CN117701585A

CN117701585A - Alfalfa MYB transcription factor MsMYB306 gene and application thereof

Info

Publication number: CN117701585A
Application number: CN202311539454.1A
Authority: CN
Inventors: 郭振飞; 黄日升; 施海帆; 蒋守臻; 吴佳璇; 王召明; 苑峰; 刘亚玲
Original assignee: Inner Mongolia Grassland Technology Innovation Center Co ltd; Nanjing Agricultural University
Current assignee: Inner Mongolia Grassland Technology Innovation Center Co ltd; Nanjing Agricultural University
Priority date: 2023-11-18
Filing date: 2023-11-18
Publication date: 2024-03-15

Abstract

The invention discloses an alfalfa MYB transcription factor MsMYB306 gene and application thereof, wherein the nucleotide sequence of the MYB transcription factor MsMYB306 gene is as follows (1) or (2): (1) a nucleotide sequence shown as SEQ ID NO. 1; (2) A nucleotide sequence having a homology of 90% or more with the nucleotide sequence of (1) and having an equivalent function. The invention provides a method for cultivating cold-resistant plants by using MsMYB306 genes, which improves the cold resistance of transgenic alfalfa after the genes are expressed in the alfalfa in a disturbing way; meanwhile, after the gene is excessively expressed in the alfalfa, the cold resistance of the transgenic alfalfa is reduced.

Description

Alfalfa MYB transcription factor MsMYB306 gene and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to an alfalfa MYB transcription factor MsMYB306 gene and application thereof.

Background

In agricultural production, how to increase the yield is particularly important. Abiotic stress severely affects the yield of agricultural production. The alfalfa planting areas are mainly distributed in areas with higher dimensionality in China, low-temperature stress is often encountered, and the yield of alfalfa is severely limited. Therefore, it is necessary to dig the gene regulating cold resistance of alfalfa.

Transcription factors refer to DNA binding proteins or related proteins capable of interacting with cis-acting elements in the promoter region of eukaryotic genes, i.e., key factors capable of specifically activating or inhibiting transcription. Most plant MYB genes encode R2R 3-type MYB proteins. The R2R3-MYB transcription factor has an N-terminal DNA binding domain (MYB domain) and an activation or inhibition domain located at the C-terminal end. In contrast to the highly conserved MYB domain, other regions of R2R3-MYB proteins are highly variable (Jiang et al 2004). Numerous studies have shown that R2R3-MYB transcription factors are involved in a variety of processes including primary and secondary metabolism, plant development, and response to biotic and abiotic stress (Dubos et al, 2010).

Low temperature stress impedes plant growth and development by severely affecting plant metabolism and gene transcription. Cold-stimulated plants rapidly induce expression of CBFs genes to activate many downstream COR genes (Liu et al, 2018). MYB transcription factors are thought to play a key role in regulating cold tolerance due to their regulation of CBFs genes. In arabidopsis, MYB15 is a negative regulator of CBFs genes. MYB15 mutant plants exhibit increased tolerance to low temperatures, whereas transgenic plants overexpressing MYB15 exhibit a cold-sensitive phenotype. MYB15 inhibits expression of CBFs genes by directly binding to MYB binding sites in CBFs promoters (Agarwal et al, 2006). MYB96 is upregulated by low temperature and increases the freezing resistance of arabidopsis by upregulating CBFs expression (Guo et al, 2013). In rice, osMYBS3 inhibits early response to low temperature stress in rice by inhibiting expression of CBFs genes, on the other hand, osMYBS3 can activate long-term low temperature signals, regulate expression of genes encoding trehalose-6-phosphatase TPPs (6-phosphate phosphatase) in the trehalose metabolic pathway to resist long-term low temperature stress (Su et al, 2010). In apples, interaction of the MdMYB2 and MdSIZ1 promoters significantly increases the tolerance of plants to cold stress (Jiang et al, 2022). Transgenic apple calli overexpressing MdMYB4d have reduced conductivity under freeze injury stress, improved survival, and positive regulation of cold tolerance of apple calli (Wu et al, 2017). MdMYB23 binds directly to the MdCBF1 and MdCBF2 promoters and activates their expression to increase cold tolerance (An et al 2018). MdMYB88 and MdMYB124 combine with the promoters of MdCSP3 (COLD SHOCK DOMAIN PROTEIN) and MdCSA 1 (CIRCADIAN CLOCK ASSOCIATED 1), respectively, to co-activate expression of MdCBF3, while indirectly promoting anthocyanin accumulation to clear ROS accumulation, thereby positively regulating cold tolerance of apples (Xie et al, 2018). In capsicum, caMYB306 inhibits the transcription negative-regulatory cold resistance of the cold-tolerant positive regulator CaCIPK13 (Ma et al 2022). However, there are few reports on the function of regulating cold resistance by MYB transcription factors in alfalfa, and it is found in Tribulus alfalfa that the transcription enhancer MtMYB61 and the transcription inhibitor MtMYB3 regulating MtMYB 4 interact with MtMYB3, impairing the ability of MtMYB3 to bind to the promoter of MtCBF4, thereby inhibiting the expression of the target gene MtCAS15 downstream of MtCBF4 and negatively regulating cold resistance of Tribulus alfalfa (Zhang et al 2016). In summary, whether the R2R3-MYB transcription factor family members still have the function of regulating cold resistance in alfalfa needs to be further studied.

Alfalfa is an important leguminous forage grass and has the characteristics of high biomass, good nutrition quality, wide cultivation range and the like. However, low temperature severely limits the growth and geographical distribution of alfalfa and reduces yield and quality. The cold-resistant gene is cloned and regulated from alfalfa, so that the gene can provide more excellent target genes for transgenic breeding of alfalfa, and is particularly important for cold-resistant molecular breeding of other leguminous crops.

Disclosure of Invention

To overcome the defects and shortcomings in the prior art, the primary purpose of the invention is to provide an alfalfa MYB transcription factor MsMYB306 gene which interferes with the expression of the gene and improves the cold resistance of plants.

Another object of the present invention is to provide the protein encoded by the alfalfa MYB transcription factor MsMYB306 gene.

It is still another object of the present invention to provide the use of the alfalfa MYB transcription factor MsMYB306 gene.

