CN108676081A - Chinese milk vetch LEAFY genes and its application - Google Patents

Abstract

The present invention provides Chinese milk vetch LEAFY genes and its applications, belong to field of plant genetic.The cDNA sequence of the Chinese milk vetch LEAFY genes is as shown in SEQ ID No.1, and coding protein sequence is as shown in SEQ ID No.2.The emergence for overexpressing LEAFY gene arabidopsis strains is averaged early 3d to bolting number of days than wild type, emergence to number of days of blooming is averaged early 2d than wild type, number of blooming is more 1.79 average than wild type, and display turns Chinese milk vetch LEAFY gene arabidopsis strain boltings number of days, number of days of blooming significantly earlier than wild type.This demonstrate Chinese milk vetch LEAFY genes with it is in close relations at spending, have the function of adjust flowering time, by the gene be applied to Chinese milk vetch character improvement, have a good application prospect.

Description

Chinese milk vetch LEAFY genes and its application

Technical field

The invention belongs to field of plant genetic, specifically, being related to Chinese milk vetch LEAFY genes, its coding egg Its application in florescence control of bletilla.

Background technology

Chinese milk vetch (Astragalussinicus L.), pulse family Astragalus gets over year raw herbaceous plant, originating in China, is important Green manure crop, while be also good leguminous forage and nectariferous plant, the Asian countries such as China, South Korea, Japan generally plant. Chinese milk vetch is important cleaning organic fertilizer resource, and quite high ratio is occupied in green manure, to optimizing the physicochemical property of soil, fertilizing Soil improves ecological environment of soil and plays great function.It is long with the development of chemical fertilizer industry since last century the eighties Phase largely uses chemical fertilizer, pesticide, the simple farming for pursuing crop yield and fertilization mode so that soil resource is faced with serious Production and ecocrisis.Sustainable use of the Chinese milk vetch to soil resource, is of great significance, and in recent years, country is for purple cloud The input of the research and production of English greatly increases.

At flower be the key that plant converts from nutrient growth to reproductive growth, while being the heterogamous center of realization Link largely determines the success or not of plant propagation.Chinese milk vetch by bloom sooner or later can dtex morning flower pattern, early blossoming Type, middle flower pattern and late flower pattern, flowering time directly determine the length of effective growing season.As green manure, the plantation of Chinese milk vetch and Harvest time depends on main crop, ensures also with the breeding time of main crop mutually to coordinate while Chinese milk vetch yield.Suitable Time promotes or avoids blooming, and breeding objective is important according to local cropping system selection and breeding florescence kind appropriate.Therefore, Flowering gene research is of great significance to Chinese milk vetch growth and development and genetic improvement.But for Chinese milk vetch grinding at flower mechanism Study carefully, both at home and abroad without in-depth study, the effect of related gene in the process of blooming is not clear.

LEAFY genes are genes specific to plant kingdom, belong to flower primordium differentiation specific gene, it is mitogenetic to control stem apex The transformation to inflorescence meristem is organized, it is not belonging to any one gene family having now known.The LEAFY bases announced Because sequence alignment is found, different plant LEAFY homologous genes encoding section lengths variations are little, and longest is arabidopsis, a length of 1275bp encodes 424 amino acid, and shortest is eucalyptus, and a length of 1080bp encodes 359 amino acid.LEAFY genes are being planted It plays a significant role in object flower differentiation, transformation and plant when blooming of the control inflorescence meristem to floral meristem Between.In flowering transition, the participation of LEAFY genes is the key that floral meristem formation.LEAFY genes are expressed in Cheng Huaqian Phyllopodium, but then inhibit the startup of phyllopodium after its expression quantity runs up to certain level, inhibit meristematic cell to battalion The transformation for supporting growth and development, to make more meristematic cells form flower primordium.LEAFY genes are maintaining floral meristem It is functioned both on during normal function, colored reverse for starting, preventing floral meristem etc..The gene is overexpressed in arabidopsis can So that side shoot is changed into flower, and advance flowering period can be made.Overexpressing the gene can make the willow that original 8-10 is bloomed through 6-7 It can bloom after the nutrient growth of the moon.In addition to this, which also plays effect in each stage of inflorescence and flower development, There is extensive expression in the nutrient growth of higher plant and each stage of reproduction, in reproductive phase, expression quantity significantly increases.

