CN114854768A

CN114854768A - Duck grass vernalization gene DgPAPS4 and application thereof

Info

Publication number: CN114854768A
Application number: CN202210642986.7A
Authority: CN
Inventors: 张新全; 杨忠富; 黄琳凯; 聂刚; 冯光燕; 李丹丹; 汪霞; 周冀琼
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2022-01-05
Filing date: 2022-06-08
Publication date: 2022-08-05
Anticipated expiration: 2042-06-08
Also published as: CN114854768B; CN114277036A

Abstract

The invention discloses a vernalization gene DgPAPS4 of dactulis glomerata and application thereof, wherein the vernalization gene DgPAPS4 is expressed in plants to adjust the flowering time of the plants, and the gene DgPAPS4 comprises a nucleotide sequence selected from the following group: A. a nucleotide sequence shown in a sequence table SeqID NO. 1; B. a nucleotide sequence complementary to the sequence A, or a nucleotide sequence with 70% homology or more; C. a nucleotide sequence encoding an amino acid sequence shown in a sequence table Seq ID No. 2; the invention also provides an expression vector containing the vernalization gene DgPAPS4, a host cell containing the overexpression vector and application of the vernalization gene DgPAPS 4. The vernalization gene DgPAPS4 provided by the invention can promote the plant flowering with vernalization effect by regulating the expression of vernalization pathway related genes, is beneficial to cultivating plant varieties with vernalization effect in different maturing stages, and simultaneously reduces the breeding workload, reduces the breeding scale, shortens the breeding time, improves the breeding efficiency and accelerates the breeding of the plant varieties with vernalization effect.

Description

Duck grass vernalization gene DgPAPS4 and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, relates to a gene influencing vernalization and flowering of plants, also relates to a protein coded by the gene, an expression vector and a host cell containing the gene, also relates to a gene primer group cloned from plant DNA, and also relates to application of the gene.

Background

Dactylis glomerata (Dactylis globosa L.) also known as orchard grass (Orchardgrass) belongs to the Poaceae family (Graminae) Poales (Pooideae) and the genus Dactylis, and is a perennial cool season type bush grass widely cultivated worldwide. The dactulis glomerata has the characteristics of high growth speed, high biological yield, high sugar content, strong shade resistance, wide application range and the like. As perennial pasture ranked four first in economic value, dactulis glomerata has important significance for the production of herbivorous animal meat and dairy products in temperate regions of the world. Besides being used as excellent pasture, the dactulis glomerata is also one of important excellent mixed-sowing grasses of grasslands and artificial grasslands under China, is mainly suitable for western returned tillering grasses and grassland construction, and has important positive significance for returned tillering grasses and forest-grass composite construction and the like. The orchard grass is mainly planted with the leguminous pasture such as alfalfa, clover and the like in a mixed mode to build a high-quality mixed-planting grassland. The flowering period of the grass seeds of the mixed sowing is required to be consistent, so that the high yield and high quality of the mixed sowing grassland can be ensured.

However, the dactulis glomerata has large variation in flowering phase, and a dactulis glomerata variety suitable for the flowering phase is lacked in actual production, so that the application of the dactulis glomerata in production is severely limited. Although many years of research have been conducted on the flowering stage of dactulis glomerata, the effect is slight.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a gene related to the vernalization pathway that can be regulated.

The second purpose of the invention is to provide a protein coded by a dactulis glomerata vernalization gene.

The invention also aims to provide a primer pair for cloning the duckweed vernalization gene.

The fourth purpose of the invention is to provide an expression vector containing a duckweed vernalization gene.

The fifth purpose of the invention is to provide a host cell containing a cymbopogon citratus vernalization gene.

The invention further aims to provide the application of the dactulis glomerata vernalization gene.

Through long-term research and trial, and many experiments and endeavors, the inventor continuously innovates and innovates, in order to solve the above technical problems, the technical scheme provided by the invention is to provide a dactulis glomerata vernalization gene DgPAPS4, which is expressed in plants to regulate the flowering time of the plants, wherein the gene DgPAPS4 comprises a nucleotide sequence selected from the following group:

A. a nucleotide sequence shown in a sequence table Seq ID No. 1;

B. a nucleotide sequence complementary to the sequence A, or a nucleotide sequence with 70% homology or more;

C. a nucleotide sequence of an amino acid sequence shown in a sequence table Seq ID No. 2.

According to one embodiment of the vernalization gene DgPAPS4 of the present invention, the plant is a vernalization plant.

