CN107779456A - M. truncatula MtWOX11 genes and its application in seed fatty acid content is improved - Google Patents

Abstract

The invention discloses a kind of M. truncatula WOX family genes Mt WUSCHEL related homeobox11 (MtWOX11), and the nucleotide sequence of the gene is as shown in SEQ ID No.1.The invention also discloses application of the gene in seeds of leguminous plant content of fatty acid is improved.By the transgenic line analytical proof obtained to the method for genetic transformation, the content of fatty acid that the gene is remarkably improved the seed of legume is overexpressed.Indicate that gene of the present invention is with a wide range of applications, it is implemented that beans quality-improving will be used for or creates new pulse family industrial crops, and the production of China beans industrial crops is significant.

Description

M. truncatula MtWOX11 genes and its application in seed fatty acid content is improved

Technical field

The present invention relates to WUSCHEL relatedhomeobox (WOX) family genes and its application, more particularly to a kind of puncture Lamb's-quarters clover WOX family gene Mt WUSCHEL relatedhomeobox 11 (MtWOX11) and its improve seeds of leguminous plant Application in content of fatty acid, belongs to genetic engineering field.

Background technology

In recent years, it is also growing day by day to the demand of soybean with the raising of China's economic level and people's living standard. Soybean is one of important pulse family industrial crops, and it can not only provide high protein, and more important is can provide oil.But The soybean quality in China is not high at present, lacks the market competitiveness, it is necessary to a large amount of imported soybeans.Therefore the big of high oily high protein is cultivated Beans new varieties have turned into current very urgent task.

At present, China's soybean yields and the research work of quality-improving are concentrated mainly on conventional breeding field, and molecule is lost Improvement is passed also in the starting stage.Its reason essentially consists in：Soybean is tetraploid, and genome is larger；Soybean is difficult to set up something lost Pass transformation system；Soybean is difficult to set up mutant library, and its building process is more complicated.Therefore beans model plant is excavated to go forward side by side Row research is the key point for solving this problem.

M. truncatula has the characteristics that small genome, self-pollination, genetic transformation are easy as model legume, therefore M. truncatula seed development mechanism is studied can not only let us be best understood from the development of seeds of leguminous plant, moreover it is possible to it is right The yield and quality for improving China soybean plays the effect of directiveness.

WUSCHEL relatedhomeobox (WOX) family gene is the distinctive a kind of transcription factor of plant.Family's base Because playing critically important regulating and controlling effect in the critical period of development of plants, include the maintenance of stem and roots and tops stem cell, embryo's hair Educate the foundation of base-apical axis polar mode and the development of lateral organ etc. (Schoof et al., 2000；van der Graaff et al.,2009；Ueda et al.,2011).WOX11 genes are a members in WOX families.Through retrieval, in arabidopsis In, WOX11 participates in the generation of regenerated root organ.In rice, WOX11 regulates and controls the growth and development of adventitious root.But retrieval hair It is existing, about (MtWOX11) nucleotide sequences of M. truncatula WOX family gene Mt WUSCHEL relatedhomeobox 11 and Amino acid sequence has not been reported, and its application in seeds of leguminous plant content of fatty acid is improved also has no report.

The content of the invention

In view of the shortcomings of the prior art, the purpose of the present invention is a kind of M. truncatula WOX family gene Mt WUSCHEL Relatedhomeobox 11 (MtWOX11) and its application in seeds of leguminous plant content of fatty acid is improved.

M. truncatula WOX family genes Mt WUSCHEL relatedhomeobox 11 of the present invention (MtWOX11), it is characterised in that：The unnamed gene is M. truncatula MtWOX11 genes, and the nucleotide sequence of the gene is such as Shown in SEQ ID No.1.

Application of the M. truncatula MtWOX11 genes of the present invention in seeds of leguminous plant content of fatty acid is improved.

Wherein：The legume preferably is selected from clover, M. truncatula, soybean, clover.