The aim of the invention is achieved by the following technical scheme:

in a first aspect, the invention claims an alfalfa MYB transcription factor MYB306 gene, where the nucleotide sequence of the MYB transcription factor MYB306 gene is (1) or (2) as follows:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) A nucleotide sequence having a homology of 90% or more with the nucleotide sequence of (1) and having an equivalent function.

In a second aspect, the present invention claims a protein encoded by the alfalfa MYB transcription factor MYB306 gene, where the amino acid sequence of the protein is (a) or (b):

(a) An amino acid sequence as shown in SEQ ID NO. 2;

(b) The amino acid sequence shown in SEQ ID NO.2 has the same function and is formed by substituting, deleting or adding one or more amino acids.

In a third aspect, the present invention claims a biological material related to the above alfalfa MYB transcription factor MYB306 gene, which is a biological material containing the MYB transcription factor MYB306 gene, or a biological material for silencing, interfering or inhibiting the MYB transcription factor MYB306 gene;

the biological material containing the MYB transcription factor MYB306 gene is at least one of the following (a) - (g):

(a) An expression cassette containing the MYB transcription factor MYB306 gene;

(b) A recombinant vector containing the MYB transcription factor MYB306 gene;

(c) A recombinant vector comprising the expression cassette of (a);

(d) A recombinant microorganism comprising the MYB transcription factor MYB306 gene;

(e) A recombinant microorganism comprising the expression cassette of (a);

(f) A recombinant microorganism comprising the recombinant vector of (b);

(g) A recombinant microorganism comprising the recombinant vector of (c);

the biological material for silencing, interfering or inhibiting the MYB transcription factor MYB306 gene is at least one of the following (I) - (IV):

interference sequences of MYB transcription factor MYB306 genes;

(II) a primer group for amplifying the MYB transcription factor MYB306 gene interference sequence of (I);

(iii) an interfering vector for the MYB transcription factor MYB306 gene;

(IV) a recombinant microorganism comprising an interfering vector as described in (III).

Further, the primer group for amplifying the MYB transcription factor MYB306 gene interference sequence comprises an MsMYB306 interference fragment sense strand amplification upstream and downstream primer and an MsMYB306 interference fragment antisense strand enzyme cutting site upstream and downstream primer;

MsMYB306 interfering fragment sense strand amplification upstream primer Y5288:

5’-ACCATGGGGCGCGCCTGGCCAAACAAGCCTTATCTGA-3’；

MsMYB306 interfering fragment sense strand amplification downstream primer Y5289:

5’-TCATCGATTGGGCGCGCCTGCTTTGTGCTCCTTCACTACA-3’；

upstream primer Y5290 of the cleavage site of the antisense strand of the MsMYB306 interfering fragment:

5’-CTTAATTAACTCTCTAGATGGCCAAACAAGCCTTATCTGA-3’；

downstream primer Y5291 of the MsMYB306 interfering fragment antisense strand cleavage site:

5’-TTGCAGGTATTTGGATCCTGCTTTGTGCTCCTTCACTACA-3’。

in a fourth aspect, the invention claims the use of the alfalfa MYB transcription factor MYB306 gene, the protein, or the biomaterial described above in (1) or (2) below:

(1) Transgenic plants with increased cold tolerance are raised or cultivated;

(2) Reducing the cold resistance of plants or cultivating cold-sensitive transgenic plants.

Further, the application of the gene can silence or interfere with the MYB306 gene of the MYB transcription factor of alfalfa in target plants to improve the cold resistance of the plants. Alternatively, over-expressing the alfalfa MYB transcription factor MYB306 gene described above in a target plant reduces the cold tolerance of the plant.

In a fifth aspect, the present invention claims a method for improving cold tolerance in a plant by silencing or interfering with the alfalfa MYB transcription factor MYB306 gene described above in the plant of interest.

Further, the process of silencing or interfering the alfalfa MYB transcription factor MYB306 gene in the target plant is as follows: the interference vector of the alfalfa MYB transcription factor MYB306 gene is constructed, and the interference vector is transferred into plants by an agrobacterium-mediated method to obtain transgenic plants with improved cold resistance.

The plant is alfalfa.

In the research process, technicians respectively adopt pCAMBIA3301 and pFGC5941 to construct an overexpression vector 35S, namely MsMYB306 and an interference vector MsMMYB 306-RNAi, and the screening pressure is glufosinate. And selecting mature alfalfa leaves subjected to cuttage for 8-12 weeks to carry out genetic transformation, and sequentially carrying out the processes of infection, co-culture, callus induction, callus regeneration, regeneration seedling rooting and the like to obtain the transgenic alfalfa.

The recombinant vector containing the alfalfa MYB transcription factor MsMYB306 gene is a recombinant expression vector, and the recombinant expression vector is obtained by connecting the nucleotide sequence of the alfalfa MYB transcription factor MsMYB306 gene with a plant expression vector; the plant over-expression vector is preferably pCAMBIA3301.

Preparing an interference vector for interfering the expression of an alfalfa MYB transcription factor MsMYB306 gene, wherein the adopted vector is preferably pFGC5941;

the preparation method of the recombinant expression vector containing alfalfa MYB transcription factor MsMYB306 gene (MsMYB 306) comprises the following steps:

(1) Primer design

Primer (1) is an upstream primer Y4752 for amplifying MsMYB306 gene fragment:

5’-ATGATGGGAAGGCCACCATGTT-3’；

primer (2) is MsMYB306 gene fragment amplification downstream primer Y4753:

5’-CTAAAAGAAATCTGAAGTAGTT-3’；

primer (3) constructs an overexpression vector for MsMYB306 to amplify the upstream primer Y4926:

5’-CACGGGGGACTCTTGACCATGGCAATGATGGGAAGGCCACCATGTT-3’；

primer (4) constructs an overexpression vector for MsMYB306 to amplify the downstream primer Y4927:

5’-CGGGGAAATTCGAGCTGGTCACCCTAAAAGAAATCTGAAGTAGTT-3’；

primer (5) is an upstream primer Y5288 for amplifying the sense strand of the MsMYB306 interference fragment:

5’-ACCATGGGGCGCGCCTGGCCAAACAAGCCTTATCTGA-3’；

primer (6) is a downstream primer Y5289 for amplifying the sense strand of the MsMYB306 interference fragment:

