CN107779456B - Medicago truncatula MtWOX11 gene and application thereof in increasing content of fatty acid in seeds - Google Patents

Abstract

The invention discloses a medicago truncatula WOX family gene Mt WUSCHEL related biomeobox 11(MtWOX11), and the nucleotide sequence of the gene is shown as SEQ ID No. 1. The invention also discloses application of the gene in improving the fatty acid content of leguminous plant seeds. Analysis of transgenic lines obtained by genetic transformation proves that the fatty acid content of seeds of leguminous plants can be obviously improved by over-expression of the gene. The gene of the invention is indicated to have wide application prospect, and the application of the gene can be used for improving the quality of beans or creating novel leguminous economic crops, and has great significance for the production of the leguminous economic crops in China.

Description

Medicago truncatula MtWOX11 gene and application thereof in increasing content of fatty acid in seeds

Technical Field

The invention relates to a WUSCHEL relatedhomeobox (WOX) family gene and application thereof, in particular to a medicago truncatula WOX family gene Mt WUSCHEL relatedhomeobox 11(MtWOX11) and application thereof in improving the fatty acid content of leguminous plant seeds, belonging to the field of genetic engineering.

Background

In recent years, with the improvement of the economic level of China and the quality of life of people, the demand of soybeans is increasing day by day. Soybean is one of the important legume commercial crops, which is capable of providing not only high protein, but more primarily oil. However, the quality of the soybean in China is not high at present, the market competitiveness is lacked, and a large amount of imported soybeans are needed. Therefore, the cultivation of new soybean varieties with high oil content and high protein content is an urgent task at present.

At present, the research work of improving the yield and the quality of soybeans in China mainly focuses on the field of conventional breeding, and molecular genetic improvement is still in the starting stage. The reason is mainly that: the soybean is tetraploid, and the genome is large; the soybean is difficult to establish a genetic transformation system; the soybean is difficult to establish a mutant library, and the construction process is complex. Therefore, the discovery and research of legume model plants are the key to solving this problem.

The medicago truncatula has the characteristics of small genome, self-pollination, easy genetic transformation and the like as leguminous model plants, so that the research on the development mechanism of the medicago truncatula seeds can not only enable people to better understand the development of the leguminous plant seeds, but also play a guiding role in improving the yield and the quality of soybeans in China.

WUSCHEL relatedhomeobox (WOX) family genes are a class of plant-specific transcription factors. The gene plays an important role in regulation and control in the key period of plant development, including the maintenance of stem and root apical stem cells, the establishment of embryonic development basal-apical axis polarity patterns, the development of lateral organs and the like (Schoof et al, 2000; van der Graaff et al, 2009; Ueda et al, 2011). The WOX11 gene is a member of the WOX family. Upon search, in Arabidopsis, WOX11 was involved in the generation of regenerated root organs. In rice, WOX11 regulates the growth and development of adventitious roots. However, the search finds that the nucleotide sequence and the amino acid sequence of the alfalfa WOX family gene Mt WUSCHEL relatedhomeobox 11(MtWOX11) are not reported, and the application of the gene in increasing the fatty acid content of seeds of leguminous plants is not reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a medicago truncatula WOX family gene Mt WUSCHEL relatedhomeobox 11(MtWOX11) and application thereof in increasing the fatty acid content of leguminous plant seeds.

The alfalfa WOX family gene Mt WUSCHEL relatedhomeobox 11(MtWOX11) of the invention is characterized in that: the gene is named as medicago truncatula MtWOX11 gene, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.

The invention relates to an application of medicago truncatula MtWOX11 gene in improving the fatty acid content of leguminous plant seeds.

Wherein: the leguminous plant is preferably selected from herba Medicaginis, herba Medicaginis Tribuli, semen glycines, and herba Trifolii Pratentis.