Using the gene constructed plant over-express vectors of M. truncatula MtWOX11 of the present invention, plant transgene is carried out Operation imports plant cell, obtains genetically modified plants.Content of fatty acid in the seed for showing to obtain genetically modified plants is detected to obtain Significantly improve., can be to the plant containing the gene M tWOX11 for the ease of being screened to genetically modified plants or cell line Expression vector (pEARLYGATE103-MtWOX11) is processed, and can such as add selected marker (GUS) or resistant Antibiotic marker (hygromycin, kanamycins, gentamicin etc.) etc..

In fact, any carrier expressed that can import foreign gene in plant can be applied, the present invention is excellent The carrier of choosing is pEARLYGATE103.

M. truncatula mutant library disclosed in present invention utilization, separation screening have obtained M. truncatula MtWOX11 genes Knockout mutations body strain, by the knockout mutations body strain of Separation Research M. truncatula MtWOX11 genes, utilize SEQ ID Primer sequence shown in No.2 and SEQ ID No.3, MtWOX11 genes are cloned into by RT-PCR technology from M. truncatula.

Retrieval display：The present invention clones the amino acid sequence of M. truncatula MtWOX11 genes and its coding first, and The transfer-gen plant for being overexpressed MtWOX11 is obtained by the method for genetic transformation.This is MtWOX11 genes first in M. truncatula In be cloned, and be overexpressed in M. truncatula.Transgenic line analytical proof, it is overexpressed the gene and is remarkably improved pulse family The content of fatty acid (Fig. 4) of the seed of plant.This proves that the gene take part in the aliphatic acid of the seed of regulation and control legume clover Content, plant gene is carried out to M. truncatula MtWOX11 genes and is overexpressed genetic transformation operation, is remarkably improved genetically modified plants The content of the aliphatic acid of seed.Indicate that gene of the present invention is with a wide range of applications.Its implementation will be used for beans Quality-improving creates new pulse family industrial crops, and the production of China beans industrial crops is significant.

Brief description of the drawings

Fig. 1：M. truncatula MtWOX11 genetic analysis.

Wherein WT is that M. truncatula wild-type plant compares, and mtwox11 is mutant.A：Illustrate M. truncatula The insertion position of Tnt1 transposons in MtWOX11 gene structure and mutant；B：The RT-PCR of M. truncatula MtWOX11 genes Interpretation of result, Actin are that gene internal reference compares.Molecular biology identification result shows that Tnt1 is inserted into MtWOX11 genes Second extron (Figure 1A), the code area CDS of M. truncatula MtWOX11 genes is 819bp, and RT-PCR results show Tnt1's Insertion causes MtWOX11 to be unable to normal expression (Figure 1B).

Fig. 2：The amino acid alignment of M. truncatula MtWOX11 gene codes.

The 29.92kDa albumen of 272 amino acid of M. truncatula MtWOX11 gene codes, there is typical HOX superfamilies " helix-loop-helix-turn-helix (helix-loop-helix-turn-helix) " domain (* institutes Show).Wherein have the MtWOX11 in M. truncatula, the AtWOX11 in arabidopsis, the GLYMA19G118400 in soybean, in rice OsWOX11；MtWOX11 and AtWOX11 amino acid similarity is 47%, MtWOX11 and GLYMA19G118400 amino The amino acid similarity that sour similarity is 60%, MtWOX11 and OsWOX11 is 47%.

Fig. 3：The expression pattern analysis of M. truncatula MtWOX11 genes.

QRT-PCR results show although MtWOX11 genes belong to constructive expression, there is expression in each organ, but Expression quantity highest in seed, next to that the expression quantity in flower, root, leaf is higher, expression quantity is relatively low in stem, petiole, terminal bud.

Fig. 4：It is overexpressed the Analysis of Fatty Acid Content of MtWOX11 transfer-gen plant seeds.