5’-TCATCGATTGGGCGCGCCTGCTTTGTGCTCCTTCACTACA-3’；

primer (7) is an upstream primer Y5290 of the antisense strand cleavage site of the MsMYB306 interference fragment:

5’-CTTAATTAACTCTCTAGATGGCCAAACAAGCCTTATCTGA-3’；

primer (8) is a downstream primer Y5291 of the antisense strand cleavage site of the MsMYB306 interference fragment:

5’-TTGCAGGTATTTGGATCCTGCTTTGTGCTCCTTCACTACA-3’；

(2) Obtaining a MsMYB306 gene fragment:

amplifying the full-length fragment of the MsMYB306 gene by using the cDNA of alfalfa as a template and using the primer (1) and the primer (2);

(3) Construction of recombinant expression vectors pCAMBIA3301-MsMYB306 and pFGC5941-MsMYB306 containing MsMMMYB 306 genes:

using a MsMYB306 gene fragment as a template, using a primer (3) and a primer (4) to lead the MsMYB306 gene fragment to introduce two restriction sites of Nco I and BstE II, and connecting the restriction sites with a plant expression vector pCAMBIA3301, thereby constructing and obtaining a recombinant expression vector pCAMBIA3301-MsMYB306 containing the MsMMYB 306 gene;

using a MsMYB306 gene fragment as a template, using a primer (5) and a primer (6) to lead the MsMYB306 gene interference sense fragment into a homology arm containing an Asc I enzyme cleavage site, using a primer (7) and a primer (8) to lead the MsMYB306 gene interference antisense fragment into a homology arm containing an Xba I enzyme cleavage site and a BamH I enzyme cleavage site, connecting the sense fragment with a plant expression vector pFGC5941 firstly, and then connecting the antisense fragment with the vector connected with the sense fragment, thereby obtaining a recombinant expression vector pFGC5941-MsMYB306 containing the MsMYB306 interference fragment;

the transgenic plant of the recombinant expression vector pCAMBIA3301-MsMYB306 containing the MsMYB306 gene and the transgenic plant of the pFGC5941-MsMYB306 interference vector are obtained by transforming alfalfa leaf tissues with the recombinant expression vector or the interference vector and culturing the transformed plant tissues.

In a specific embodiment of the present invention, the plant is preferably alfalfa.

The preparation method of the transgenic plant for expressing alfalfa MsMYB306 comprises the following steps:

(1) Introducing the recombinant expression vectors pCAMBIA3301-MsMYB306 and the interference vectors pFGC5941-MsMYB306 into agrobacterium tumefaciens EHA105 by using an electric shock transformation method to respectively obtain positive agrobacterium colonies containing the recombinant expression vectors pCAMBIA3301-MsMYB306 and the interference vectors pFGC5941-MsMYB306;

(2) The leaves of alfalfa are impregnated with the activated agrobacterium EHA105 containing the recombinant expression vector pCAMBIA3301-MsMYB306 and the interference vector pFGC5941-MsMYB306, and the transgenic alfalfa is obtained through co-culture and glufosinate resistance screening.

The invention clones cDNA sequence MsMYB306 of MyB transcription factor from alfalfa, connects the obtained sense segment and antisense segment of MsMYB306 gene with plant expression vector, constructs over-expression vector and interference vector, transforms alfalfa leaf with the constructed over-expression vector and interference vector, and then cultures transgenic alfalfa. The MsMYB306 gene is subjected to low-temperature induction and down-regulated expression; the invention provides a method for regulating plant cold resistance by using MsMYB306 gene, which reduces the cold resistance of transgenic alfalfa after the gene is excessively expressed in the alfalfa; meanwhile, after the gene is interfered in the alfalfa, the cold resistance of the transgenic alfalfa is improved.

Compared with the prior art, the invention has the following advantages and effects:

(1) The cDNA sequence of MsMYB306 is cloned from alfalfa, and the expression of the gene is inhibited by low temperature induction to down regulate the expression.

(2) The cDNA full length of the MsMYB306 is connected with a plant over-expression vector to construct an over-expression vector suitable for alfalfa, the sense segment and the antisense segment of the MsMYB306 gene are connected with a plant interference vector to construct an interference expression vector suitable for alfalfa, the alfalfa is transformed, the transgenic alfalfa over-expressing the MsMYB306 obviously reduces the cold resistance, and the transgenic alfalfa interfering with the expression of the MsMYB306 obviously improves the cold resistance.

(3) The invention provides a method for culturing cold-resistant alfalfa by using RNAi interference technology to down-regulate MsMYB306 expression.

(4) The invention provides a method for cultivating stress-tolerant plants by using MsMYB306 genes.

Drawings

FIG. 1 is a schematic representation of the expression cassette of the overexpression vector pCAMBIA3301-MsMYB 306.

FIG. 2 is a schematic representation of insertion sites for sense and antisense fragments of the interfering expression vector pFGC5941-MsMYB 306.

FIG. 3 is a diagram of agarose gel electrophoresis of PCR amplification of the open reading frame sequence of the MsMYB306 gene;

wherein M is a standard DNA molecule; lane 1 is an amplified fragment of the MsMYB306 gene.

FIG. 4 is a diagram of PCR identification agarose gel electrophoresis of the over-expression vector pCAMBIA3301-MsMYB306;

wherein M is a standard DNA molecule; lanes 1, 2, 3, 4, 5, 6, 7 and 8 are positive recombinants.

FIG. 5 is a diagram of agarose gel electrophoresis of PCR amplification of RNAi interference fragment of MsMYB306 gene;

wherein M is a standard DNA molecule; lane 1 is the amplification of the sense fragment of the MsMYB306 gene and lane 2 is the amplification of the antisense fragment of the MsMYB306 gene.

FIG. 6 is an illustration of insertion PCR identification agarose gel electrophoresis of sense and antisense fragments of the interfering expression vector pFGC5941-MsMYB306;

wherein M is a standard DNA molecule; lanes 1, 2, 3, 4, 5, 7 and 8 are positive sense fragment recombinants, lanes 9, 10, 11, 12, 13, 14, 15 and 16 are positive antisense fragment recombinants; lane 6 is a negative sense fragment recombinant.