Invention content

The object of the present invention is to provide Chinese milk vetch LEAFY genes and its applications.

Present invention firstly provides Chinese milk vetch LEAFY genes, have：

1) nucleotide sequence shown in SEQ ID No.1；Or

2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or increases one or several nucleotide；Or

3) nucleotide sequence hybridized under strict conditions with the DNA sequence dna 1) limited.

The present invention provides the albumen of above-mentioned Chinese milk vetch LEAFY gene codes, have：

1) amino acid sequence as shown in SEQ ID No.2；Or

2) amino acid sequence shown in SEQ ID No.2 is substituted, lacks and/or increases one or more amino acid and tool There is the same active protein derived from 1).

The present invention provides the biomaterial containing above-mentioned coding Chinese milk vetch LEAFY genes, the biomaterial is expression Carrier, host cell or expression cassette.

Biomaterial the present invention provides above-mentioned Chinese milk vetch LEAFY genes or its albumen encoded or containing the gene exists Plant is promoted to do sth. in advance the application in bolting.

Biomaterial the present invention provides above-mentioned Chinese milk vetch LEAFY genes or its albumen encoded or containing the gene exists Promote the application in plant Blooming.

Biomaterial the present invention provides above-mentioned Chinese milk vetch LEAFY genes or its albumen encoded or containing the gene exists Increase the application in flowering of plant number.

Biomaterial the present invention provides above-mentioned Chinese milk vetch LEAFY genes or its albumen encoded or containing the gene exists Application in prepare transgenosis plant.

Biomaterial the present invention provides above-mentioned Chinese milk vetch LEAFY genes or its albumen encoded or containing the gene exists Application in plant germplasm resource improvement.

The plant is Chinese milk vetch, arabidopsis.

Chinese milk vetch LEAFY gene orders provided by the invention are as shown in SEQ ID No.1, coding protein sequence such as SEQ Shown in ID No.2.Arabidopsis is infected by agrobacterium-mediated transformation, which is transferred in arabidopsis, obtains turning Chinese milk vetch LEAFY The arabidopsis strain of gene is bloomed in advance as a result, it has been found that the arabidopsis strain for turning Chinese milk vetch LEAFY genes bolting occurs and shifts to an earlier date, Bolting number of days is averaged early 3d than wild type, and number of days of blooming is averaged early 2d than wild type, and number of blooming is more 1.79 average than wild type. This demonstrate Chinese milk vetch LEAFY genes with it is in close relations at spending, have the function of adjust flowering time.The gene is applied to Plant trait is improved, and is had a good application prospect.Theoretical foundation is provided at colored technological means for exploitation regulation and control, while to divide Sub- breeding improvement Chinese milk vetch character lays the foundation.

Description of the drawings

Fig. 1 is 1 target fragment electrophoretogram of embodiment, wherein right swimming lane is marker DL 2000；Piece for the purpose of left swimming lane Section.

Fig. 2 is 5 ' RACE experiment purpose fragment electrophoretic figures.

Fig. 3 is 3 ' RACE experiment purpose fragment electrophoretic figures.

Fig. 4 is LEAFY protein secondary structure predictions and functional site annotated map.

Fig. 5 is (number is confidence using the NJ systematic evolution trees of the LEAFY gene amino acid sequences built of MEGA 5.2 Degree).Bootstrep is set as 1000, using Jones-Thorton-Taylor model construction NJ trees.

Fig. 6 is that expression quantity of the LEAFY genes in Chinese milk vetch Different Organs compares figure.