The alfalfa MtWOX11 gene of the invention is used for constructing a plant overexpression vector, and plant transgenic operation is carried out to introduce the vector into plant cells, so as to obtain transgenic plants. Detection shows that the content of fatty acid in the obtained seeds of the transgenic plants is obviously improved. In order to facilitate the selection of transgenic plants or cell lines, plant expression vectors (pEARLYGATE103-MtWOX11) containing the gene MtWOX11 may be processed, for example, selection markers (GUS, etc.) or antibiotic markers having resistance (hygromycin, kanamycin, gentamicin, etc.) may be added.

Practically, any vector that can introduce a foreign gene into a plant for expression can be used, and a preferred vector of the present invention is pEARLYGATE 103.

The invention utilizes the disclosed medicago truncatula mutant library to obtain a knockout mutant strain of the MtWOX11 gene of the medicago truncatula through separation and screening, and utilizes primer sequences shown in SEQ ID No.2 and SEQ ID No.3 to clone the MtWOX11 gene from the medicago truncatula through RT-PCR technology by separating and researching the knockout mutant strain of the MtWOX11 gene of the medicago truncatula.

And (3) retrieval and display: the invention clones the medicago truncatula MtWOX11 gene and the coded amino acid sequence thereof for the first time, and obtains a transgenic plant over-expressing MtWOX11 by a genetic transformation method. This is the first time that the MtWOX11 gene was cloned and overexpressed in medicago truncatula. Transgenic line analysis proves that the fatty acid content of seeds of leguminous plants can be remarkably improved by over-expressing the gene (figure 4). The gene is proved to participate in regulating and controlling the fatty acid content of the seeds of the alfalfa of the leguminous plant, and the fatty acid content of the seeds of the transgenic plant can be obviously improved by carrying out the plant gene overexpression genetic transformation operation on the MtWOX11 gene of the alfalfa of tribulus. The gene of the invention is indicated to have wide application prospect. The application of the method can be used for improving the quality of beans or creating novel leguminous economic crops, and has great significance for the production of the leguminous economic crops in China.

Drawings

FIG. 1: and (3) analyzing MtWOX11 gene of medicago truncatula.

Wherein WT is a wild type plant of Medicago truncatula and mtwox11 is a mutant. A: illustrating the genetic structure of Medicago truncatula MtWOX11 and the insertion position of Tnt1 transposon in the mutant; b: RT-PCR result analysis of the medicago truncatula MtWOX11 gene, and Actin is used as an intragenic reference control. Molecular biological identification results show that Tnt1 is inserted into the second exon of the MtWOX11 gene (FIG. 1A), the CDS of the coding region of the MtWOX11 gene of the medicago truncatula is 819bp, and RT-PCR results show that the insertion of Tnt1 causes the MtWOX11 not to be normally expressed (FIG. 1B).

FIG. 2: alignment of amino acid sequences encoded by the Medicago truncatula MtWOX11 gene.

The Medicago truncatula MtWOX11 gene encodes a 29.92kDa protein of 272 amino acids with a "helix-loop-helix-turn-helix" domain (shown by the symbol) typical of the HOX superfamily. Wherein the plant comprises MtWOX11 in Medicago truncatula, AtWOX11 in Arabidopsis, GLYMA19G118400 in soybean, and OsWOX11 in rice; the amino acid similarity of MtWOX11 and AtWOX11 is 47%, the amino acid similarity of MtWOX11 and GLYMA19G118400 is 60%, and the amino acid similarity of MtWOX11 and OsWOX11 is 47%.

FIG. 3: and analyzing the expression pattern of the Medicago truncatula MtWOX11 gene.

The qRT-PCR result shows that the MtWOX11 gene is constitutively expressed and is expressed in each organ, but the expression level in seeds is highest, the expression level in flowers, roots and leaves is higher, and the expression level in stems, petioles and terminal buds is lower.

FIG. 4: and analyzing the fatty acid content of the seeds of the transgenic plants over expressing MtWOX 11.