FIG. 7 is a bar graph of the analysis of the results of induction of MsMYB306 gene expression at low temperature 4 ℃;

the letters a, b, c and d above the bars represent significant differences between the different treatments (P < 0.05), i.e. the same letters above the data bars represent no significant differences and the different letters above the data bars represent significant differences.

FIG. 8 agarose gel electrophoresis of PCR identification results of transgenic alfalfa introduced with over-expression vector pCAMBIA3301-MsMYB306 and interference expression vector pFGC5941-MsMYB306;

wherein WT represents a wild type; the numbers 1-5 represent different overexpressed transgenic alfalfa strains, and the numbers 6-9 represent different RNAi-interfering transgenic alfalfa strains.

FIG. 9 is a graph of the analysis of the results of real-time quantitative PCR of 5 transgenic alfalfa introduced with the overexpression vector pCAMBIA3301-MsMYB306;

wherein WT represents a wild type; the numbers 1 to 5 represent different transgenic alfalfa strains; letters a, b, c, d and e above the columns indicate that the difference between the different materials is significant (P < 0.05).

FIG. 10 is a graph of analysis of the results of real-time quantitative PCR of 4 transgenic alfalfa introduced with the recombinant expression vector MsMYB 306-RNAi;

wherein WT represents a wild type; the numbers 6-9 represent different transgenic alfalfa strains; the letters a, b and c above the columns represent significant differences between the different materials (P < 0.05).

FIG. 11 is an analysis chart of the results of freeze resistance detection of transgenic alfalfa into which the recombinant expression vector MsMYB306-RNAi was introduced;

wherein, (A) is a photograph of each strain after freezing injury treatment under non-cold acclimation and cold acclimation conditions; (B) Low temperature semi-lethal temperature of each strain under non-cold acclimation and cold acclimation conditions; (C) Survival rate of each strain under non-cold acclimation and cold acclimation conditions; letters a, b, c, d and e above the columns indicate that the difference between the different materials is significant (P < 0.05).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Alfalfa (Medicago sativa l.) variety Regen-SY4D (Yu et al 2022), supplied by the grass biotechnology and breeding laboratory at the university of tokyo agriculture.

Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105: purchased from beijing tianen gene technologies limited.

The pCAMBIA3301 overexpression vector and the pFGC5941 RNAi vector, supplied by the grass biotechnology and breeding laboratory at the university of Nanjing agriculture (Yu et al 2022), are common vectors disclosed in the prior art.

Coli DH 5. Alpha: purchased from beijing, department of biotechnology, inc. The above biological materials are all conventional biological materials known to those skilled in the art.

EXAMPLE 1 cloning of the MsMYB306 Gene

1. Preparation of alfalfa cDNA template

Alfalfa was taken from the white horse test base of the university of Nanjing agriculture, transplanted into a mixed culture medium (peat soil: vermiculite: perlite=2:2:1 (volume ratio), cultivated in an incubator at 22 ℃/20 ℃ (day/night) with photoperiod of 16h/8h (light/dark), humidity 75%, light intensity 200 μm ^-2 s ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Applying a proper amount of compound fertilizer (N: P: K=15:15:15) solution to the mixture periodically; after the materials grow uniformly, the materials are used for test treatment, mature leaves of alfalfa are taken, and thenCell/Tissue Total RNA Isolation Kit (Vazyme) kit the RNA of plant tissue was extracted, and PrimeScript RT reagent Kit (Takara) was used to synthesize long fragment cDNA for subsequent gene cloning, using the RNA as template, with reference to the procedure of the instructions provided by Takara.

2. Design and amplify MsMYB306 gene primer

Primers were designed to amplify the open reading frame of alfalfa MsMYB306 cDNA (synthesized by Beijing qing biosciences Co., ltd.) based on the cDNA sequence of the genomic "Xinjiang big leaf" alfalfa variety MsMYB306 gene. The sequence of the upstream primer Y4752 for amplifying MsMYB306 is as follows: 5'-ATGATGGGAAGGCCACCATGTT-3'; the sequence of the downstream primer Y4753 for amplifying MsMYB306 is as follows: 5'-CTAAAAGAAATCTGAAGTAGTT-3';

3. amplification to obtain MsMYB306 full-length ORF

PCR amplification was performed using the template prepared in the first step and the primers designed in the second step:

PCR reaction System (50. Mu.L): KOD-FX-DNA polymerase (TOYOBO Co., ltd., 1U. Mu.L) ^-1 )1μL，2×buffer 25μL，dNTPs(10mmol·L ^-1 ) 10. Mu.L, upstream primer (10. Mu. Mol.L) ^-1 ) 1.5. Mu.L, downstream primer (10. Mu. Mol.L) ^-1 ) 1.5. Mu.L, first strand cDNA 1. Mu.L, ddH were reverse transcribed ₂ O is added to 50 mu L;

PCR reaction procedure: 94 ℃ for 2min;98℃10s,55℃30s,68℃1min,34 cycles; 68 ℃ for 10min; detecting the amplified product by agarose gel electrophoresis with the mass fraction of 1%;

a sequence of about 1000bp in length was obtained (FIG. 3), and the obtained PCR product was recovered by using a DNA gel recovery kit (AXYGEN Co.).

Example 2 construction of the overexpression vector pCAMBIA3301-MsMYB306 and the interference vector pFGC5941-MsMYB306

1. Construction of the overexpression vector pCAMBIA3301-MsMYB306

Amplification primers with Nco I and BstE II cleavage sites were designed based on the MsMYB306 open reading frame sequence obtained in example 1:

MsMYB306 construction of the over-expression vector amplification upstream primer Y4926:

5’-CACGGGGGACTCTTGACCATGGCAATGATGGGAAGGCCACCATGTT-3’；

MsMYB306 construction of the overexpression vector amplification downstream primer Y4927:

