetic background gene does not include encoding functional SEQ ID NO.3 and SEQ ID The gene or homologous gene of albumen shown in NO.4 sequence.

3. a kind of method for improving plant polyunsaturated fatty acid yield, which is characterized in that method, which is included in plant, to be carried out FAD2 transgenosis, the gene for causing FAD2 transgene silencing in the plant is knocked or silencing, the gene To encode functional SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ The gene or homologous gene of any albumen shown in ID NO.6 and SEQ ID NO.7；Alternatively, the gene is that coding is active The gene or homologous gene of albumen shown in SEQ ID NO.3 and SEQ the ID NO.4 sequence of energy.

4. improving the method for plant content of polyunsaturated fatty acid as claimed in claim 3, which is characterized in that the plant is Oil crops.

5. improving the method for plant content of polyunsaturated fatty acid as claimed in claim 3, which is characterized in that more insatiable hungers It is linoleic acid or/and linolenic acid with fatty acid.