Grey bar graph shows the ratio of fatty acid content in MtWOX11 mutant versus wild type seeds, black bar graph shows the ratio of fatty acid content in over-expressed MtWOX11 transgenic plants versus wild type seeds. Indicates significant difference, Anova Single Factor p <0.05, indicates very significant difference, Anova Single Factor p < 0.01. The results show that the total fatty acid content in the seeds of the overexpression transgenic plants is increased by 12.2168% compared with the wild type, and particularly, the content of fatty acids such as C15.0 (pentadecanoic acid), C15.1 (cis-10-pentadecanoic acid), C16.0 (palmitic acid), C18.2N6C (linoleic acid), C20.2 (cis-11, 14-eicosadienoic acid), C20.3N6 (cis-8, 11, 14-eicosatrienoic acid), C20.4N6 (arachidonic acid), C20.5N3 (cis-5, 8, 11, 14, 17-eicosapentaenoic acid) and C24.0 (tetracosanoic acid) is reduced in the mutants, and is increased in the overexpression MtWOX11 transgenic lines, wherein the content of pentadecanoic acid is obviously increased.

Detailed Description

Example 1 cloning and expression analysis of the Medicago truncatula MtWOX11 Gene

1.1 obtaining and identification of Medicago truncatula mtwox11 mutants

Screening a mutant library marked by medicago truncatula Tnt1 through a public website https:// media-mutated. noble. org/mutant/database. php, and screening out mutant strains Tnt1 inserted into the MtWOX11 gene from 22,000 mutant strains by using a Thermal asymmetric intercalary-PCR (TAIL-PCR) technology.

1.1.1 extraction of Total RNA of Medicago truncatula mtwox11

(1) Placing 0.1g of tissue material into a mortar precooled by liquid nitrogen, and fully grinding the tissue material into powder in the liquid nitrogen;

(2) after the liquid nitrogen had evaporated to dryness, the mixture was immediately transferred to a 2ml centrifuge tube, about 1ml of TRIzol extract from Invitrogen was added to each 100mg of the material, and after thawing, the sample was repeatedly sucked and blown with a sample gun, and the sample was vigorously shaken and mixed to be sufficiently lysed, and then allowed to stand at room temperature for 5 minutes.

(3) Adding 0.2ml of chloroform, violently shaking and uniformly mixing for 15 seconds, and standing for 10 minutes at room temperature;

(4) centrifuging at 12000rpm for 15 min at 4 ℃;

(5) carefully sucking out the upper aqueous phase by using a pipette, adding the upper aqueous phase into a new centrifugal tube of 1.5ml, adding 500 mu l of isopropanol (1:1 volume), fully mixing the mixture, and precipitating the mixture at the temperature of minus 20 ℃ for 30min or overnight;

(6) centrifuging at 4 deg.C and 12000rpm for 10min, and carefully discarding the supernatant;

(7) the RNA pellet was washed with 1ml of 75% ethanol; centrifuging at 4 deg.C and 8000rpm for 10min, and collecting precipitate;

(8) washing the RNA precipitate once by using 75% ethanol repeatedly;

(9) removing supernatant, air drying RNA precipitate on sterile operating platform for about 10-15 min, making RNA be transparent, adding RNase-free water with appropriate volume (30-50 μ l), and dissolving thoroughly (can be stored at-80 deg.C for a long time);

(10) an ultraviolet spectrophotometer and 1% Agrose gel electrophoresis are used for detecting the concentration and the quality of RNA.

Note: a) the RNA yield was measured by UV spectrophotometer at 260nm absorbance of 40. mu.g/ml at 1 OD. According to the absorbance values at 260nm and 280nm, the purity of the RNA is detected, and the OD260/OD280 ratio of the pure RNA is close to 2.0 (the ratio is preferably between 1.9 and 2.1).

b) The quality and size of the RNA was examined by electrophoresis on a 1% Agrose gel. Mu.l of RNA was aspirated, 3. mu.l of RNase-free water was added, and 1. mu.l of loading buffer was added to denature at 65 ℃ for 5 minutes. After electrophoresis, the cells were stained with EB, and 3. mu.l of 1kb DNA Marker was used as a control.