5’-CGGGGAAATTCGAGCTGGTCACCCTAAAAGAAATCTGAAGTAGTT-3’；

the MsMYB306 full-length DNA fragment with homology arms is amplified by PCR, and pCAMBIA3301 expression vector is respectively cut by restriction enzymes Nco I (NEB) and BstE II (NEB) in a double enzyme way, and the gene fragment and the vector fragment are respectively cut according to a molar ratio of 1:2, and the reaction system is as follows: 5 XCE II Buffer 4. Mu.L, exnase II 2. Mu.L, recovered target fragment 0.06pmol, water make up to 20. Mu.L, connection at 37℃for 30min, and cooling on ice to obtain a connection product;

the ligation product is thermally shock transformed into escherichia coli: the ligation product was added to 100. Mu.L DH 5. Alpha. Competent cells (purchased from Beijing engine biotechnology Co., ltd.) and left in ice for 5min; after heat shock for 45s at 42 ℃, placing in ice for 2min; the heat-shocked cells were added to 0.5mL of LB liquid, and cultured with shaking at 200rpm on a constant temperature shaking table at 37℃for 0.5h. After brief centrifugation, 500. Mu.L of supernatant was removed, leaving 100. Mu.L of resuspended bacteria liquid to spread on LB solid (containing 50. Mu.g/mL Kana) medium and cultured in 37℃incubator upside down overnight;

screening, purifying and sequencing of recombinant plasmid pCAMBIA3301-MsMYB 306: single colonies were picked and PCR was performed using the upstream primer Y4926 and the downstream primer Y4927, and the results of the gel electrophoresis experiments showed that a single band was present at about 1000bp, indicating that the MsMYB306 gene was contained on the recombinant vector (FIG. 4). The positive monoclonal is submitted to Beijing qing biological science and technology Co-Ltd for sequencing, and the result shows that the sequence of the insert fragment is completely consistent with the sequence of the MsMYB306 coding region, and the enzyme cutting sites at the two ends of the insert fragment are also completely correct, so that the recombinant expression vector pCAMBIA3301-MsMYB306 is successfully constructed, and the schematic diagram of the expression frame of the recombinant expression vector is shown in figure 1.

2. Construction of interference vector pFGC5941-MsMYB306

According to the MsMYB306 open reading frame sequence obtained in example 1, a band with a size of about 276bp is selected, an amplification primer with an Asc I (NEB) cleavage site is designed for PCR amplification to obtain a sense fragment containing a homology arm (FIG. 5), a vector pFGC5941 is subjected to single cleavage by using Asc I (Takara Co.), and a target fragment is connected with the vector fragment by adopting a homologous recombination method after the PCR product and the cleavage product are recovered.

MsMYB306 interfering fragment sense strand amplification upstream primer Y5288:

5’-ACCATGGGGCGCGCCTGGCCAAACAAGCCTTATCTGA-3’；

5’-TCATCGATTGGGCGCGCCTGCTTTGTGCTCCTTCACTACA-3’；

the recovery method, the homologous recombination method and the method for transforming the plasmid into the escherichia coli are the same as the above methods.

By colony PCR detection, the forward primer Y5288 and the downstream primer Y5289 are amplified by using the MsMYB306 interference fragment sense strand to obtain fragments with correct sizes, the band size is about 276bp (figure 6), and the result shows that the recombinant vector contains a 276bp sense fragment (shown as SEQ ID NO. 3). Further sequencing of the insert showed that the sequence of the insert was identical to the sequence of the selected sense fragment and that the cleavage sites at both ends of the insert were also completely correct, thus proving successful construction of the vector plasmid containing the sense strand fragment.

The double enzyme cutting sites with Xba I (NEB) and BamH I (NEB) are further designed according to the connection rule and method of the interference vector, the gene fragment of MsMYB306 obtained in example 1 is used as a template for PCR amplification to obtain an antisense fragment containing a homology arm, meanwhile, the vector containing the sense fragment obtained in the above step is subjected to double enzyme cutting by using Xba I and BamH I, and the target fragment is connected with the vector fragment by adopting a homologous recombination method after the recovery of the PCR product and the enzyme cutting product.

5’-CTTAATTAACTCTCTAGATGGCCAAACAAGCCTTATCTGA-3’；

5’-TTGCAGGTATTTGGATCCTGCTTTGTGCTCCTTCACTACA-3’；

By colony PCR detection, the antisense strand of MsMYB306 interference fragment was used to amplify the upstream primer Y5290 and the downstream primer Y5291 to obtain fragments with correct sizes, the band size was about 276bp (FIG. 6), and the result shows that the recombinant vector contains an antisense fragment with the size of 276bp (shown as SEQ ID NO. 4). Further sequencing of the insert showed complete identity of the insert sequence to the selected antisense sequence and complete correct cleavage sites at both ends of the insert, thus proving successful construction of vector plasmids containing both sense and antisense strand fragments. Wherein FIG. 2 is a schematic diagram of insertion sites of sense and antisense fragments of recombinant expression vector pFGC5941-MsMYB 306.

Example 3 down-regulated expression of MsMYB306 by Low temperature inhibition

1. Obtaining low-temperature treated alfalfa templates

Alfalfa was taken from the white horse test base of the university of Nanjing agriculture, transplanted into a mixed culture medium (peat soil: vermiculite: perlite=2:2:1 (volume ratio), cultivated in an incubator at 22 ℃/20 ℃ (day/night) with photoperiod of 16h/8h (light/dark), humidity 75%, light intensity 200 μm ^-2 s ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Applying a proper amount of compound fertilizer (N: P: K=15:15:15) solution to the mixture periodically; after the materials grow uniformly and uniformly, the materials are used for low-temperature 4 ℃ test treatment, the materials are respectively treated for 0h, 1h, 2h, 6h and 12h at normal temperature and 4 ℃ and then mature leaves are taken out, the mature leaves are packaged by tinfoil paper and then are put into liquid nitrogen for rapid freezing, and the materials are used for the production of the composite materialThe Cell/Tissue Total RNA Isolation Kit (Vazyme) kit is used for extracting RNA from plant tissues, and HiScript III RT SuperMix for qPCR Kit (Vazyme) is used for synthesizing cDNA by taking the RNA as a template for subsequent qRT-PCR experiments.