Fig. 7 is the PCR positive detections for overexpressing LEAFY gene Arabidopsis plants, and swimming lane is purpose base successively from left to right Because of P1233CheckF/P1233R primer detections, swimming lane 12 is negative control.

Fig. 8 is the Arabidopsis plant picture grown after 32d, and left side is wild type, and right side is to turn LEAFY gene arabidopsis.

Fig. 9 be overexpress Chinese milk vetch LEAFY gene arabidopsis bolting number of days, bloom number of days and bloom number statistics.

Specific implementation mode

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..

Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art, Biochemical reagents used in embodiment, material are commercially available.

The acquisition of 1 Chinese milk vetch LEAFY genes of embodiment

1. the fresh Chinese milk vetch tissue samples of test material.

2.RNA extracts the synthesis with the first chains of cDNA

Conventional method extracts RNA, uses the RevertAid First Strand cDNA of Fermentas companies Synthesis Kit synthesize the first chains of cDNA.

3. the design and sequence of primer

The design that confirmatory experiment primer is carried out using 5.0 softwares of Primer Premier, by raw work bioengineering (Shanghai) Limited liability company is synthesized.

1 Primer of table and sequence

4.5 ' RACE are tested

The design and sequence of 4.1 primers

Using transcript profile sequence verification as a result, using 5.0 Software for Design of Primer Premier, two 5 ' RACE of specificity Primer is shown in Table 1.It is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

The synthesis of 4.2 the first chain of target gene cDNA

The conjunction of the first chain of target gene cDNA is carried out to total serum IgE using SUPERSCRIPT II RT enzymes and primer GSP-1 At carrying out RNA processing to the cDNA of synthesis using RNase Mix.

4.3 use DNA Purification System:GLASSMAX DNA isolation spincartridges To being purified through the processed cDNA of RNAase.

4.4 carry out end using TdT enzymes and dCTP to cDNA after purification adds poly C

4.5 use the bridging rivet primer AAP of band inside primer GSP-2 and kit to having added the cDNA of dC tails to carry out The PCR first round expands.

4.6 carry out nest-type PRC second using the bridging universal amplification primer AUAP of band inside primer GSP-3 and kit takes turns Amplification.

The recovery purifying of 4.7 target fragments

Second wheel PCR product is subjected to electrophoresis and gel extraction purifying is carried out to purpose band, step presses QIAquick Gel Extraction Kit Specification carries out, and electrophoresis detection result is shown in Fig. 2.

The clone of 4.8 target fragments and sequencing

PCR product after purification is attached with pMD18T, and positive colony is sequenced after conversion, as a result sees SEQ ID NO.11。

5.3 ' RACE are tested

The design and sequence of 5.1 primers

Using transcript profile sequence verification as a result, using 5.0 Software for Design of Primer Premier, two 3 ' RACE of specificity Primer is shown in Table 1, is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

5.2 experimental methods and result

1) reverse transcriptase SMARTScribe is used^TM3 ' CDS primer A of Reverse Transcriptase and primer Reverse transcription is carried out to total serum IgE and synthesizes cDNA.

2) primer rLFY-F1 and UPM (5'- are used CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'(long)；5'- CTAATACGACTCACTATAGGGC-3 ' (short)), carry out first round PCR amplification by template of the cDNA of aforementioned synthesis.

3) first round pcr amplification product is diluted 50 times, then carrying out the second wheel PCR with primer rLFY-F2 and UPM expands Increase.

4) the second wheel PCR product is subjected to electrophoresis and gel extraction purifying is carried out to purpose band, electrophoresis result is shown in Fig. 3.

5) PCR product after purification is attached with pMD18T, is sent after conversion to Shanghai and is given birth to work direct Sequencing.

6. according to transcript profile sequence verification, 5 ' RAEC and 3 ' RACE are as a result, be spliced into the full-length cDNA of experiment purpose gene Then sequence compares starting and the terminator codon position that analysis predicts gene by NCBI.As a result see SEQ ID NO.1, The protein amino acid sequence that it is encoded is as shown in SEQ IDNO.2.