1.1.2cDNA reverse transcription

Reverse transcriptase: transcriptor Reverse Transcriptase (Roche).

(1)13 μ l reaction System

(2) Denaturation at 65 ℃ for 10min, rapid insertion into ice, followed by addition of:

(3) mixing the mixture lightly, and reacting for 10min at 25 ℃; reacting at 55 ℃ for 30 min;

(4) diluted with ultrapure water to the appropriate concentration. As a template for PCR.

1.1.3RT-PCR

(1) PCR reaction (20. mu.l):

(2) the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 30sec, renaturation at 55 deg.C for 30sec, extension at 72 deg.C for 1min, and circulation for 30 times; extension at 72 ℃ for 10 min.

1.2. Cloning of Medicago truncatula MtWOX11 gene

The MtWOX11 gene is cloned from medicago truncatula by RT-PCR technology by using the primer sequences shown in SEQ ID No.2 and SEQ ID No. 3. The nucleotide sequence of the medicago truncatula MtWOX11 gene is shown as SEQ ID No. 1.

The full-length coding region (CDS) sequence of the MtWOX11 gene was amplified using the primers shown in SEQ ID No.2 and SEQ ID No. 3. The CDS sequence was ligated into pEARLEYGATE103 vector using Gateway technology and then sequenced to verify the correctness of the cloned sequence.

1.2.1 Total RNA of wild plant seeds of medicago truncatula is extracted by the same method.

1.2.2 reverse transcription to generate cDNA, as described above.

1.2.3 cloning and sequencing of the open reading frame

The primer sequence is as follows: see the attached figures SEQ ID No.2 and SEQ ID No.3

PCR reaction (50. mu.l):

the PCR reaction program is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 deg.C for 30sec, renaturation at 63.5 deg.C for 10sec, extension at 72 deg.C for 1min, and circulation for 35 times; extension at 72 ℃ for 7 min.

The amplified fragment was recovered, ligated with pENTR/D-TOPO vector and transformed into E.coli DH 5. alpha. and sequencing was carried out by Pidaemon Biotechnology (Shanghai) Co., Ltd.

pENTR/D-TOPO vector connection reaction system:

(1) the method comprises the following steps:

DNA (ng) connecting fragment length (bp) × 0.007

Salt solution：1μl

ddH 2O: add to a final volume of 50. mu.l

(2) Mu.l of pENTR/D-TOPO vector is added into 2.5. mu.l of the system.

(3) The above systems were ligated for 1 hour at 22 ℃.

(4) The above system was transformed into E.coli DH5 α.

1.3. Sequence information of Medicago truncatula MtWOX11 gene

Sequence information fromhttp://www.medicagogenome.org/And (5) obtaining the website. The nucleotide sequence of the medicago truncatula MtWOX11 gene is shown as SEQ ID No. 1.

1.4. Expression analysis of Medicago truncatula MtWOX11 gene

Extracting RNA from root, stem, leaf, flower, seed, petiole and terminal bud of wild alfalfa (R108) to perform tissue expression analysis.

1.4.1 extraction of RNA

After the medicago truncatula seeds germinate, the root, stem, leaf, flower, seed, petiole and terminal bud parts of the seedlings which grow for 16 weeks are taken to extract RNA by the same method.

1.4.2 Reverse Transcription (RT) to generate cDNA

1. Reverse transcription generates cDNA as described above.

PCR reaction and data processing method

(1) PCR was performed using cDNA as a template.

(2) And (3) PCR system:

(3) PCR procedure:

10min at 95 ℃; 10S at 95 ℃, 15S at 59 ℃, 30S at 72 ℃ and 41 cycles; melting curves are made at intervals of 0.5 ℃ at 65-95 ℃; storing at 10 deg.C.