2. Designing specific detection primer

Based on the MsMYB306 gene cDNA sequence and alfalfa action sequence, quantitative primers for detection were designed using on-line software (https:// sg.idtdna. Com/pages/tools/primequestrinurl=% 2FPrimerQuest% 2F):

upstream quantitative primer Y4928 of MsMYB306 gene: 5'-GTTCGCAGCTTCCGAATAAAG-3';

downstream quantitative primer Y4929 of MsMYB306 gene: 5'-ACCAAGGGTTGTAGGGTTATG-3';

upstream quantitative primer Y358 of action gene: 5'-CCCACTGGATGTCTGTAGGTT-3';

downstream quantitative primer Y359 of action gene: 5'-AGAATTAAGTAGCAGCGCAAA-3';

3. quantitative PCR detection of expression differences

Diluting the template cDNA prepared in the step one by 30 times for quantifying the template for PCR, wherein the reaction system is 10 mu L: 2X ChamQ SYBR qPCR Master Mix (Vazyme) 5. Mu.L, 0.2. Mu.L each of the upstream and downstream primers (10. Mu.M), 4. Mu.L of cDNA template, 0.6. Mu.L of sterile water; mini Option Real-Time PCR System manufactured by Bio-Rad Co., ltd, the PCR reaction conditions were set as follows: 95 ℃ for 30s;95 ℃ for 10s,60 ℃ for 30s,40 cycles; 95℃15s,60℃60s,95℃15s. 3 repeats are set for each sample, and housekeeping gene action is taken as an internal reference gene; after the reaction is finished, a dissolution curve analysis is carried out, the PCR amplification efficiency is more than 95%, and the relative expression quantity of the genes is automatically calculated by using Bio-Rad CFX Manager (Version 1.6) software.

The results of real-time quantitative PCR showed that MsMYB306 expression was inhibited from down-regulating at 2h of low temperature treatment and maintained low levels all the way to 12h (fig. 7). The result shows that the low temperature inhibits the down-regulated expression of the MsMYB306 gene.

Example 4 Generation and molecular detection of transgenic alfalfa

1. Production of transgenic alfalfa

1. Introduction of recombinant expression vector MsMYB306-RNAi into Agrobacterium tumefaciens EHA105

Preparation of EHA105 competence Lin et al (Lin JJ. Electrotransformation of agrobacteria. Methods Mol biol.1995, 47:171-8.) and improvements: the monoclonal was picked and inoculated in 5mL of YEP (75 mg/L rifampicin or other antibiotics) broth, 28℃at 200rpm, and shake cultured overnight. The bacterial solution was transferred to 200mL of YEP (75 mg/L rifampicin or other antibiotics) medium at 1:100, and cultured with shaking at 28℃and 200rpm until OD=0.4 (about 4-5 h). Transfer to a 250mL sterile centrifuge bottle, centrifuge at 4000rpm for 10min at 4℃and discard the supernatant. Resuspension with 150mL of pre-chilled ultrapure water, centrifugation at 4000rpm at 4℃for 10min, and supernatant discarded. The steps 4 and 5 were repeated 2-3 times (the volume of ultrapure water could be reduced in a gradient, as appropriate) and finally resuspended in 10% (v/v) glycerol in a 2mL ice bath. 100. Mu.L of each tube was dispensed into 1.5mL sterile EP tubes (pre-chilled in a-80℃refrigerator) and stored at-80 ℃. Thawing competent cells on ice, pre-cooling a 0.2cm inner diameter electric shock cup on ice, adding 1.5 mu L of pCAMBIA3301-MsMYB306 or pFGC5941-MsMYB306 plasmid (20 ng/. Mu.L) into 100 mu L of thawed competent cells in an ultra-clean workbench, uniformly mixing the walls of the flick tube, transferring to the electric shock cup after ice bath for 1min, placing between electrodes of an electric shock instrument (Micropulser, bio-RAD company), and selecting a program Agr for electric shock; after the electric shock is finished, rapidly pouring 1mL of YEP liquid culture medium into an electric shock cup in an ultra-clean workbench, transferring the electric shock cup into a fungus shaking tube by using a pipetting gun, and carrying out gentle shaking culture for 2 hours at 28 ℃; sucking 0.3mL of bacterial liquid, coating on a YEP plate (containing 35mg/L rifampicin and 50mg/L kanamycin), and culturing in an incubator at 28 ℃ for 48 hours in an inversion way;

2. identification of Agrobacterium Positive colonies containing recombinant expression vectors pCAMBIA3301-MsMYB306 or pFGC5941-MsMYB306

PCR positive colonies were picked into 3mL of YEP liquid medium (containing 35mg/L rifampicin and 50mg/L kanamycin) and cultured with shaking at 28℃for 40h. 1 mu L of bacterial liquid is sucked for PCR detection, and PCR detection primers of the recombinant expression vector pCAMBIA3301-MsMYB306 are as follows: the PCR detection primers of the upstream primer Y4926 and the downstream primer Y4927, pFGC5941-MsMYB306 are as follows: upstream primer Y5288 and downstream primer Y5289.PCR reaction System (10. Mu.L): 2X Taq Plus Master Mix II (Vazyme) 5. Mu.L, upstream primer (10. Mu. Mol.L) ^-1 ) 1. Mu.L, downstream primer (10. Mu. Mol.L) ^-1 ) 1. Mu.L, 1. Mu.L of bacterial liquid and ddH ₂ O is added to 10 mu L; PCR reaction procedure: PCR reaction procedure: 3min at 95 ℃; 15s at 95 ℃,20 s at 60 ℃,20 s at 72 ℃ and 34 cycles; 72 ℃ for 10min; the amplified products were detected by agarose gel electrophoresis with a mass fraction of 1% (m/v, g/100 ml); determining that the positive clone contains a plant expression vector pCAMBIA3301-MsMYB306 or pFGC5941-MsMYB306; 0.8mL of agrobacterium liquid is taken, 0.2mL of glycerin with the volume fraction of 80% (v/v) is added, and the mixture is uniformly mixed and stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for standby.