The analysis of biological information of 2 Chinese milk vetch LEAFY genes of embodiment

1, ORF is identified

RACE technologies obtain LEAFY genes (SEQ ID NO.1) full-length cDNA length be 1400p, contain there are one The length of the open reading frame of 1191bp long, 5 ' and 3 ' noncoding regions is respectively 33bp and 176bp, and polyA tails are located at 1386- At 1400bp.See Fig. 4.

2, sequence component and analysis of physical and chemical property use

ProtParam carries out protein amino acid content and analysis of physical and chemical property.The protein of LEAFY gene codes contains 396 amino acid, molecular weight are 44723 dalton, and theoretical isoelectric point is 6.41, and chemical constituent is as shown in table 2.The albumen Containing 59 negatively charged amino acid residues and 56 positively charged amino acid residues, its N-terminal is originated with methionine.It should Albumen contains 6222 atoms, wherein carbon atom 1956, hydrogen atom 3075, nitrogen-atoms 579, oxygen atom 594, sulphur altogether 15, atom, aliphatic index 72.20, hydrophobicity index are -0.651, and unstability index is 51.52.In summary Data, the albumen are classified as unstability albumen.It was found that Chinese milk vetch LEAFY albumen contains, there are one typical Floricaula/ Leafyprotein structural domains (IPR002910).

The chemical constituent of table 2LEAFY gene coded proteins

Amino acid	Quantity	Quantity accounting	Quality accounting	Amino acid	Quantity	Quantity accounting	Quality accounting
								Ala(A)	35	8.80%	6.02%	Lys(K)	20	5.10%	5.64%
Arg(R)	36	9.10%	12.10%	Met(M)	8	2.00%	2.30%
								Asn(N)	16	4.00%	4.08%	Phe(F)	12	3.00%	3.82%
Asp(D)	21	5.30%	5.39%	Pro(P)	22	5.60%	4.89%
								Cys(C)	7	1.80%	1.64%	Ser(S)	22	5.60%	4.46%
Gln(Q)	13	3.30%	3.67%	Thr(T)	14	3.50%	3.22%
								Glu(E)	38	9.60%	10.79%	Trp(W)	6	1.50%	2.36%
Gly(G)	31	7.80%	4.49%	Tyr(Y)	14	3.50%	4.89%
								His(H)	10	2.50%	2.99%	Val(V)	26	6.60%	5.88%
Ile(I)	16	4.00%	4.05	Pyl(O)	0	0.00%	0.00%
								Leu(L)	29	7.30%	7.34	Sec(U)	0	0.00%	0.00%

3, phosphorylation site is predicted

The secondary structure prediction of LEAFY albumen is carried out using CLC Genomics Workbench, the albumen is by 396 ammonia Base acid forms, including 22 α spirals and 5 β-pleated sheets.Amidation site and N- glycosylation sites in amino acid sequence are being schemed It is marked in 4.The phosphorylation site that serine, threonine and tyrosine in protein are predicted with NetPhos, in LEAFY protein In predict 8 Ser-phosphorylation sites, 2 threonine phosphorylation sites and 2 tyrosine phosphorylation sites (table 3) altogether.

3 phosphorylation site prediction result of table

4, chadogram builds

Analysis is compared with other 9 species homologous proteins to the LEAFY albumen of Chinese milk vetch using BlastP programs, As a result show between species there is certain similitude (table 4).Wherein, Chinese milk vetch and M. truncatula (Medicago Truncatula) and the similarity of the LEAFY albumen of alfalfa (Medicago sativa) reaches 85.43%, with chick-pea The similarity of (Cicer arietinum) LEAFY albumen has respectively reached 84.67%, this is the result shows that Chinese milk vetch LEAFY eggs There is certain homology with these species in vain.The structure of different plant species LEAFY albumen chadograms is carried out with 5.2 softwares of MEGA, As a result Chinese milk vetch and chick-pea (Cicer arietinum), M. truncatula (Medicagotruncatula), alfalfa are shown (Medicago sativa) and pea (Pisum sativum) gather for a kind of (Fig. 5), may have similar function.Using CLC Genomics Workbench softwares carry out Multiple Sequence Alignment.