Mtwox11 mutant shown in grey histogram and content of fatty acid in wild type seeds ratio, black histogram The ratio of shown overexpression MtWOX11 transfer-gen plants and content of fatty acid in wild type seeds.* significant difference, Anova are represented Single Factor p<0.05, * * represents pole significant difference, Anova Single Factor p<0.01.As a result show, mistake Total fatty acid content adds 12.2168%, particularly C15.0 (15 compared with wild type in express transgenic plant seed Alkanoic acid), C15.1 (cis- 10- pentadecylenic acids), C16.0 (palmitic acid), C18.2N6C (linoleic acid), C20.2 (cis- 11,14- bis- Eicosadienoic acid), C20.3N6 (cis- 8,11,14- eicosatrienoic acids), C20.4N6 (arachidonic acid), C20.5N3 it is (suitable Formula-EPA), under the content of the aliphatic acid such as C24.0 (lignoceric acid) has in mutant Drop, and have rising in MtWOX11 transgenic lines are overexpressed, wherein, the content of pentadecanoic acid significantly improves.

Embodiment

The M. truncatula MtWOX11 gene cloning and expressions of embodiment 1 are analyzed

The acquisition and identification of 1.1 M. truncatula mtwox11 mutant

Pass through open website https://medicago-mutant.noble.org/mutant/database.php is screened The mutant library of M. truncatula Tnt1 marks, and utilize Thermal asymmetric interlaced-PCR (TAIL- PCR) technology, the mutant strain that Tnt1 is inserted into MtWOX11 genes is filtered out in 22,000 mutant strains.

1.1.1 M. truncatula mtwox11 total serum IgE is extracted

(1) 0.1g organization materials are put into the mortar of Liquid nitrogen precooler, powder is fully ground into liquid nitrogen；

(2) treat that liquid nitrogen volatilization is dry, be immediately transferred in 2ml centrifuge tube, about add 1ml's per 100mg materials The TRIzol extract solutions of Invitrogen companies, after thawing, are inhaled and blown repeatedly with sample loading gun, and acutely vibration mixes sample, makes sample Fully cracking, room temperature are placed 5 minutes.

(3) 0.2ml chloroforms are added, acutely vibration is mixed 15 seconds, and room temperature is placed 10 minutes；

(4) 4 DEG C, 12000rpm is centrifuged 15 minutes；

(5) upper strata aqueous phase is carefully suctioned out with pipettor, added in new 1.5ml centrifuge tube, add 500 μ l isopropanol (1:1 volume), fully mix, -20 DEG C, precipitate 30min or stay overnight；

(6) 4 DEG C, 12000rpm centrifugation 10min, careful abandoning supernatant；

(7) RNA precipitate is washed with 1ml 75% ethanol；4 DEG C, 8000rpm centrifugations 10min collects precipitation；

(8) repeat to washed once RNA precipitate with 75% ethanol；

(9) supernatant is removed, for RNA precipitate in being dried on aseptic operating platform about 10-15 minutes, RNA shows slightly transparent, adds appropriate bulk The RNase-free water of product (30-50 μ l) fully dissolves and (can be placed on -80 DEG C long-term preserve)；

(10) ultraviolet specrophotometer and 1%Agrose detected through gel electrophoresis RNA concentration and quality.

Note：A) UV spectrophotometer measuring RNA yield, the absorbance at 260nm, 1OD=40 μ g/ml are used.Root According to the light absorption value at 260nm and 280nm, RNA purity is detected, the OD260/OD280 ratios of pure rna should be close to 2.0 (ratios Preferably between 1.9~2.1).

B) with 1%Agrose gel electrophoresises inspection side RNA quality and size.Draw the RNase- that 1 μ l RNA add 3 μ l Free water, 65 DEG C of 1 μ l sample-loading buffers are added to be denatured 5 minutes.Dyed after electrophoresis with EB, separately take 3 μ l 1kb DNA Marker to make For control.

1.1.2cDNA reverse transcription

Reverse transcriptase：Transcriptor Reverse Transcriptase(Roche).

(1) 13 μ l reaction systems

(2) 65 DEG C of denaturation 10min, are rapidly inserted into ice, then add:

(3) gently mix, 25 DEG C of reaction 10min；55 DEG C of reaction 30min；

(4) it is diluted to suitable concn with ultra-pure water.As pcr template.

1.1.3RT-PCR

(1) PCR reaction systems (20 μ l)：

(2) PCR response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30sec, 55 DEG C of renaturation 30sec, 72 DEG C extend 1min, circulate 30 times；72 DEG C of extension 10min.