3. Obtaining transgenic alfalfa plants

(1) Preparation of reagents and Medium

(1) 2,4-D stock (0.5 mg/mL), 6-BA stock (1 mg/L), 100mM AS, timentin antibiotic stock (200 mg/mL), cephalosporin antibiotic stock (200 mg/mL), basta stock (10 mg/mL);

(2) KT stock (1 mg/L): weighing 100mg of KT powder, dissolving the KT powder with 2mL of 95% ethanol, and then, fixing the volume to 100mL by distilled water and preserving the KT powder at 4 ℃;

(3) 100mM AS: weighing 0.1962g of AS powder, dissolving in 10mL of DMSO, filtering with 0.22 μm organic filter membrane, sterilizing, and packaging at-20deg.C;

(4) amectin antibiotic mother liquor (200 mg/mL): weighing 20g of timentin powder, dissolving in 80mL of distilled water, finally fixing the volume to 100mL, filtering and sterilizing by using a 0.22 mu m water-based filter membrane, and subpackaging and preserving at-20 ℃;

(5) cephalosporin mother liquor (200 mg/mL): weighing 20g of cephalosporin powder, dissolving in 80mL of distilled water, finally, fixing the volume to 100mL, filtering and sterilizing with a 0.22 μm water-based filter membrane, and subpackaging at-20 ℃ for storage;

(6) basta mother liquor: diluting 200g/L Basta liquid to 10mg/mL stock solution, filtering with 0.22 μm water system filter membrane, sterilizing, and packaging at-20deg.C;

(7) SM4 callus-retaining medium (pH 5.8, antibiotic and screening agent added, basta:2mg/L, timentin: 200mg/L, cephalosporin: 200 mg/L)

(8) MSBK medium (pH 5.85, antibiotic and screening agent added, basta:1mg/L, timentin: 200mg/L, cephalosporin: 200 mg/L)

(9) MSS medium (pH 5.8, antibiotic and screening agent added, basta:1mg/L, timentin: 200mg/L, cephalosporin: 200 mg/L)

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(2) Identification of alfalfa leaves and positive plants infested with Agrobacterium-mediated methods

(1) 2days before infection, single colonies on a target gene transformed agrobacterium plate are selected, cultured overnight in a liquid medium containing kanamycin (50 mg/L) and rifampicin (35 mg/L) and positive clones are identified;

(2) what needs to be prepared 1 day before infection (one transformation): tweezers, a cutter, an empty dish, 200mL of YEP, 200mL of SM4+AS solid medium, 300mL of SM4 liquid medium, 200mL of sterilized distilled water, 100mL of sodium hypochlorite disinfectant, 2 empty bottles of filter paper and 250mL, 2 wide-mouth bottles and 1 sharp-bottomed centrifuge tube of 50 mL. And according to 1: inoculating the bacterial liquid into YEP containing the same antibiotics according to the proportion of 500;

(3) on the day of transformation, shearing alfalfa Regen-SY4D leaves growing 4-6 weeks by using sterilized scissors, cleaning surface impurities by using tap water, sterilizing the surfaces by using 6.25% NaClO for 40min, cleaning the surfaces by using sterilized distilled water for 4-5 times, removing NaClO, cutting off leaf edges of leaf stalks, and manufacturing a plurality of wounds on the leaves for transformation experiments;

(4) the OD of the target bacterial liquid of 28 ℃ shaking culture is kept ₆₀₀ Centrifuging at 22deg.C and 3000rpm for 13min at 0.6-0.8, suspending the precipitate with 100mL of SM4 (containing 100 μm acetosyringone) to obtain an aggressive dye solution;

(5) putting the cut leaves into an invasion dye liquor, fully mixing, vacuumizing after 40Hz ultrasonic wave for 3-5min (the water temperature is lower than 20 ℃), and vacuumizing for 10min at-0.1 pka (more than 0.09 pka); 28 ℃,80rpm,1.5h, keep dark;

(6) taking out the slice, removing agrobacterium on the surface of the slice on sterilized filter paper as much as possible, transferring to SM4 solid culture medium containing 100 mu M acetosyringone, ensuring that the leaf is right-side-up, and culturing in dark for 2days;

(7) transferring the slice after co-culture to SM4 solid medium (ensuring the right side of the leaf to be upward) containing 20mg/L Basta, 200mg/L timentin and 200mg/L cephalosporin, culturing under light for 5-6 weeks, and changing the plate every 2 weeks;

(8) transferring the callus to MSBK culture medium with 20mg/L Basta halving for culture, and changing the plate every 3weeks until forming a prophase embryo; about 20-30 days, the pre-embryo develops into a true embryo;

(9) after 2 to 3weeks, the embryo starts to grow into plantlets and grows out; transferring the plantlet to MSS culture medium, and transferring to tissue culture tube for seedling strengthening after the plantlet grows out;

transferring the robust plants into a greenhouse, defining seedlings grown from the same callus as the same transformation event, carrying out different Line numbers, culturing, extracting DNA, and carrying out transgenic identification by using Bar gene primers (sequences shown in the attached table 1) on a vector, and subsequently carrying out stress resistance function analysis.

PCR detection of double transgenic alfalfa

1. Method for extracting alfalfa total DNA

The 2 XCTAB extract is placed in a water bath kettle at 65 ℃ in advance for preheating. A proper amount of leaf plant samples are placed in a 2mL centrifuge tube with a steel ball, quickly frozen with liquid nitrogen and ground into powder by a grinder (40 Hz,1 min). 500. Mu.L of the preheated extract was added to the centrifuge tube, and the mixture was mixed in a water bath at 65℃for 30min, upside down, and once every 10min. An equal volume (500. Mu.L) of chloroform/isoamyl alcohol (24:1, V/V) was added, and after shaking vigorously, the mixture was allowed to stand for 5min, and centrifuged at 12000rpm at 4℃for 10min. Transfer 350. Mu.L supernatant to a new 1.5mL centrifuge tube, add equal volume of isopropanol, mix upside down, stand in-20deg.C refrigerator for more than 30 min. Centrifugation was performed at 12000rpm at 4℃for 10min, at which time a white precipitate (DNA) was present at the bottom of the tube. The supernatant was discarded, 1mL of 75% ethanol was added, the mixture was inverted several times, and the mixture was allowed to stand at 4℃for 5 minutes and centrifuged at 12000rpm for 10 minutes. The supernatant was discarded, the liquid in the centrifuge tube was dried, and 50. Mu.L of sterilized water preheated at 65℃was added to dissolve the DNA.