(4) And judging the product unicity and the dissolution temperature by using the melting curve, and calculating the relative content of the target gene in different samples by using a delta Ct algorithm by using the given Ct value.

Example 2 construction of plant expression vectors

The full-length cDNA sequence of MtWOX11 was constructed into pENTR/D-TOPO intermediate vector, and then the full-length CDS sequence of MtWOX11 was ligated into pEarleyGate103 overexpression vector by LR recombination. Obtaining the plant expression vector which drives the overexpression of the MtWOX11 gene by using a CaMV35S promoter.

2.1MtWOX11 full-length CDS sequence was constructed into pENTR/D-TOPO vector as above.

2.2 recombinant ligation to pEarleyGate103 overexpression vector.

(1) Reaction system:

pENTR/D-TOPO vector 1. mu.l

1 μ l of pEarleyGate103 overexpression vector

2X Fuzyme MasterMix:0.5μl

(2) The reaction mixture was incubated at 25 ℃ for 2-3 hours.

(3) The above system was transformed into E.coli DH5 α.

2.3 identification of recombinants

(1) PCR validation of plasmids

Single colonies are selected and respectively inoculated in 5ml LB liquid culture medium containing Kan to be shaken and cultured at 37 ℃ overnight, plasmids are extracted by an alkaline denaturation method, and PCR amplification is carried out by gene specific primers.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; 30sec at 94 ℃, 30sec at 55 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min. The PCR product was identified by electrophoresis on a 1.0% agarose gel.

(2) And (5) sequencing and identifying.

Example 3 preparation and transformation of Agrobacterium competence

3.1 preparation of Agrobacterium AGL1/EHA105 competence

1. A single colony of Agrobacterium tumefaciens was picked from a YEP plate (containing 50. mu.g/ml rifampicin), inoculated into a YEP liquid medium (containing 50. mu.g/ml rifampicin), cultured at 200rpm/min at 28 ℃ overnight.

2. Inoculating 2ml of overnight culture medium into 50ml of YEP liquid medium containing the same antibiotic, and culturing under the same conditions to OD₆₀₀Up to 0.5.

3. The bacterial liquid is subjected to ice bath for 30min, centrifuged at 4 ℃ and 5000rpm for 10min, and the thalli are collected.

4. The cells were resuspended in 10ml of 0.15mol/L NaCl in an ice bath, and the cells were collected by centrifugation.

5. Resuspended in 1ml of 20mmol/L ice-cooled CaCl₂In the solution, the bacterial suspension was dispensed into a 1.5ml Eppendorf tube at 200. mu.l/tube, and frozen in liquid nitrogen for 1min and stored at-70 ℃ for further use.

3.2 Freeze thawing method for transformation of Agrobacterium tumefaciens EHA105

1. The agrobacterium competent cells were thawed at room temperature, 1 μ g of expression vector plasmid DNA was added, mixed well and ice-cooled for 30 min.

2. Quickly freezing in liquid nitrogen for 1min, and rapidly transferring to 37 deg.C and maintaining for 3 min.

3. Add 800. mu.l of YEP without antibiotics, shake-incubate for 3 hours at 28 ℃.

Cells were collected by centrifugation at 4.7000rpm for 30s, plated on YEP plates containing rifampicin at 50. mu.g/ml and Kan at 50. mu.g/ml, and cultured in the reverse dark at 28 ℃ for 2-3 days.

3.3 PCR identification of the cells

Single colonies were picked and transferred to a PCR system (without DNA template) as described above at 2.3 and PCR amplified with gene specific primers. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; 30sec at 94 ℃, 30sec at 55 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min. The PCR product was identified by electrophoresis on a 1.0% agarose gel.

Example 4 transgenic functional validation-Medicago truncatula transformation screening

Agrobacterium EHA105 containing the plant expression vector was used to impregnate leaves of Medicago truncatula using tissue culture (a published general procedure). Then callus formation and embryo formation are induced. After the seedlings grow out, extracting DNA and carrying out PCR screening to obtain a homozygous transgenic plant line.