1.2. the clone of M. truncatula MtWOX11 genes

Using the primer sequence shown in SEQ ID No.2 and SEQ ID No.3, by RT-PCR technology from M. truncatula It is cloned into MtWOX11 genes.The nucleotide sequence of M. truncatula MtWOX11 genes is as shown in SEQ ID No.1.

Utilize the primer shown in SEQ ID No.2 and SEQ ID No.3, the full length coding region of amplification MtWOX11 genes (CDS) sequence.CDS sequences are connected into pEARLEYGATE103 carriers using Gateway technologies, sequencing is then carried out, tests Demonstrate,prove the correctness of cloned sequence.

1.2.1 the Total RNA of M. truncatula WT lines seed are extracted, method is same as above.

1.2.2 reverse transcription produces cDNA, and method is same as above.

1.2.3 the clone of ORFs and sequencing

Primer sequence：See accompanying drawing SEQ ID No.2 and SEQ ID No.3

PCR reaction systems (50 μ l)：

PCR response procedures are：98 DEG C of pre-degeneration 2min；98 DEG C of denaturation 30sec, 63.5 DEG C of renaturation 10sec, 72 DEG C extend 1min, circulate 35 times；72 DEG C of extension 7min.

It is connected after amplified fragments recovery with pENTR/D-TOPO carriers and converts bacillus coli DH 5 alpha, is sequenced still biological by platinum Technology (Shanghai) Co., Ltd. completes.

PENTR/D-TOPO carrier coupled reaction systems:

(1) system：

DNA(ng):Junction fragment length (bp) × 0.007

Salt solution：1μl

ddH2O：Add to the μ l of final volume 50

(2) the pENTR/D-TOPO carriers for taking the μ l of above system 2.5 to add 0.5 μ l.

(3) by the connection 1 hour of 22 DEG C of above system.

(4) above system is converted into bacillus coli DH 5 alpha.

1.3. the sequence information of M. truncatula MtWOX11 genes

Sequence information fromhttp://www.medicagogenome.org/Website obtains.M. truncatula MtWOX11 genes Nucleotide sequence as shown in SEQ ID No.1.

1.4. the expression analysis of M. truncatula MtWOX11 genes

Root, stem, leaf, flower, seed, petiole, the terminal bud position extraction RNA of the wild type (R108) of M. truncatula is taken to carry out group Knit expression analysis.

1.4.1RNA extraction

After M. truncatula seed is sprouted, the root of seedling of growth 16 weeks, stem, leaf, flower, seed, petiole, terminal bud position are taken, is carried RNA is taken, method is same as above.

1.4.2 reverse transcription (RT) produces cDNA

1. reverse transcription produces cDNA, method is same as above.

2.PCR reacts and data processing method

(1) using cDNA as template, performing PCR reaction is entered.

(2) PCR system：

(3) PCR programs：

95℃10min；95 DEG C of 10S, 59 DEG C of 15S, 72 DEG C of 30S, 41 circulations；65 DEG C -95 DEG C, melted every 0.5 DEG C Curve；10 DEG C of preservations.

(4) judge product unicity and solution temperature using melting curve, calculated using the Ct values provided using Δ Δ Ct Relative amount of the target gene in different samples is calculated in method.

The structure of embodiment 2, plant expression vector

MtWOX11 full length cDNA sequences are building up in pENTR/D-TOPO intermediate carriers, then being recombinated by LR will MtWOX11 total length CDS sequences are connected in pEarleyGate103 over-express vectors.Obtain driving with CaMV35S promoters The plant expression vector of MtWOX11 gene overexpressions.

Into pENTR/D-TOPO carriers, method is same as above 2.1MtWOX11 total length CDS sequence constructs.

2.2LR restructuring is connected to pEarleyGate103 over-express vectors.

(1) reaction system：

PENTR/D-TOPO carriers:1μl

PEarleyGate103 over-express vectors:1μl

2X Fuzyme MasterMix:0.5μl

(2) reaction mixture is incubated 2-3 hours at 25 DEG C.