According to one embodiment of the dactulis glomerata vernalization gene DgPAPS4, the plant comprises dactulis glomerata or arabidopsis thaliana.

The invention also provides a protein coded by the Dactylis glomerata vernalization gene DgPAPS4, and the amino acid sequence of the protein is selected from the amino acid sequence shown in the sequence table Seq ID No. 2.

The invention also provides a primer pair for cloning the dactulis glomerata vernalization gene DgPAPS4, wherein the base sequence of the primer pair is as follows:

an upstream primer F: 5'-atggtaccatgacgacctctaataagccgatat-3' the flow of the air in the air conditioner,

a downstream primer R: 5'-atcgctagcctacaaactaatgcatggccatttc-3' are provided.

The invention also provides an expression vector containing the duckweed vernalization gene DgPAPS4, wherein the full-length duckweed vernalization gene DgPAPS4 is inserted into the vector pCambia1300, and the vector contains a CaMV35S promoter, so that an over-expression vector pCambia1300-35S-DgPAPS4 is obtained.

The invention also provides a host cell containing the overexpression vector, and the overexpression vector pCambia1300-35S-DgPAPS4 is transferred into the agrobacterium tumefaciens strain to obtain the host cell.

The invention also provides application of the dactulis glomerata vernalization gene DgPAPS4 to cultivation of new varieties of dactulis glomerata in different mature periods.

Compared with the prior art, one of the technical solutions has the following advantages:

a) the vernalization gene DgPAPS4 provided by the invention can promote the plant flowering with vernalization effect by regulating the expression of vernalization pathway related genes, is beneficial to cultivating plant varieties with vernalization effect in different maturing stages, and simultaneously reduces the breeding workload, reduces the breeding scale, shortens the breeding years, improves the breeding efficiency and accelerates the breeding of the plant varieties with vernalization effect.

b) The cocksfoot blossoming is a character with complex control network control, the molecular mechanism of cocksfoot blooming period control is not clear at present, and related genes and sites are difficult to apply in cocksfoot molecular assisted breeding. Secondly, the gene capable of regulating and controlling the flowering of the dactulis glomerata is not verified through functional analysis in the dactulis glomerata. At present, the related genes and sites of the orchard grass in the flowering period are excavated and positioned only through transcriptome, QTL positioning and the like, so that the molecular breeding process of the orchard grass is slow. The invention discovers a gene DgAGL20 regulated and controlled by DNA methylation based on the whole genome bisulfite sequencing combined with the transcriptome sequencing result, and discovers an upstream regulatory gene DgPAPS4 by further research, and the transgenic result shows that the expression of AtAGL20 in a transgenic material is regulated and controlled by DgPAPS4, and the development and application of the regulatory gene DgPAPS4 are beneficial to cultivating new varieties of cocksfoot in different maturity and shortening the breeding period.

Drawings

FIG. 1 is a graph comparing the methylation levels of the promoter of DgAGL 20.

FIG. 2 shows the DMR region of DgAGL 20.

FIG. 3 is a graph comparing the expression of DgAGL20 in different vernalization periods of Dactylis glomerata.

FIG. 4 is a graph comparing the expression of DgPAPS4 in different vernalization periods of Dactylis glomerata.

FIG. 5 is a graph comparing the phenotype of the methyl booster and inhibitor treated Dactylis glomerata (A) with the expression of DgAGL20 from Dactylis glomerata (B).

FIG. 6 is a comparison graph of phenotype (A), flowering time (B) and relative expression of AtAGL20 in Arabidopsis at different vernalization stages (C) of Arabidopsis transgenic with DgPAPS4 gene, and the McrBC-qPCR analysis shows that methylation level of the Arabidopsis AtAGL20 promoter region is lower as the qPCR signal is higher (D). .

Detailed Description

The following description is made with reference to the accompanying drawings and a specific embodiment.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. In the present embodiment, any conventional means is used unless otherwise specified.

