CN108676081B - Astragalus sinicus LEAFY gene and application thereof - Google Patents

Abstract

The invention provides a milk vetch LEAFY gene and application thereof, belonging to the technical field of plant genetic engineering. The cDNA sequence of the astragalus sinicus LEAFY gene is shown in SEQ ID No.1, and the coding protein sequence is shown in SEQ ID No. 2. The days from seedling emergence to bolting of the over-expression LEAFY gene Arabidopsis line are 3 days earlier than the average days of the wild type, the days from seedling emergence to flowering are 2 days earlier than the average days of the wild type, the flowering number is 1.79 more than the average days of the wild type, and the days of bolting and flowering of the astragalus membranaceus LEAFY gene Arabidopsis line are obviously earlier than the days of the wild type. The fact shows that the astragalus sinicus LEAFY gene is closely related to flowering, has the function of adjusting flowering time, and has good application prospect when being applied to character improvement of astragalus sinicus.

Description

Astragalus sinicus LEAFY gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a milk vetch LEAFY gene, a coding protein thereof and application thereof in florescence regulation.

Background

Astragalus sinicus L, a perennial herb of Astragalus membranaceus, is an important green manure crop in China, is also a high-quality leguminous pasture and honey source plant, and is commonly planted in Asian countries such as China, Korea, Japan, etc. Milk vetch is an important clean organic fertilizer source, occupies a quite high proportion in green manure, and plays a great role in optimizing the physical and chemical properties of soil, fertilizing the soil and improving the ecological environment of the soil. Since the eighties of the last century, with the development of the fertilizer industry, a large amount of fertilizers and pesticides are used for a long time, and the cultivation and fertilization mode of crop yield is simply pursued, so that soil resources face serious production and ecological crisis. The astragalus sinicus has important significance for sustainable utilization of soil resources, and in recent years, the national investment for scientific research and production of the astragalus sinicus is greatly increased.

The flower formation is the key conversion of the plant from vegetative growth to reproductive growth, is also the central link for realizing generation alternation, and determines the success of plant propagation to a great extent. The astragalus sinicus can be distinguished into early flower type, middle flower type and late flower type according to the early and late blossoming, and the blossoming time directly determines the length of the effective growing season. As green manure, the planting and harvesting time of the milk vetch depends on the main crop, and the milk vetch is coordinated with the growth period of the main crop while the yield of the milk vetch is ensured. Promote or avoid flowering in proper time, and the breeding of proper variety in flowering phase according to local cultivation system is an important breeding target. Therefore, the research of the flowering gene has important significance on the growth and development and genetic improvement of the astragalus sinicus. However, the research on the mechanism of astragalus sinicus flowering has not been deeply studied at home and abroad, and the role of the flowering related gene in the process is not clear.

The LEAFY gene is a gene specific to the plant kingdom, belongs to a floral primordium differentiation specific gene, controls the conversion of a shoot apical meristem to an inflorescence meristem, and does not belong to any gene family known at present. The published sequence comparison of LEAFY genes shows that the length of coding regions of LEAFY homologous genes of different plants is not changed greatly, the longest coding region is arabidopsis thaliana, the longest coding region is 1275bp, 424 amino acids are coded, the shortest coding region is eucalyptus, the length is 1080bp, and 359 amino acids are coded. The LEAFY gene plays an important role in the process of plant flowering, and controls the transformation of inflorescence meristems into flower meristems and the flowering time of plants. The involvement of the LEAFY gene in floral transitions is critical for floral meristem formation. The LEAFY gene is expressed in a leaf primordium before flowering, but when the expression level is accumulated to a certain level, the initiation of the leaf primordium is inhibited, and the transformation of meristematic cells to vegetative growth and development is inhibited, so that more meristematic cells form the flower primordium. The LEAFY gene plays a role in maintaining the normal function of the floral meristem, starting the flower, preventing the reversal of the floral meristem and the like. Overexpression of the gene in Arabidopsis can convert lateral branches into flowers and can advance the flowering phase. The overexpression of the gene can lead poplar which originally blooms in 8-10 years to bloom after 6-7 months of vegetative growth. In addition, the gene plays a role in various stages of inflorescence and flower development, has wide expression in various stages of vegetative growth and reproduction of higher plants, and obviously increases the expression amount in the reproduction stage.