4.1 alfalfa leaf transformation

The agrobacterium EHA105 has the capability of infecting plants and transferring genes, so the constructed plant expression vector of the MtWOX11 gene coding region is transferred into agrobacterium and then PCR verification is carried out. Agrobacterium EHA105 containing the plant expression vector was used to impregnate leaves of Medicago truncatula using tissue culture (a published general procedure). Then callus formation and embryo formation are induced. After the seedlings grow out, extracting DNA and carrying out PCR screening to obtain a homozygous transgenic plant line.

4.1.1 tissue culture method

(1) The healthy and bigger leaves are taken and the petioles are removed and placed in the tube. Washing with sterile water containing 20% NaClO and 0.1% Tween-20 for 15 min, and washing with sterile water for 3-5 times.

(2) The leaves were cut into small pieces to create wounds.

(3) The Agrobacterium was shaken to an OD of 0.6-0.8. The cells were collected by centrifugation and then resuspended to an OD of 0.1-0.2 using SH3a medium.

(4) Pouring the prepared bacterial liquid into the cut leaves. Vacuumizing for 10-15 min and slowly shaking for 10-15 min. The leaves were plated out as densely as possible on SH3a medium which was not resistant. Culturing at room temperature in dark.

(5) And washing the mixture for three times overnight with deionized water until the mixture is clear, and then washing the mixture with cafe-added deionized water.

(6) Transferred to a resistant SH3a, cultivated in a greenhouse for 4-6 weeks in the absence of light, and then the callus was transferred to SH9a medium. Transferring to 1/2MS culture medium after callus is differentiated into bud. And transplanting to soil after seedling emergence.

4.1.2 transgenic shoot detection

DNA was extracted and PCR detected (same procedure as above).

4.2 molecular characterization of transgenic plants

And detecting the gene expression level of the transgenic plant. Respectively extracting RNA of the transgenic plant and the wild plant, carrying out RT-PCR amplification after reverse transcription, and analyzing the gene expression difference of the over-expressed plant and the wild plant. The expression level of MtWOX11 in the over-expression plants is obviously higher than that of wild plants.

RT-PCR results show that the MtWOX11 gene is constitutively expressed and is expressed in all organs, but the expression level is highest in seeds, higher in flowers, roots and leaves and lower in stems, petioles and terminal buds. (see FIG. 3).

And a strain with the highest MtWOX11 expression amount is used for subsequent work.

RNA extraction, reverse transcription, RT-PCR real-time quantitative PCR (same method as above).

Example 5 transgenic functional verification-functional analysis of seed fatty acid quality

The analysis of mtwox11 mutant and overexpressed transgenic plant seed quality was performed by sumisco caminot biotechnology limited.

The analysis result of the fatty acid content of the seeds of the transgenic plant overexpressing MtWOX11 is shown in FIG. 4.

Wherein: grey bar graph shows the ratio of fatty acid content in MtWOX11 mutant versus wild type seeds, black bar graph shows the ratio of fatty acid content in over-expressed MtWOX11 transgenic plants versus wild type seeds. Indicates significant difference, Anova Single Factor p <0.05, indicates very significant difference, Anova Single Factor p < 0.01.

The results show that the total fatty acid content in the seeds of the overexpression transgenic plants is increased by 12.2168% compared with the wild type, and particularly, the content of fatty acids such as C15.0 (pentadecanoic acid), C15.1 (cis-10-pentadecanoic acid), C16.0 (palmitic acid), C18.2N6C (linoleic acid), C20.2 (cis-11, 14-eicosadienoic acid), C20.3N6 (cis-8, 11, 14-eicosatrienoic acid), C20.4N6 (arachidonic acid), C20.5N3 (cis-5, 8, 11, 14, 17-eicosapentaenoic acid) and C24.0 (tetracosanoic acid) is reduced in the mutants, and is increased in the overexpression MtWOX11 transgenic lines, wherein the content of pentadecanoic acid is obviously increased.