The comparative analysis of table 4 Chinese milk vetch LEAFY albumen and other species LEAFY albumen

5, homologous modeling and tertiary protein structure prediction

The prediction of protein three-dimensional structure is the I-TASSER online softwares (http based on homologous modeling:// Zhanglab.ccmb.med.umich.edu/I-TASSER/) prediction obtains.The similitude of formwork structure and pre- geodesic structure by TM values and RMSD values are weighed.The overlay chart of two structures is by Pymol softwares (http:// Pymol.sourceforge.net it) obtains.Predict that obtained structure total quality quality is weighed by C values.C values are by threading What the parameter of template sequence and the model configuration polymerization used was calculated, it is worth generally between -5 to 2.The higher explanation of C values is pre- The structure measured is more credible, and vice versa.The results show that Chinese milk vetch LEAFY and arabidopsis LEAFY have 396 and 420 respectively Amino acid forms.Arabidopsis LEAFY albumen is 2vy1 in the ID of PDB lane databases.The three-dimensional structure of Chinese milk vetch LEAFY albumen is It is predicted on I-TASSER softwares as template using 2vy1.Chinese milk vetch LEAFY albumen is from red carbon teminal to the nitrogen end of blue.In advance The protein structure C values of survey are -0.98, indicate that it is very similar in terms of folding and secondary structure with the A chains of 2vy1.Display target DNA solution temperatures (melting temperature, TM) value of the structural similarity of albumen and template protein be 0.59 ± 0.14, root-mean-square-deviation (root mean square deviation, RMSD) value is

Relative expression levels' detection that 3 Chinese milk vetch LEAFY genes of embodiment are respectively organized in Chinese milk vetch

1, Total RNAs extraction

Chinese milk vetch is the vegetable material of chamber planting, is acquired made of seed culture with common Chinese milk vetch plant material Fresh Chinese milk vetch tissue samples are planted in Zhejiang Academy of Agricultural Science greenhouse.

5 sample rna content of table and purity

2, fluorescence quantification PCR primer design and synthesis

Quantification PCR primer design is carried out using Primer Premier 6.0 and 7.8 softwares of Beacon designer, so It is synthesized afterwards by Sangon Biotech (Shanghai) Co., Ltd., fluorogenic quantitative detection primer and internal control primer sequence are shown in Table 1.

3, Real-Time PCR amplification systems and reaction condition

6 quantitative PCR reaction system of table and condition

4, Real-Time pcr genes differential expression statistical analysis

In triplicate, the relative expression levels of each gene are with 2 for each sample^{(Ct reference gene-Ct target gene)}It is for statistical analysis. It the results are shown in Table 7.

Table 7

Using 18S rRNA as internal reference, using expression of the qRT-PCR detection LEAFY genes in Different Organs, the results showed that, LEAFY genes have expression, expression quantity to be followed successively by bud, flower, leaf, leaf bud, root, stem and pod from high to low in each organ.Through SPSS softwares analyze, between Different Organs, Chinese milk vetch LEAFY genes in bud, spend in differential expression significantly (P ＜ 0.05), Difference is not notable (Fig. 6) in leaf, leaf bud, root, stem and pod.Thus the expression that LEAFY genes can be explained has tissue specificity, It may be played an important role during Chinese milk vetch flower development.

The culture of 4 transgenic arabidopsis of embodiment and its phenotypic analysis

1, contain the structure of the recombinant plasmid of Chinese milk vetch LEAFY genes of the present invention (SEQ ID NO.1).