Disclosure of Invention

The invention aims to provide a astragalus sinicus LEAFY gene and application thereof.

The present invention firstly provides a milk vetch LEAFY gene, which has:

1) a nucleotide sequence shown as SEQ ID No. 1; or

2) The nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides; or

3) Nucleotide sequences which hybridize under stringent conditions with the DNA sequences defined in 1).

The invention provides a protein coded by the astragalus sinicus LEAFY gene, which comprises the following components in part by weight:

1) an amino acid sequence shown as SEQ ID No. 2; or

2) Protein which is derived from the protein 1) and has the same activity and is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

The invention provides a biological material containing the astragalus sinicus LEAFY gene, which is an expression vector, a host cell or an expression cassette.

The invention provides an application of the astragalus sinicus LEAFY gene or a protein coded by the same or a biological material containing the same in promoting early bolting of plants.

The invention provides the application of the astragalus sinicus LEAFY gene or the protein coded by the same or a biological material containing the same in promoting the early flowering of plants.

The invention provides the application of the astragalus sinicus LEAFY gene or the protein coded by the gene or the biological material containing the gene in increasing the flowering number of plants.

The invention provides the application of the astragalus sinicus LEAFY gene or the protein coded by the same or a biological material containing the same in preparing transgenic plants.

The invention provides the application of the astragalus sinicus LEAFY gene or the protein coded by the gene or the biological material containing the gene in the improvement of plant germplasm resources.

The plants are astragalus sinicus and arabidopsis thaliana.

The astragalus sinicus LEAFY gene sequence provided by the invention is shown as SEQ ID No.1, and the coding protein sequence is shown as SEQ ID No. 2. Infecting arabidopsis thaliana by an agrobacterium-mediated method, transferring the gene into arabidopsis thaliana to obtain an arabidopsis thaliana strain of the astragalus membranaceus LEAFY gene, and finding out that the arabidopsis thaliana strain of the astragalus membranaceus LEAFY gene is early in bolting and flowering, wherein the bolting days are 3d earlier than the wild type on average, the flowering days are 2d earlier than the wild type on average, and the flowering number is 1.79 more than the wild type on average. This indicates that the astragalus sinicus LEAFY gene is closely related to flowering and has the function of regulating flowering time. The gene is applied to the improvement of plant traits and has good application prospect. Provides a theoretical basis for developing a technical means for regulating and controlling flowering, and lays a foundation for improving the character of the astragalus sinicus by molecular breeding.

Drawings

FIG. 1 is an electrophoretogram of a fragment of the object of example 1, wherein the right lane is marker DL 2000; the left lane is the target fragment.

FIG. 2 is an electrophoretogram of a fragment of interest of a 5' RACE experiment.

FIG. 3 is an electrophoretogram of a fragment of interest of a 3' RACE experiment.

FIG. 4 is a graph of LEAFY protein secondary structure prediction and functional site annotation.

Fig. 5 is an NJ phylogenetic tree (numbers are confidence) of the amino acid sequence of the LEAFY gene constructed using MEGA 5.2. bootstrep is set to 1000, and an NJ tree is constructed by adopting a Jones-Thorton-Taylor model.

FIG. 6 is a graph showing the comparison of expression levels of LEAFY gene in different organs of milk vetch.

FIG. 7 shows PCR positive detection of an Arabidopsis plant with overexpression LEAFY gene, from left to right, detection of a primer of the target gene P1233CheckF/P1233R is shown, and lane 12 is a negative control.

FIG. 8 is a picture of an Arabidopsis plant grown for 32d, with wild type on the left and LEAFY transgenic Arabidopsis on the right.