The results prove that the MtWOX11 gene participates in regulating and controlling the content of the fatty acid in the seeds of leguminous plants, and the content of the fatty acid in the seeds can be obviously improved by over-expressing the gene. The MtWOX11 gene is predicted to have an important function in increasing the fatty acid content of leguminous seeds.

Sequence listing

<110> Shandong university

<120> Medicago truncatula MtWOX11 gene and application thereof in increasing content of fatty acid in seeds

<141>2017-11-20

<160>3

<210>1

<211>3627

<212>DNA

<213> Artificial sequence

<220>

<223> Medicago truncatula WUSCHEL-related homoeobox 11 genome nucleotide sequence

<400>1

aggtttcagt ttcatttcaa gtttcaacaa aacatatttt gttatttctt ctatctatag 60

tgcattggaa tggaagataa gatgcaacat gaccccccca acaacactcc aacccaacat 120

ggttccgaga aaactgaacc ggttaggtca aggtggacac caaaaccaga acaaattctc 180

atacttgagt ccatcttcaa cagtggcatg gtgaatcccc caaaagaaga aactatcaaa 240

ataagaaaac ttcttgagaa attcggcaac gtcggtgacg ccaacgtctt ctactggttc 300

caaaaccgcc ggtctagatc tcgccgccga caacgccaga tgcagcaggc cacacttgat 360

cagcaaagaa atcagatggc tatgatgcag cctcagcaag ttgttaacga tggtgcaagt 420

gcaattcctt gtgatatggt tcaaaccaac ccaaccatgg tttttggtgg ttcctcttct 480

tgtttaaatg attcttccgg ttcttcctct tcatcatgtg gtggtgttct tagtggtcaa 540

caaggcatgg atggtttgtt ttcagtttct tctcaaatgg gttttcttgg agttgatcaa 600

actttagctg caccatcact tttgtgccct tctctttctc ctaatttcaa ctatcactct 660

ggtaattggc tgcatgtctt ttatttattt attatttatt ttatcactaa aaaatttatt 720

ttaaaatcca tggagcactc caattcatac taaaatcaca aaagattatt tgttcaaaat 780

catttagaat ctccacaaat aattttgtat tattcaaaaa agctaactta aaattatttt 840

ttcaatattt ttttttataa atacacgaga tatactccaa catattacat caaagcctaa 900

tccgattttg ataaatttca aacccactag aaatttgttt attatatgca tggttgtgtt 960

tatcaatttt attttacttt ttatgagatt tatttgtaaa aatataaaaa ataagattag 1020

agaaagcata tacaaagata agagatcgta ataaaaagtt aaaagagttt cattttttaa 1080

taattttctt tttttggtat ccctaaccgt gcaccggtta gcgtcaccct taacattttc 1140

attggatatt tatggtgaaa tttacatata gcaaatcaat tgccgaattc tggctttgaa 1200

ccaaagttta aatatgtgat atgaaagatc caatcatatg tgtcaattac gatcgatcat 1260

tttttatgtg ctttgagctg gaatatttcc tcctattcaa tatatatttt cttgcataaa 1320

ttctaaatct gatgctacaa aatttatttt cttatcagtt gttattaatg catatttgaa 1380

ttcatagttg gttcttcaca aaaataaaat aattcatcca tgctcttagt ttaaaaaagt 1440

tcatgtttat ggaatgattg attaagtttg aggatgggta cagaataaag tgggtagctt 1500

ttccttgatt tgacagttcc gcgtttgaat tgttgcatga aagttgttct ttccggaaaa 1560

acatgaacgc tactttccgg ctgcagtttc tgttctttcc tttttattct ctcacttttt 1620

tcgactttct tcaatagtaa tactcctgct tctgcacaaa aaatttcaaa ccctagatta 1680

tgtttttatt tttatttttt tatatcatta tcatttcatc tgatggttac cctcttagta 1740

aatattcacc atacattact agtttttttc catcattttt ataaaattat tcaactttac 1800