(3) above system is converted into bacillus coli DH 5 alpha.

The identification of 2.3 recons

(1) the PCR checkings of plasmid

Picking single bacterium colony is inoculated in 37 DEG C of shaken cultivations in LB fluid nutrient mediums of the 5ml containing Kan and stayed overnight respectively, alkaline denaturation Plasmid is extracted, entering performing PCR with gene specific primer expands.

PCR reaction conditions：94 DEG C of 3min of pre-degeneration；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 35 circulations；72℃ Extend 10min.PCR primer is identified with 1.0% agarose gel electrophoresis.

(2) sequencing identification.

The preparation and conversion of embodiment 3, Agrobacterium competence

The preparation of 3.1 Agrobacterium AGL1/EHA105 competence

1. from picking Agrobacterium tumefaciems single bacterium colony on YEP flat boards (containing 50 μ g/ml rifampins), YEP Liquid Cultures are inoculated in In base (containing 50 μ g/ml rifampins), 200rpm/min, 28 DEG C of overnight incubations.

2. take 2ml to be incubated overnight liquid to be inoculated in YEP fluid nutrient mediums of the 50ml containing identical antibiotic under the same conditions Cultivate to OD₆₀₀Up to 0.5.

3. bacterium solution ice bath 30min, 5000rpm centrifugation 10min, collects thalline by 4 DEG C.

4. thalline is resuspended in the 10ml 0.15mol/L NaCl of ice bath, thalline is collected by centrifugation.

5. it is resuspended in the CaCl of 1ml 20mmol/L ice precoolings₂In solution, bacterium solution is divided in 1.5ml with 200 μ l/ pipes In Eppendorf pipes, quick-frozen 1min in liquid nitrogen is put, -70 DEG C save backup.

3.2 freeze-thaw methods conversion Agrobacterium tumefaciems EHA105

1. melting Agrobacterium competent cell at room temperature, 1 μ g expression vector DNAs, ice bath after mixing are added 30min。

2. putting liquid nitrogen flash freezer 1min, 37 DEG C of insulation 3min are moved to rapidly.

3. add the YEP 800 μ l of antibiotic-free, 28 DEG C of concussion and cultivates 3 hours.

4.7000rpm centrifugation 30s collect thalline, be applied to containing 50 μ g/ml rifampins, 50 μ g/ml Kan YEP flat boards on, 28 DEG C are inverted light culture 2-3 days.

3.3 thalline PCR are identified

Picking single bacterium colony is transferred in as previously described 2.3 PCR system (being not added with DNA profiling), is entered with gene specific primer Performing PCR expands.PCR reaction conditions：94 DEG C of 3min of pre-degeneration；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 35 circulations；72 DEG C extension 10min.PCR primer is identified with 1.0% agarose gel electrophoresis.

Embodiment 4, transgenosis functional verification-M. truncatula transformation and selection

Using tissue culture method (a kind of disclosed universal method), soak the Agrobacterium EHA105 containing plant expression vector Contaminate M. truncatula blade.Then callus induction formation, embryogenesis.After it grows seedling, extraction DNA enters performing PCR screening, produces To Transgenic wheat line.

4.1 M. truncatula blades convert

Agrobacterium EHA105 has the ability of plant and metastatic gene of infecting, therefore by the MtWOX11 gene coding regions of structure Plant expression vector is transferred to Agrobacterium, then enters performing PCR checking.Using tissue culture method (a kind of disclosed universal method), make Agrobacterium EHA105 containing plant expression vector contaminates M. truncatula blade.Then callus induction formation, embryogenesis.Treat its length After going out seedling, extraction DNA enters performing PCR screening, that is, obtains Transgenic wheat line.

4.1.1 tissue culture method

(1) blade for taking health larger goes petiole to be placed in pipe.With the Tween-20 containing 20% NaClO and 0.1% Sterilizing wash 15 minutes, then with sterilizing wash 3-5 times.

(2) blade is cut into small pieces, creates wound.