FIG. 9 shows statistics of bolting days, flowering days and flowering numbers of Arabidopsis thaliana over-expressing the milk vetch LEAFY gene.

Detailed Description

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

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the biochemical reagents and materials used in the examples are commercially available.

Example 1 obtaining the Astragalus sinicus LEAFY Gene

1. Test materials fresh milk vetch tissue samples.

RNA extraction and first strand cDNA Synthesis

RNA was extracted by a conventional method, and the First Strand cDNA was synthesized using the RevertAID First Strand cDNAsSynthesis Kit from Fermentas.

3. Design and sequence of primers

Design of experimental primers was verified by using Primer Premier 5.0 software, and synthesized by Biotechnology engineering (Shanghai) GmbH.

TABLE 1 primer names and sequences

4.5' RACE experiment

4.1 design and sequence of primers

Using the transcriptome sequencing validation results, two specific 5' RACE primers were designed using Primer Premier 5.0 software, see Table 1. Synthesized by Biotechnology engineering (Shanghai) Co., Ltd.

4.2 Synthesis of first Strand cDNA of the Gene of interest

Total RNA was subjected to first strand cDNA synthesis of the target gene using SUPERSCRIPT II RT enzyme and primer GSP-1, and the synthesized cDNA was subjected to RNA removal treatment using RNase Mix.

4.3 Purification of the RNAase-treated cDNA Using DNA Purification System GLASSMAX DNA isolation encapsulating tags.

4.4 terminating the purified cDNA with poly-C using TdT enzyme and dCTP

4.5 first round PCR amplification of dC tailed cDNA was performed using primer GSP-2 and bridging rivet primer AAP in the inner band of the kit.

4.6 nested PCR second round amplification was performed using primer GSP-3 and bridging universal amplification primer AUAP in the inner band of the kit.

4.7 recovery and purification of the fragment of interest

And (3) performing electrophoresis on the second round PCR product, cutting the gel, recovering and purifying the target band, wherein the steps are performed according to the specification of the recovery kit, and the electrophoresis detection result is shown in figure 2.

4.8 cloning and sequencing of fragments of interest

The purified PCR product was ligated with pMD18T, and positive clones were sequenced after transformation, the results are shown in SEQ ID NO. 11.

5.3' RACE experiment

5.1 design and sequence of primers

Using the results of transcriptome sequencing verification, two specific 3' RACE primers were designed using Primer Premier 5.0 software, as shown in Table 1, and synthesized by Biotechnology engineering (Shanghai) Inc.

5.2 Experimental methods and results

1) Using reverse transcriptase SMARTScribe^TMReverse transcription of the total RNA and primer 3' CDS primer A was performed to synthesize cDNA.

2) Primers rLFY-F1 and UPM (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3' (long); 5'-CTAATACGACTCACTATAGGGC-3' (short)), the first round of PCR amplification was performed using the previously synthesized cDNA as a template.

3) The first round of PCR amplification products were diluted 50-fold and then a second round of PCR amplification was performed with primers rLFY-F2 and UPM.

4) The second round PCR product was electrophoresed and the target band was recovered and purified by cutting gel, and the result of electrophoresis is shown in FIG. 3.

5) The purified PCR product was ligated with pMD18T, transformed and sent to the Shanghai Producer for direct sequencing.

6. According to the transcriptome sequence verification, 5 'RAEC and 3' RACE results, the full-length cDNA sequence of the target gene of the experiment is spliced, and then the positions of the start codon and the stop codon of the gene are predicted through NCBI comparison analysis. The result is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

Example 2 bioinformatic analysis of Astragalus sinicus LEAFY Gene

1. ORF identification

The full-length cDNA of LEAFY gene (SEQ ID NO.1) obtained by RACE technology has a length of 1400p, contains an open reading frame with a length of 1191bp, the lengths of 5 'and 3' non-coding regions are 33bp and 176bp respectively, and the polyA tail is positioned at 1386-1400 bp. See fig. 4.