Example 1

This example describes a vernalization gene, DgPAPS4, which can be expressed in Dactylis glomerata to regulate the flowering time of Dactylis glomerata. The vernalization gene DgPAPS4 comprises a nucleotide sequence selected from the group consisting of:

A. a nucleotide sequence shown in a sequence table Seq ID No. 1.

B. And (b) a nucleotide sequence complementary to the sequence of A. Or a nucleotide sequence having more than 70% homology, for example, a nucleotide sequence having more than 80% homology, a nucleotide sequence having more than 90% homology, a nucleotide sequence having more than 95% homology.

Example 2

This example describes a protein whose amino acid sequence is shown in Seq ID No. 2. The sequence is encoded by Duck grass vernalization gene DgPAPS4(A) in example 1.

Example 3

This example describes primer pairs for cloning a duckweed vernalization gene PAPS4, the base sequences of the primer pairs are respectively an upstream primer F shown in sequence table Seq ID No. 3: 5'-atggtaccatgacgacctctaataagccgatat-3', a downstream primer R shown in a sequence table Seq ID No. 4: 5'-atcgctagcctacaaactaatgcatggccatttc-3' are provided. By using the primer pair, the nucleotide sequence shown in the sequence table Seq ID No.1 can be extracted from the DNA of the dactylicapnos.

Example 4

This example describes the use of the vernalization gene DgPAPS4 for breeding new varieties of dactulis glomerata in different mature stages by using genetic breeding work such as partial or complete knock-out, transformation, and gene transfer of allogeneic plants of the vernalization gene DgPAPS 4.

Example 5

This example further describes the process of obtaining the duckweed vernalization gene DgPAPS4, and synchronously describes an expression vector containing the duckweed vernalization gene PAPS4, and a host cell containing the expression vector. In this example, the sequence of DgPAPS4 is a nucleotide sequence shown in sequence table Seq ID No. 1. The specific process is as follows.

1. Preparing a dactulis glomerata sequencing material.

The dactulis glomerata diploid line 2006-1 is propagated through tillering, 2 tillering are carried out on each part, and the dactulis glomerata materials are transplanted and then grow for one month in 10 hours in the daytime/14 hours at night and in short day at the temperature of 22 ℃. The dactulis glomerata plants grown for one month under short-day conditions are transferred to a plant growth chamber for vernalization under the conditions of 10 hours in the day/14 hours at night and 8 weeks under the low-temperature short-day condition at the temperature of 4 ℃, and samples are taken before transfer to low temperature (non-vernalized NV), 4 weeks (V4) and 8 weeks (V8) respectively for subsequent transcriptome and whole genome bisulfite sequencing.

2. And (3) sequencing the orchard grass whole genome bisulfite.

Genomic DNA was extracted from Dactylis glomerata leaves of V0, V4 and V8 using the Cetyl Trimethyl Ammonium Bromide (CTAB) method. A bisulfite sequencing library was then prepared. Samples were obtained from Guangzhou Didyo using Illumina HiSeq ^TM The 2500 platform was used for sequencing. Briefly, 1. mu.g of genomic DNA was fragmented into 100-300 bp by sonication (Covaris, Massachusetts, USA) and purified using the MiniElute PCR purification kit (QIAGEN, MD, USA). The fragmented DNA ends are paired, and an "a" is added and a linker is ligated. Adaptor-ligated fragments were converted using the Methylation-Gold kit (ZYMO, CA, USA) according to the manufacturer's instructions. Finally, the transformed DNA fragments were PCR amplified by kyoto, guangzhou and sequenced using Illumina NovaSeq6000 platform.