2, Agrobacterium competence is converted

Correct recombinant plasmid transformed Agrobacterium competence will be sequenced.Bacterium colony PCR identifications show that vector plasmid successfully turns Enter into Agrobacterium.

3, transformation of Arabidopsis thaliana flow (inflorescence dip method)

(1) it plants：Select good water absorption, soil property soft to stone cooperation Nutrition Soil (1：1/2) it is used as arabidopsis planting soil Earth.The flowerpot of diameter 9cm sows 100-150 per basin.The film in flowerpot upper cover later is sowed, is provided to the growth of plant The environment of one moistening.

(2) it transplants：Sowing 10-15 days, waits for that Arabidopsis thaliana Seedlings were grown to four leaf periods and starts to transplant, per 4-5, basin.

(3) it goes to push up：Bud is cut when arabidopsis is bloomed for the first time, the hyperplasia of the more sprays of side shoot can be promoted.It is suitble to There is no siliques that is ripe, being also fertilized without generation for the flowers of transformed plant.

(4) dip dyeing liquid for shell is prepared：The Agrobacterium after conversion is resuspended in 5% sucrose solution makes OD=0.8, in order to keep sugarcane Sugar juice it is fresh, can be now with the current, without sterilizing.100-200ml disseminates 2-3 small basin plant, and 400-500ml disseminates, 2-3 flowerpot (9cm) plant.Surfactant is added before dip dyeing to concentration 0.05% (500ul/L).

(5) it disseminates：The flower surface portion of full-bloom stage arabidopsis is impregnated into 20-30s in agrobacterium suspension in post-conversion, It gently rotates simultaneously.

(6) light culture：Plant bagging after dip dyeing is kept into the moisture state darkroom culture of height for 24 hours.

(7) it is cultivated after disseminating：It waters every other day, ensures moisture abundance.

(8) seed collection：Seed maturity, can be with sowing after silique natural cracking.

(9) transgenic seed screens：The gained seed after culture dip dyeing on the tablet containing hygromycin antibiotic.40mg's About 200, seed is in vernalization 2 days on 0.5 × MS culture mediums of the μ of 10-50 containing hygromycin g/ml, later in continuous light condition Lower culture 7-10 days.Determine whether transgenic seed according to upgrowth situation.The seed for being successfully transferred to recombinant plasmid can be anti- Property culture and upper normal growth go out 4 or more true leaves.Non-transgenic seed is unable to normal growth, is only capable of growing 2 cotyledons, root Growth is also heavily suppressed, death after general sprouting 10 days.

(10) transfer-gen plant turns soil cultivation.It, will be positive after transgenic seed sprouts 2 weeks on MS+ hygromycin tablets Plant is transferred to soil and continues to cultivate.

(11) PCR is identified：It takes positive plant blade to carry out the extraction of genomic DNA and uses objective gene sequence and carrier 35S promoter aligning primer carries out PCR verifications, primer sequence P1233CheckF：5'-CTAGACGAGGAGGTGTC-3' (SEQ ID NO.17)；P1233R：5'-CCGCTCGAGGAAAGGAAGATGGGCACTTC-3’(SEQID NO.18).Detection knot Fruit sees Fig. 7.Detect transgenic positive plant.It is the detection of selectable plant marker hygromycin gene successively from left to right.Hygromycin F： GAGCATATACGCCC

GGAGTC, hygromycin R：GTCTCCGACCTGATGCAGCTCTCGG.

By LEAFY genetic transformation to Agrobacterium C5C81, arabidopsis is infected using inflorescence method.By arabidopsis kind to be detected Son is planted on resistance culture base, convert successful plant can normal growth, and unconverted successful plant be yellow death Success that plant LEAFY genes are unconverted.It will continue to cultivate in seedling replanting to culturing pot that primary dcreening operation obtains, when growing to 10~12 When piece, clip blade carries out the extraction of genomic DNA, and is drawn using target fragment amplimer and carrier 35S promoter sequence Object carries out PCR detections, and electrophoresis result purpose band occurs, shows that LEAFY genes have been integrated into arabidopsis gene group.