(3) it is 0.6-0.8 Agrobacterium to be shaken into OD values.Thalline is collected by centrifugation, thalline then is resuspended extremely with SH3a culture mediums OD values 0.1-0.2.

(4) ready bacterium solution is poured into the blade cut.10-15 minutes are vacuumized, shake 10-15 minutes slowly.By leaf Piece is laid on the SH3a culture mediums of non-resistant, is spread as far as possible close.Lucifuge incubated at room temperature.

(5) it is overnight, deionization wash three times, until clarification, then with plus caf é deionization washing.

(6) go on the SH3a for adding resistance, lucifuge hot-house culture 4-6 weeks, then callus is transferred to SH9a training Support in base.Treat that callus differentiation and bud formation is returned again in 1/2MS culture mediums.When being moved to after emergence in soil.

4.1.2 transgenic seedling detects

Extract DNA, PCR detections (method is same as above).

4.2 transfer-gen plant Molecular Identifications

The detection of gene expression dose is carried out to above-mentioned transfer-gen plant.Transfer-gen plant and WT lines are extracted respectively RNA, RT-PCR amplifications are carried out after reverse transcription, analysis is overexpressed the gene expression difference of plant and WT lines.MtWOX11 Expression quantity in plant is overexpressed is all apparently higher than WT lines.

RT-PCR results show although MtWOX11 genes belong to constructive expression, there is expression in each organ, but Expression quantity highest in seed, next to that the expression quantity in flower, root, leaf is higher, expression quantity is relatively low in stem, petiole, terminal bud. (see Fig. 3)

Follow-up work is carried out using a MtWOX11 expression quantity highest strain.

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

Embodiment 5, transgenosis functional verification-Fatty Acids in Seeds quality function analysis

Analysis to mtwox11 mutant and overexpression transfer-gen plant seed quality, had by Suzhou section inscription biotechnology Limit company completes.

The Analysis of Fatty Acid Content result for being overexpressed MtWOX11 transfer-gen plant seeds is shown in Fig. 4.

Wherein：Mtwox11 mutant shown in grey histogram and content of fatty acid in wild type seeds ratio, black post The ratio of MtWOX11 transfer-gen plants and content of fatty acid in wild type seeds is overexpressed shown in shape figure.* significant difference is represented, Anova Single Factor p<0.05, * * represents pole significant difference, Anova Single Factor p<0.01.

As a result show, be overexpressed total fatty acid content in transfer-gen plant seed and added compared with wild type 12.2168%, particularly C15.0 (pentadecanoic acid), C15.1 (cis- 10- pentadecylenic acids), C16.0 (palmitic acid), C18.2N6C (linoleic acid), C20.2 (cis- 11,14- eicosadienoic acids), C20.3N6 (cis- 8,11,14- eicosatrienoic acids), C20.4N6 (arachidonic acid), C20.5N3 (20:5OMEGA-3), C24.0 (lignoceric acid) Content Deng aliphatic acid has decline in mutant, and has rising in MtWOX11 transgenic lines are overexpressed, wherein, The content of pentadecanoic acid significantly improves.

The above results prove that MtWOX11 genes take part in the seed fatty acid content of regulation and control legume, be overexpressed the base Cause, the content of aliphatic acid in seed can be significantly improved.Indication MtWOX11 genes have in leguminous seeds content of fatty acid is improved Important function.

Sequence table

<110>Shandong University

<120>M. truncatula MtWOX11 genes and its application in seed fatty acid content is improved

<141>2017-11-20

<160>3

<210>1

<211>3627

<212>DNA

<213>Artificial sequence

<220>

<223>M. truncatula WUSCHEL-related homeobox11 genome nucleotide sequences

<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. a kind of M. truncatula WOX family genes Mt WUSCHEL related homeobox 11 (MtWOX11), its feature exist In：The unnamed gene is M. truncatula MtWOX11 genes, and the nucleotide sequence of the gene is as shown in SEQ ID No.1.

2. application of the M. truncatula MtWOX11 genes described in claim 1 in seeds of leguminous plant content of fatty acid is improved.

3. application according to claim 2, it is characterised in that：The legume be selected from clover, M. truncatula, soybean, Clover.