3. Whole genome bisulfite sequencing data analysis

Double ended sequencing reads containing linker, low mass (q ≦ 20), and poly-N reads were removed. Clean data reads for each sample were compared to https:// www.ncbi.nlm.nih.gov/genome/? the reference genomes for Dactylis disclosed by term ═ Dactylis were aligned, with parameters set by default. Cytosine methylation (mC) was identified using BSMAP software (v2.90) and genome-wide methylation levels were calculated as mC/(mC + non-mC). In addition, the methylation level of the gene and its flanking 2-kb region was calculated from the averaged level per 100 bp. The Differentially Methylated Regions (DMR) between the two samples were identified using methylit tool (v1.10.0) according to the following criteria: (1) mean methylation level fold difference > 2; (2) the Pearson's chi-square test coefficient P is less than or equal to 0.05; (3) the length of the DMR is 40 bp-10 kb; (4) the distance between adjacent methylation sites is less than 200 bp; (5) each methylated cytosine has more than 4 reads coverage, and each cytosine has more than 10 reads coverage; (6) at least one sample has more than 5 methylated cytosines. Through analysis, referring to fig. 1, fig. 1 shows that the methylation level of the AGL20 promoter CHH in dactulis glomerata is significantly increased, and a DMR overlapping with the flowering gene DgAGL20 is found in the V0-V4 comparison group; referring to FIG. 2, FIG. 2 is an IGV graph of AGL20 promoter methylation levels during different vernalization stages of Dactylis glomerata vernalization, DG3C00078 is the flowering gene DgAGL20, and the region shown in box A in FIG. 2 is the promoter differential methylation region.

4. Transcriptome sequencing.

The 12 individual plants used for whole genome methylation sequencing (WGBS) were combined into three independent biological replicates, each replicate containing four individuals. Total RNA was isolated from Dactylis glomerata using Trizol kit (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's protocol. RNA-seq library, PCR amplification and sequencing using Illumina HiSeq4000 were performed by Diao, Calif. To obtain high quality clean data, reads containing adapters, poly-N reads, and low quality bases (q ≦ 20) were removed using fastp (v0.18.0). Clean data was then mapped to the dactulis glomerata genome by HISAT2(v2.2.4) with the parameters: "-rn-strandness RF" and other parameters are set to default values. String Tie (v1.3.1) is used to assemble the map read. String Tie calculates gene expression levels as FPKM (number of fragments per million bases). Referring to fig. 3, fig. 3 shows the expression of the agm 20 of dactylicapnos at different vernalization stages, and the expression level of AGL20 was found to be significantly higher than that of V0 in the V4 sample by analyzing the transcriptome data. In combination with the whole gene bisulfite sequencing data, it is speculated that the expression level of AGL20 may be under methylation regulation. Further analysis of transcriptome data revealed that, referring to fig. 4, fig. 4 shows the expression level of DgPAPS4 in different vernalization stages of dactylicapnos dactylicapni, and that DgPAPS4 and DgAGL20 have the same expression pattern in the vernalization prophase, and that DgPAPS4 is a gene related to the RdDM pathway, and the expression pattern in the vernalization prophase is consistent with the methylation change, indicating that DgPAPS4 may participate in the regulation of DgAGL20 through methylation. The inventors have conducted further analysis and study on the gene.

5. And (3) verifying the methylation level in the process of vernalization of dactylicapnos.

The cymbopogon citratus plant clones were vernalized in a growth chamber under short light (8h/16h, day/night) at 4 ℃. In the treatment, 500. mu.M of the DNA methylation facilitator Methyl Triflate (MTFMS) (TCI, T2029) and the inhibitor zebularine (Zeb) (Selleck, S71130) were dissolved in ddH containing 0.05% Silwet L-77(Solarbio, S9430) ₂ O and directly sprayed on the leaves of Dactylis glomerata. With ddH containing 0.05% Silwet L-77 ₂ O-treated Dactylis glomerata plants were used as control (Mock). All treatments were sprayed 1 time per 2d until vernalization was complete, at least 10 plants per treatment. Use of

qRT-PCR was performed by EvaGreen 2X qPCR Master Mix to analyze the expression of DgAGL20 in each treatment.

TABLE 1 qRT-PCR reaction systems Table

The primers are as follows:

an upstream primer DgAGL20-F:5'-agctgttgcgggagaacttaga-3' shown in a sequence table Seq ID No.5,

a downstream primer DgAGL 20-R5'-tcgccgttcctcctcctcat-3' shown in a sequence table Seq ID No.6,

internal reference primers:

an upstream primer DgGAPDH-F:5'-tctgaccgttagacttgagaagg-3' shown in the sequence table Seq ID No.7,

a downstream primer DgGAPDH-R:5'-cttgagcttaccctcagactcct-3' shown in the sequence table Seq ID No. 8.