2. Sequence composition and physicochemical property analysis

ProtParam was used for protein amino acid content and physicochemical property analysis. The protein encoded by LEAFY gene contains 396 amino acids, has the molecular weight of 44723 daltons, the theoretical isoelectric point of 6.41, and the chemical components are shown in Table 2. The protein contains 59 negatively charged amino acid residues and 56 positively charged amino acid residues, the N-terminus of which starts with methionine. The protein contains 6222 atoms in total, wherein the carbon atom 1956, the hydrogen atom 3075, the nitrogen atom 579, the oxygen atom 594 and the sulfur atom 15, the aliphatic index is 72.20, the hydrophobicity index is-0.651, and the instability index is 51.52. Combining the above data, the protein was classified as an unstable protein. The astragalus LEAFY protein was found to contain a typical floricila/leafyprotein domain (IPR 002910).

TABLE 2 chemical composition of the protein encoded by LEAFY Gene

Amino acids

Number of

Ratio of number to number

Mass ratio of

Amino acids

Number of

Ratio of number to number

Mass ratio of

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 prediction

Secondary structure prediction of LEAFY protein was performed using CLC Genomics Workbench, which consists of 396 amino acids, containing 22 α helices and 5 β folds the amidated and N-glycosylated sites in the amino acid sequence have been labeled in FIG. 4 NetPhos was used to predict the phosphorylation sites of serine, threonine and tyrosine in the protein, which was predicted to be 8 serine phosphorylation sites, 2 threonine phosphorylation sites and 2 tyrosine phosphorylation sites in LEAFY protein (Table 3).

TABLE 3 phosphorylation site prediction results

4. Construction of an evolutionary tree

The LEAFY protein of Astragalus sinicus was analyzed by comparison with the homologous proteins of 9 other species using the BlastP program, and the results showed some similarity between the species (Table 4). The result shows that the astragalus sinicus LEAFY protein has certain homology with the species, wherein the similarity of the astragalus sinicus, the LEAFY protein of Medicago truncatula (Medicago truncatula) and Medicago sativa (Medicago sativa) reaches 85.43 percent, and the similarity of the astragalus sinicus, the Medicago truncatula (Medicago sativa) and the LEAFY protein of chickpea (Cicer arietinum) respectively reaches 84.67 percent. The construction of LEAFY protein evolutionary trees of different species was performed by MEGA 5.2 software, and the results showed that milk vetch and chickpea (Cicer arietinum), Medicago truncatula (Medicago truncatula), Medicago sativa (Medicago sativa) and pea (Pisum sativum) were clustered into one class (FIG. 5), possibly with similar functions. CLC Genomics Workbench software is used for multi-sequence alignment.

TABLE 4 comparative analysis of Astragalus sinicus LEAFY protein with LEAFY proteins of other species

5. Homology modeling and protein tertiary structure prediction

The prediction of the three-dimensional structure of the protein is obtained based on the prediction of I-TASSER online software (http:// zhangglab. ccmb. med. umich. edu/I-TASSER /) of homologous modeling. The similarity of the template structure to the predicted structure is measured by the TM and RMSD values. An overlay of the two structures was obtained by Pymol software (http:// Pymol. The overall quality of the structure obtained by prediction is measured by the C value. The C value is calculated from the template sequence used for threading and the parameters simulating the structural aggregation, and is generally between-5 and 2. Higher values of C indicate more confidence in the predicted structure and vice versa. The results show that astragalus sinicus LEAFY and arabidopsis thaliana LEAFY consist of 396 and 420 amino acids, respectively. The Arabidopsis LEAFY protein has an ID of 2vy1 in the PDB database. The three-dimensional structure of the astragalus sinicus LEAFY protein is predicted on an I-TASSER software by taking 2vy1 as a template. The milk vetch LEAFY protein goes from the red carbon end to the blue nitrogen end. The predicted protein structure C value was-0.98, indicating that it is very similar to the A chain of 2vy1 in terms of folding and secondary structure. The DNA dissolution Temperature (TM) value showing structural similarity between the target protein and the template protein was 0.59. + -. 0.14, and the Root Mean Square Deviation (RMSD) value was