cgttaaaaaa aagctcatta atctctcaat tgtttggtta actacgatgt taattactgg 1860

ttaaaagaaa tccataattt tcatatcttt tgttattttt tttaacaaac caaaatggaa 1920

tatattaaga aaaaaggagg cttctaataa gcagtgtgcc aaaaagacct caaaaatata 1980

caaggaaata aatcacaagg gaaccaaaac aaaaagaatt tatccgacgc ccaaacaaac 2040

aaatgggttc gaccactgca tatgaaagtt tagcctaatg tggtcatcaa agaaaacttg 2100

ctttgttcat tgcatgcgtt acatattgtt ttcttgcatt tttttcagtg attgagatat 2160

gaaattcagg agtaaattga gattcaaagt gtttgctgaa acattaatga gatcaacttc 2220

ttttatgctt taatttttgt atttgaccaa tatgtaaggt gatgttctat tttttatttt 2280

tttttggtca agttctaaca atattttggt attattaagg atttggaggt gctagtactg 2340

taacaggatt ggcaacagtg tttatcaatg ggattgcaac agaaattcaa gcagggccac 2400

tagacatcaa aacagtgttt ggagaagatg tgatgttagt tcattcctct ggtgtgccag 2460

ttcccacaaa tgaacatggc atcttgattc agagcttgca ccatggtgaa agctactttc 2520

tggtacttgc cacataccat catacatctt caaatcaatt gtttctattc taaatattgt 2580

gttgtcacat cttaatcatt gttctttatt ttcttttcaa tccttatgtc tactaatttg 2640

aaaattaatt cacacacatg tgcacatatg tgtaggtaca atcattttaa aatgtattca 2700

acgatttgtt gtcacatcaa taaatgaata tgaaaatcac atagattttc gtattcatta 2760

attgatgtga caatacatta tttgatgcat gtgtaaaata attgtatatt tagagcatcc 2820

ataatggaaa ccctaaattt agagttctta aactggtctc acgtgaacac gtcactcttt 2880

aataatattt taataacagt atctaataaa cactcaatca ctacaataga gatcctctaa 2940

caaaatgtct aaaccggtcc caccactaac tcttcatcat ttacatttca acaatacaac 3000

gattatttta ataaaaagta gtgtggagcc cacttaagaa tggggtttta aatgattcca 3060

acacccaaaa aaaaaatctc caataaagat acctattagg tgccggatct taaatgatga 3120

agacctcacc atatgagatg gtcttacaaa acatattaat taaactcttg aaaatatcga 3180

gcatatcata catacaataa atgtaaaaca attttacaca tatccaagaa aacaatttta 3240

cacatatatc taaattgttc aatatgttat tgtatagaaa tatatttttt agagttattg 3300

tatagaaaca tgttatatga aaacattcac ttcaaaatgt tttgggagtg atcacattga 3360

actaaagcta cttaggattt atctactcct atgttctaat cttgtcatat aaaaaatagt 3420

ctactcctat gttttaattc actaaacata atatttttca cttacattat tttgaattcc 3480

tactactcca gtaattaagg agtttttttg tgtatcatga ttattaaact ttggtttaca 3540

ggtatcaaag tcagcacaag tttgaactga ccatgctcta atgctggagt tggaaggtcc 3600

ctatgcgtgt gctcctctct ttttaac 3627

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>2

atggaagata agatgcaaca tg 22

<210>3

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>3

aacttgtgct gactttgata ccaga 25

Claims (3)

1. An application of medicago truncatula MtWOX11 gene in improving the fatty acid content of leguminous plant seeds, wherein the nucleotide sequence of the medicago truncatula MtWOX11 gene is shown as SEQ ID No. 1.

2. Use according to claim 1, characterized in that: the leguminous plant is selected from alfalfa, soybean, clover.

3. Use according to claim 2, characterized in that: and selecting the medicago truncatula.

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