Analysis found that in the methyl promoter treated samples, see figure 5, the expression level of DgAGL20 was significantly higher at vernalization 4 weeks than the control and methyl inhibitor treated materials, indicating that expression of DgAGL20 was promoted by hypermethylation. FIG. 5 shows the results of methyl promoter (MTFMS) and inhibitor (Zeb) treatment experiments during the vernalization of Dactylis glomerata; FIG. 5, panel A, shows the phenotype of dactulis glomerata after treatment for 20 days of growth in warm (22 ℃) long day (16h light/8 h dark); b is the expression quantity of AGL20 in dactulis glomerata treated differently. Specifically, FIG. 5 is a comparison graph of the phenotype of Dactylis glomerata and the expression level of DgAGL20 in Dactylis glomerata treated with the methyl enhancer and inhibitor, wherein the respective treatments are: V4M: vernalization treated 4-week control, V4 Zeb: vernalization treatment for 4 weeks + methyl inhibitor Zeb treatment, V4 MTFMS: vernalization treatment for 4 weeks + methyl promoter MTFMS treatment.

6. The construction of Duck grass DgPAPS4 gene transfer Arabidopsis thaliana overexpression strain and the statistics of flowering time.

The full-length dgdgdpaps 4 was inserted into the vector pCambia1300, which contained the CaMV35S promoter. To generate transgenic plants overexpressing DgPAPS4, Agrobacterium tumefaciens strain EHA105 harboring pCambia1300-35S-DgPAPS4 was used for transformation. Inflorescence dipping method was used for Arabidopsis transformation. Homozygous lines (generation T3) were used for further functional validation. For arabidopsis vernalization, seeds were germinated on 1/2MS plates until the first pair of rosette leaves appeared, and seedlings were transferred from the plates to soil. Subsequently, the plants were pre-grown for 10d under long-day conditions (16h/8h, light/dark, 22 ℃) and then were vernalized for 4 weeks under short-day conditions (8h/16h, light/dark), 4 ℃ and returned to long-day conditions (16h/8h, light/dark, 22 ℃) after vernalization. Flowering time was calculated as the number of days of flowering after cold treatment, from when plants were moved to 22 ℃ to bolting, but not including pre-growth or spent time in cold (fig. 6A, B). WT representationArabidopsis thaliana Col-0 wild type, OE1, OE3, OE4 represent three Arabidopsis thaliana strains overexpressing DgPAPS4, respectively. Use of

qRT-PCR was performed by EvaGreen 2X qPCR Master Mix to analyze the expression level of AtAGL20 in Arabidopsis thaliana. The DNA of 4 weeks of purified Arabidopsis thaliana was extracted using the cetyltrimethylammonium bromide (CTAB) method and 500ng of DNA was used in the McrBC kit (NEB, M0272) according to the manufacturer's protocol for 4 hours of digestion; the McrBC enzyme needs the existence of GTP when cutting methylated DNA sites, the reaction without adding GTP is used as a negative control, and the methylation level of the promoter region of the Arabidopsis AtAGL20 is analyzed by qRT-PCR after the enzyme cutting is finished. FIG. 6 shows the results of the vernalization assay of the transgenic Arabidopsis thaliana with DgPAPS 4; wherein A is the phenotype of growth under warm (22 ℃) long sunlight (16h light/8 h dark) after vernalization of transgenic Arabidopsis and wild type for 4 weeks; b is statistics of flowering time under warm (22 ℃) long sunlight (16h light/8 h dark) after vernalization of transgenic Arabidopsis and wild type; c is the expression level of AGL20 in Arabidopsis at different vernalization stages; d is the methylation level of the Arabidopsis AGL20 promoter region after 4 weeks of vernalization analyzed by McrBC-qPCR, with the higher the qPCR signal, the lower the methylation level.