Example 3 detection of the relative expression level of the Astragalus sinicus LEAFY Gene in various tissues of Astragalus sinicus

1. Total RNA extraction

Milk vetch is a plant material planted in a greenhouse, is a fresh milk vetch tissue sample which is obtained by collecting seeds and culturing the seeds of a common milk vetch plant material and is planted in the greenhouse of agricultural academy of sciences in Zhejiang province.

TABLE 5 RNA content and purity of samples

2. Fluorescent quantitative PCR primer design and synthesis

The Primer Premier 6.0 and Beacon designer 7.8 software were used for quantitative PCR Primer design, and then synthesized by Biotechnology engineering (Shanghai) GmbH, and the sequences of the fluorescence quantitative detection Primer and the internal reference Primer are shown in Table 1.

3. Real-Time PCR amplification system and reaction conditions

TABLE 6 quantitative PCR reaction System and conditions

4. Real-Time PCR gene expression difference statistical analysis

Each sample was replicated three times, and the relative expression levels of the respective genes were calculated as 2^{(Ct internal reference gene-Ct target gene)}Statistical analysis was performed. The results are shown in Table 7.

TABLE 7

The expression of the LEAFY gene in different organs is detected by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction) with 18S rRNA as an internal reference, and the results show that the LEAFY gene is expressed in all organs, and the expression levels of the LEAFY gene are flower buds, flowers, leaves, leaf buds, roots, stems and pods from high to low. Through SPSS software analysis, the expression difference of the astragalus sinicus LEAFY gene in flower buds and flowers is obvious (P is less than 0.05) and the difference in leaves, leaf buds, roots, stems and pods is not obvious (figure 6). Therefore, the expression of the LEAFY gene has tissue specificity and may play an important role in the development process of the astragalus sinicus flowers.

Example 4 culture of transgenic Arabidopsis thaliana and phenotypic analysis thereof

1. Construction of a recombinant plasmid containing the Astragalus sinicus LEAFY gene (SEQ ID NO. 1).

2. Transformed agrobacterium competence

And transforming the recombinant plasmid with correct sequencing into agrobacterium tumefaciens competence. Colony PCR identification shows that the vector plasmid has been successfully transferred into agrobacterium.

3. Arabidopsis transformation procedure (inflorescence dip-dyeing method)

(1) Planting: the nutrient soil (1: 1/2) matched with the soft soil and the fine soil with good water absorption is selected as the planting soil of the arabidopsis thaliana. The diameter of the flowerpot is 9cm, and each pot is seeded with 100-150 seeds. After sowing, the flowerpot is covered with a film to provide a moist environment for the growth of plants.

(2) Transplanting: sowing for 10-15 days, and beginning to transplant when the arabidopsis seedlings grow to four leaves, wherein 4-5 seedlings are planted in each pot.

(3) Removing the top: when arabidopsis flowers for the first time, buds are cut off, and the proliferation of more flower branches on lateral branches can be promoted. Flowers suitable for transformation of the plants did not mature and did not produce fertilized siliques.

(4) Preparing a dip dyeing solution: the transformed agrobacterium was resuspended in 5% sucrose solution to make OD 0.8, and the sucrose solution was ready for use without sterilization in order to keep the freshness. 100-. The surfactant was added to a concentration of 0.05% (500ul/L) prior to padding.

(5) Dip dyeing: the surface part of the flower of Arabidopsis thaliana in full-bloom stage was soaked in the transformed Agrobacterium suspension for 20-30s while gently swirling.

(6) Dark culture: and covering the soaked plants with bags, keeping the plants in a high wet state, and culturing for 24 hours in a dark room.