4, transgenic arabidopsis phenotypic analysis

Transgenic arabidopsis T2 generations are sowed, the situation of blooming of transgenic line is recorded.Bolting is calculated since after planting, is opened Required number of days is spent, and number of blooming, the results are shown in Table 8 and Fig. 8, Fig. 9.

Table 8

Compared with wild-type Arabidopsis plants, transgenic arabidopsis is to show early blossoming phenotype.To transgenic arabidopsis And the bolting number of days of wildtype Arabidopsis thaliana, bloom number of days and several counted of blooming.The results show that overexpression LEAFY genes are quasi- The bolting number of days of southern mustard strain is averaged early 3d than wild type, and number of days of blooming is averaged early 2d than wild type, and number of blooming is more flat than wild type It is 1.79 more.This shows that bolting of the overexpression to arabidopsis of LEAFY genes, florescence, number of blooming have an impact.LEAFY bases The expression of cause can promote flowering of plant.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Sequence table

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Asn Asn Asn Ser Gly Gly Gly Gly Gly Gly Gly Val Cys Glu Arg Gln

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Arg Glu His Pro Phe Ile Val Thr Glu Pro Gly Glu Val Ala Arg Gly

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Lys Leu Arg Gln Leu Cys His Ala Glu Arg Asn Ser Ala Ala Ala Ser

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<213>Artificial sequence (Artificial Sequence)

<400> 5

catgttataa gccacttgtg gca 23

<210> 6

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

tatgtgccta ccaagctccg tcagc 25

<210> 7

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

cgtcaaggtt gggatattga tgc 23

<210> 8

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

cttccaacag aaacggaact agaag 25

<210> 9

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

cgcggtaatt ccagctccaa tag 23

<210> 10

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

cgacccaacc caaggtccaa c 21

<210> 11

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

cgtcaaggtt gggatattga tgc 23

<210> 12

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

cttccaacag aaacggaact agaag 25

Claims (10)

1. Chinese milk vetch LEAFY, which is characterized in that it has：

1) amino acid sequence as shown in SEQ ID No.2；Or

2) amino acid sequence shown in SEQ ID No.2 is substituted, lacks and/or increases one or more amino acid and with same The protein derived from 1) of isoreactivity.

2. the encoding gene of Chinese milk vetch LEAFY described in claim 1, which is characterized in that it has：

1) nucleotide sequence shown in SEQ ID No.1；Or

2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or increases one or several nucleotide；Or

3) nucleotide sequence hybridized under strict conditions with the DNA sequence dna 1) limited.

3. the biomaterial containing the encoding gene of Chinese milk vetch LEAFY described in claim 2, the biomaterial is that expression carries Body, expression cassette, host cell or engineering bacteria.

4. the encoding gene described in Chinese milk vetch LEAFY described in claim 1 or claim 2 or the life described in claim 3 The application interim in regulation and control plant flowers of object material.

5. the encoding gene described in Chinese milk vetch LEAFY described in claim 1 or claim 2 or the life described in claim 3 Application of the object material in promoting plant to do sth. in advance bolting.

6. the encoding gene described in Chinese milk vetch LEAFY described in claim 1 or claim 2 or the life described in claim 3 Application of the object material in promoting plant Blooming.

7. the encoding gene described in Chinese milk vetch LEAFY described in claim 1 or claim 2 or the life described in claim 3 Application of the object material in increasing flowering of plant number.

8. the encoding gene described in Chinese milk vetch LEAFY described in claim 1 or claim 2 or the life described in claim 3 Application of the object material in prepare transgenosis plant.

9. the encoding gene described in Chinese milk vetch LEAFY described in claim 1 or claim 2 or the life described in claim 3 Application of the object material in plant germplasm resource improvement.

10. the application as described in claim 4-9 is any, which is characterized in that the plant is Chinese milk vetch, arabidopsis.