The primers used were:

an upstream primer AtAGL20-F:5'-caagcagacaagtgactttctc-3' shown in a sequence table Seq ID No.9,

a downstream primer AtAGL20-R:5'-gagctggcgaattcataaagtt-3' shown in a sequence table Seq ID No.10,

the internal reference primer is as follows:

an upstream primer AtTUB2-F shown in a sequence table Seq ID No. 11: 5'-gccttgtacgatatttgcttcaggac-3' the flow of the air in the air conditioner,

a downstream primer AtTUB2-R:5'-cggaggtcagagttgagttgac-3' shown in a sequence table Seq ID No.12,

primers used for McrBC-qPCR:

an upstream primer AtAGL20-F shown in a sequence table Seq ID No. 13: 5'-tcaaacattcggtgacgagtaagca-3' the flow of the air in the air conditioner,

an upstream primer AtAGL20-R:5'-ggacgagcagattaagttggtggaa-3' shown in a sequence table Seq ID No. 14.

After analysis, it was found that the expression level in the transgenic lines was significantly higher than that of the wild type after vernalization treatment (FIG. 6C). FIG. 6 shows phenotypes of DgPAPS 4-transgenic Arabidopsis thaliana, flowering-time (flowering-time is the number of days of transfer to flowering under warm long-day conditions after vernalization treatment, excluding the time of vernalization treatment), relative expression levels of Arabidopsis AtAGL20 at different vernalization stages of Arabidopsis thaliana, and methylation levels of Arabidopsis thaliana AtAGL20 promoter region after vernalization. Wherein, NV: not vernalized; V4W: vernalization for 4 weeks.

Experiments prove that the DgPAPS4 gene can be used for cultivating new varieties of dactulis glomerata in different maturity stages and shortening the breeding period by regulating the methylation level of an AtAGL20 promoter region in a transgenic material and further regulating the expression of AtAGL20 in the transgenic material in vernalization.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Sequence listing

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His Gly Ile Ser Ile Asp Leu Leu Tyr Ala Ser Val Ser Leu Leu Val

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Arg Val Phe Ala Gln Trp His Trp Pro Thr Pro Val Met Leu Cys Ala

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Pro Arg Asp Arg Thr His His Met Pro Ile Ile Thr Pro Ala Tyr Pro

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Cys Met Asn Ser Ser Tyr Asn Val Ser Thr Ser Thr Leu Arg Val Met