(7) And (3) culturing after dip dyeing: watering every other day to ensure sufficient water.

(8) Seed collection: the seeds are mature, and the seeds can be harvested after the siliques are naturally cracked.

(9) Screening transgenic seeds: the seeds obtained after the dip-dyeing are cultured on plates containing hygromycin antibiotics. About 200 seeds of 40mg were vernalized in 0.5 × MS medium containing 10-50 μ g/ml hygromycin for 2 days, followed by culture under continuous light for 7-10 days. And judging whether the seeds are transgenic seeds according to the growth condition. The seeds successfully transferred into the recombinant plasmid can normally grow more than 4 true leaves in resistance culture. The non-transgenic seeds can not grow normally, only 2 leaves can grow, the growth of roots is also severely inhibited, and the seeds die after germinating for 10 days.

(10) And (5) cultivating the transgenic plant in soil. After the transgenic seeds germinate on the MS + hygromycin plate for 2 weeks, positive plants are transferred into soil for continuous culture.

(11) And (3) PCR identification: taking positive plant leaves to extract genome DNA and carrying out PCR verification by using a target gene sequence and a vector 35S promoter sequence primer, wherein the primer sequence is P1233 CheckF: 5'-CTAGACGAGGAGGTGTC-3' (SEQ ID NO. 17); P1233R: 5'-CCGCTCGAGGAAAGGAAGATGGGCACTTC-3' (SEQ ID NO. 18). The results are shown in FIG. 7. Detecting the transgenic positive plants. The plant selection marker hygromycin gene detection is performed from left to right. Hygromycin F: GAGCATATACGCCC

GGAGTC, hygromycin R: GTCTCCGACCTGATGCAGCTCTCGG are provided.

The LEAFY gene is transformed into agrobacterium C5C81, and arabidopsis thaliana is infected by an inflorescence method. The arabidopsis seeds to be detected are planted on a resistance culture medium, the successfully transformed plants can grow normally, and the unsuccessfully transformed plants are yellow dead plants without successfully transforming LEAFY genes. Transplanting the primarily screened seedlings into a culture pot for continuous culture, when 10-12 seedlings grow, cutting leaves for extracting genome DNA, performing PCR detection by using a target fragment amplification primer and a carrier 35S promoter sequence primer, and indicating that the LEAFY gene is integrated into the arabidopsis genome as a result of electrophoresis, wherein a target band appears.

4. Phenotypic analysis of transgenic Arabidopsis

Transgenic Arabidopsis T2 generations were sown and flowering of the transgenic lines was recorded. The number of days required for bolting and flowering, and the number of flowering were counted from the start of sowing, and the results are shown in table 8, fig. 8, and fig. 9.

TABLE 8

The transgenic arabidopsis line exhibited an early flowering phenotype compared to the wild type arabidopsis plant. And counting the bolting days, flowering days and flowering numbers of the transgenic arabidopsis thaliana and the wild arabidopsis thaliana. The results show that the number of bolting days of the Arabidopsis line over-expressing the LEAFY gene is 3 days earlier than that of the wild type on average, the number of flowering days is 2 days earlier than that of the wild type on average, and the number of flowering days is 1.79 more than that of the wild type on average. This indicates that overexpression of LEAFY gene has influence on bolting, flowering period and flowering number of Arabidopsis. Expression of LEAFY gene can promote plant flowering.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

Astragalus sinicus LEAFY gene and application thereof

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Met Asp Pro Asp Ala Phe Thr Ala Ser Leu Phe Lys Trp Asp Pro Arg