305 310 315 320

Ile Glu Gln Phe Gln Phe Gly Asn Lys Ile Cys Gln Glu Ile Glu Met

325 330 335

Asn Lys Ala Lys Trp Ser Ala Leu Phe Glu Pro Phe Asn Phe Phe Glu

340 345 350

Ala Tyr Lys Asn Tyr Leu Gln Val Asp Ile Ile Ala Glu Asp Asp Gln

355 360 365

Asp Leu Arg Leu Trp Lys Gly Trp Val Glu Ser Arg Leu Arg Gln Leu

370 375 380

Thr Leu Lys Ile Glu Arg Asp Thr Tyr Gly Lys Leu Gln Cys His Pro

385 390 395 400

Tyr Pro Ser Asp Tyr Ala Asp Pro Ser Arg Gln Cys Ala His Cys Ala

405 410 415

Phe Phe Met Gly Leu Ser Arg Lys Glu Gly Val Lys Ile Gln Glu Gly

420 425 430

Gln Gln Phe Asp Ile Arg Gly Thr Val Asp Glu Phe Arg His Glu Ile

435 440 445

Asn Met Tyr Met Phe Trp Lys Pro Gly Met Glu Leu Ala Val Ser His

450 455 460

Val Arg Arg Lys Glu Ile Pro Ala Tyr Val Phe Pro Glu Gly Tyr Arg

465 470 475 480

Arg Pro Arg Pro Gln Arg His Val Asn His Gln Gln Gln Ser Asn Lys

485 490 495

Asn Ala Thr Glu Asn Ser Pro Ser Leu Thr Gly Ser Pro Asp Ser Gln

500 505 510

Leu Lys Arg Lys His Ala Ser Ala Gly Met Asp Asp Thr Glu Pro Cys

515 520 525

Arg Ser Val Lys Arg Ala Ser Val Ser Pro Val His Thr Lys Thr Ser

530 535 540

Ser Pro Leu Ser Gly Asn Val Ser Asp Glu Pro Thr Ser Asn Asn Gln

545 550 555 560

Thr Lys Leu Thr Ser Asn Ala Ser Gly Gly Ser Gln Asn Ser Pro Ser

565 570 575

Ile Gly Asn Leu Glu His Ala Lys Cys Ser Gly Ser Pro Gln Ala Ser

580 585 590

Glu Lys Ser Val Asp Ser Ile Ala Ser Gly Ser Lys Cys Val Lys Val

595 600 605

Glu Ala Val Cys Ser Gly Asp Val Thr Ser Lys His Val Asn Cys Ile

610 615 620

Ser His Val Lys Asp Ser Thr Ala Pro Ala Val Ala Val Ser Thr Thr

625 630 635 640

Leu Lys Arg Val Ala Glu Lys Val Val Leu Glu Leu Val Gly Ser Glu

645 650 655

Ser Leu Gly Gly Asn Asn Ala Asp Leu Leu Gln Ile Thr Glu Lys Asp

660 665 670

Met Gly Ser Val Leu Ala Glu Lys Val His Phe Gly Gly Asn Arg Val

675 680 685

Ser Gln Ser Gly Leu His Glu Glu Leu Glu Val Ser Ala Ser Gly Ala

690 695 700

Phe Ser Pro Phe Leu Lys Trp Pro Cys Ile Ser Leu

705 710 715

<210> 3

<211> 33

<212> DNA

<213> Dactylis glomerata (Dactylis globerata)

<400> 3

atggtaccat gacgacctct aataagccga tat 33

<210> 4

<211> 34

<212> DNA

<213> Dactylis glomerata (Dactylis globerata)

<400> 4

atcgctagcc tacaaactaa tgcatggcca tttc 34

<210> 5

<211> 22

<212> DNA

<213> Dactylis glomerata (Dactylis globerata)

<400> 5

agctgttgcg ggagaactta ga 22

<210> 6

<211> 20

<212> DNA

<213> Dactylis glomerata (Dactylis globerata)

<400> 6

tcgccgttcc tcctcctcat 20

<210> 7

<211> 23

<212> DNA

<213> Dactylis glomerata (Dactylis globerata)

<400> 7

tctgaccgtt agacttgaga agg 23

<210> 8

<211> 23

<212> DNA

<213> Dactylis glomerata (Dactylis globerata)

<400> 8

cttgagctta ccctcagact cct 23

<210> 9

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 9

caagcagaca agtgactttc tc 22

<210> 10

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 10

gagctggcga attcataaag tt 22

<210> 11

<211> 26

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 11

gccttgtacg atatttgctt caggac 26

<210> 12

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 12

cggaggtcag agttgagttg ac 22

<210> 13

<211> 25

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 13

tcaaacattc ggtgacgagt aagca 25

<210> 14

<211> 25

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 14

ggacgagcag attaagttgg tggaa 25

Claims

1. A duckweed vernalization gene DgPAPS4 expressed in a plant to regulate flowering time of said plant, wherein said gene DgPAPS4 comprises a nucleotide sequence selected from the group consisting of:

A. a nucleotide sequence shown in a sequence table Seq ID No. 1;

2. The duckweed vernalization gene DgPAPS4 according to claim 1, wherein the plant is a vernalization plant.

3. The dactulis glomerata vernalization gene DGPAPS4 according to claim 1, wherein the plant comprises dactulis glomerata or arabidopsis thaliana.

4. A protein encoded by the dactulis glomerata vernalization gene DgPAPS4 according to any one of claims 1 to 3, wherein the amino acid sequence of the protein is selected from the amino acid sequences shown in sequence listing Seq ID No. 2.

5. A primer pair for cloning a duckweed vernalization gene DgPAPS4 is characterized in that the base sequences of the primer pair are as follows:

a downstream primer R: 5'-atcgctagcctacaaactaatgcatggccatttc-3' are provided.

6. An expression vector containing a duckweed vernalization gene DgPAPS4, wherein the full-length duckweed vernalization gene DgPAPS4 is inserted into a vector pCambia1300, wherein the vector contains a CaMV35S promoter, and an over-expression vector pCambia1300-35S-DgPAPS4 is obtained.

7. A host cell comprising the overexpression vector of claim 6, wherein the overexpression vector pCambia1300-35S-DgPAPS4 is transformed into Agrobacterium tumefaciens strain to obtain the host cell.

8. The use of the duckweed vernalization gene DgPAPS4 according to claim 1 for breeding new varieties of dactulis glomerata in different mature periods.