15 10 15

Thr Val Leu Pro Pro Ala Pro Pro Pro Arg Pro Pro Leu Leu Glu Tyr

20 25 30

Thr Met Ser Pro Ala Thr Ala Pro Val Pro Tyr His Pro Val Arg Ala

35 40 45

Pro Arg Glu Leu Gly Gly Leu Glu Glu Leu Phe Gln Ala Tyr Gly Ile

50 55 60

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

65 70 75 80

Thr Leu Ile Asp Met Lys Asp Glu Glu Leu Asp Asp Met Met Asn Ser

85 90 95

Leu Ser Gln Ile Phe Arg Trp Asp Leu Leu Val Gly Glu Arg Tyr Gly

100 105 110

Ile Lys Ala Ala Ile Arg Ala Glu Arg Arg Arg Val Asp Asp Glu Glu

115 120 125

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

130 135 140

Leu Ser Gln Glu Gly Leu Ser Glu Glu Pro Val Met Gln Arg Asp Lys

145 150 155 160

Glu Val Val Gly Ser Gly Gly Gly Asn Thr Trp Glu Val Val Ala Ala

165170 175

Glu Glu Arg Arg Lys Gln Arg Arg Arg Arg Ser Arg Met Lys Val Asn

180 185 190

Val His Gly Asp Glu Asn Glu Glu Ala Glu Asp Glu Glu Gly Glu Asp

195 200 205

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

210 215 220

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

225 230 235 240

Lys Lys Asn Gly Leu Asp Tyr Leu Phe His Leu Tyr Glu Gln Cys Arg

245 250 255

Glu Phe Leu Ile Gln Val Gln Thr Ile Ala Lys Asp Arg Gly Glu Lys

260 265 270

Cys Pro Thr Lys Val Thr Asn Gln Val Phe Arg Tyr Ala Lys Lys Ala

275 280 285

Gly Ala Ser Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys

290 295 300

Tyr Ala Leu His Cys Leu Asp Glu Glu Val Ser Asn Glu Leu Arg Arg

305 310 315 320

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

325330 335

Tyr Lys Pro Leu Val Ala Ile Ala Gly Arg Gln Gly Trp Asp Ile Asp

340 345 350

Ala Ile Phe Asn Ala His Ser Arg Leu Ser Ile Trp Tyr Val Pro Thr

355 360 365

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

370 375 380

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

385 390 395

<210>3

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ctctgacagg atgatatggc accg 24

<210>4

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ctgttgccgg agacatagtg tattc 25

<210>5

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

catgttataa gccacttgtg gca23

<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 (9)

1. The astragalus sinicus LEAFY is characterized in that the amino acid sequence is shown as SEQ ID No. 2.

2. The Astragalus sinicus LEAFY coding gene as claimed in claim 1, wherein the nucleotide sequence is as shown in SEQ ID No. 1.

3. A biomaterial containing the coding gene of Astragalus sinicus LEAFY as claimed in claim 2, which is an expression vector, an expression cassette or an engineered bacterium.

4. Use of the milk vetch LEAFY of claim 1 or the coding gene of claim 2 or the biological material of claim 3 for modulating flowering phase in plants; the plant is astragalus sinicus or arabidopsis thaliana.

5. Use of the astragalus sinicus LEAFY of claim 1, the encoding gene of claim 2 or the biological material of claim 3 for promoting early bolting of plants; the plant is astragalus sinicus or arabidopsis thaliana.

6. Use of the astragalus sinicus LEAFY of claim 1 or the coding gene of claim 2 or the biological material of claim 3 for promoting premature flowering in a plant; the plant is astragalus sinicus or arabidopsis thaliana.

7. Use of the astragalus sinicus LEAFY of claim 1 or the encoding gene of claim 2 or the biological material of claim 3 for increasing the number of flowers in a plant; the plant is astragalus sinicus or arabidopsis thaliana.

8. Use of the astragalus sinicus LEAFY of claim 1 or the coding gene of claim 2 or the biological material of claim 3 for the preparation of a transgenic plant; the plant is astragalus sinicus or arabidopsis thaliana.

9. Use of the astragalus sinicus LEAFY of claim 1, the coding gene of claim 2 or the biological material of claim 3 for the improvement of plant germplasm resources; the plant is astragalus sinicus or arabidopsis thaliana.

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