2. PCR detection of transgenic alfalfa

Since the vectors pCAMBIA3301 and pFGC5941, which infect plants, also carry Bar resistance genes in their left and right border regions, primers Y953 (5'-ATGAGCCCAGAACGACGC-3') and Y954 (5'-TCAAATCTCGGTGACGGG-3') which specifically recognize the genes were used to detect whether the gene of interest was successfully transferred into the plant genome; the DNA obtained in the step is used as a template for PCR amplification, and wild alfalfa is used as a negative control. Reaction system (10 μl): 2X Taq Plus Master Mix II (Vazyme Co.) 5. Mu.L, upstream primer Y953 (10. Mu. Mol. L) ^-1 ) 1. Mu.L, downstream primer Y954 (10. Mu. Mol. L) ^-1 ) 1. Mu.L, 1. Mu.L of bacterial liquid and ddH ₂ O is added to 10 mu L;

PCR reaction procedure: 3min at 95 ℃; 15s at 95 ℃,30 s at 55 ℃,20 s at 72 ℃ and 34 cycles; 72 ℃ for 10min; the amplified products were detected by agarose gel electrophoresis with a mass fraction of 1% (FIG. 8);

the expression level of the MsMYB306 gene in the over-expressed strain and the RNAi interference strain was detected by real-time quantitative PCR, and the result shows that the mRNA level of the MsMYB306 gene in the over-expressed transgenic alfalfa was significantly increased (fig. 9) and the mRNA level of the MsMYB306 gene in the RNAi transgenic alfalfa was significantly decreased (fig. 10) compared to the wild alfalfa.

Example 5 identification of the freezing resistance of transgenic alfalfa

1. Low temperature semi-lethal temperature measurement

And (3) taking alfalfa leaves, subtracting redundant leaf handles, cleaning the surfaces of the leaves by deionized water, and sucking the water on the surfaces of the leaves by filter paper. Every three individual leaves were placed in pre-chilled glass tubes and a small piece of ice was carefully added and three replicates were set for each sample. The test tube is placed in a low-temperature freezing circulation instrument, and is subjected to cooling treatment at 2 ℃ per hour after cold acclimation from 0 ℃ (minus 2 ℃), each temperature treatment is carried out for 1 hour, 5 temperatures are set for each treatment, the test tube at the temperature point is taken out every 2 ℃ and is placed at 4 ℃ for overnight thawing. 5mL of deionized water was added to each tube, the conductivity of the water was noted as C0, and the conductivity was measured after shaking on a horizontal shaker at room temperature until the electrolyte was impermeable. The test tube was treated in a boiling water bath for 40min to release all electrolytes from the leaves, cooled to room temperature and the conductivity C2 was determined. The formula was calculated with reference to the former study (Yu et al, 2022) to determine the semi-lethal temperature.

2. Determination of survival Rate after alfalfa Freeze treatment

The alfalfa freeze injury treatment experimental procedures were primarily referred to the methods of the former study (Yu et al 2022) and modified on the basis of this in connection with the growth conditions of the plant material. The alfalfa strip with at least 2 knots is cut in a square plastic basin with a side length of 9 cm and containing mixed nutrient soil (peat soil: vermiculite: perlite=3:1:1 (v/v)), grown in long sunlight (16 h of illumination, 8h of darkness), placed in a 24 ℃ illumination incubator for 40-50d, respectively placed in a-5 ℃ low temperature illumination incubator for 6h, then transferred to 4 ℃ for thawing treatment, the growth is resumed in a long sunlight, 22 ℃ incubator, and the survival rate is counted after two weeks. The low-temperature domesticated alfalfa is required to grow for about 40-50 days, long sunlight is moved, the alfalfa is subjected to cold domestication for one week in a 4 ℃ illumination incubator, then the alfalfa is placed in the-7 ℃ low-temperature illumination incubator for 6 hours, and the subsequent operation is the same as that of the non-cold domesticated material. Survival rate formula: survival = number of surviving plants/total number of plants 100%.

As a result, as shown in fig. 11A-C, the low temperature half-lethal temperature of the overexpressed transgenic alfalfa, whether not it was subjected to cold-acclimation at 4 ℃ or cold-acclimation at 4 ℃ for 7 days, was significantly higher than that of the wild type, while the survival rate was significantly lower than that of the wild type, whereas the low temperature half-lethal temperature of the transgenic alfalfa of the RNAi strain was significantly lower than that of the wild type, while the survival rate was significantly higher than that of the wild type. This suggests that transgenic alfalfa that interfered with the expression of MsMYB306 had significantly higher freezing resistance than the wild type, whereas transgenic alfalfa that overexpressed MsMYB306 had significantly lower freezing resistance than the wild type.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

SEQ ID NO.1

Alfalfa (Medicago sativa L.)

Nucleotide sequence for encoding alfalfa MYB transcription factor MsMYB306

SEQ ID NO.2

Alfalfa (Medicago sativa L.)

Encoding alfalfa MYB transcription factor MsMYB306

SEQ ID NO.3

Alfalfa (Medicago sativa L.)

Alfalfa MYB transcription factor MsMYB306 sense fragment for RNAi vector construction

SEQ ID NO.4

Alfalfa (Medicago sativa L.)

Alfalfa MYB transcription factor MsMYB306 antisense fragment for RNAi vector construction

SEQ ID NO.5 (MsMYB 306 gene fragment amplification upstream primer Y4752):

5’-ATGATGGGAAGGCCACCATGTT-3’；

SEQ ID NO.6 (MsMYB 306 gene fragment amplification downstream primer Y4753):

5’-CTAAAAGAAATCTGAAGTAGTT-3’；

SEQ ID NO.7 (MsMYB 306 construction of the overexpression vector amplification upstream primer Y4926):

5’-CACGGGGGACTCTTGACCATGGCAATGATGGGAAGGCCACCATGTT-3’；

SEQ ID NO.8 (MsMYB 306 constructs the overexpression vector amplification downstream primer Y4927):

5’-CGGGGAAATTCGAGCTGGTCACCCTAAAAGAAATCTGAAGTAGTT-3’；

SEQ ID NO.9 (MsMYB 306 interference fragment sense strand amplification upstream primer Y5288):

5’-ACCATGGGGCGCGCCTGGCCAAACAAGCCTTATCTGA-3’；

SEQ ID NO.10 (MsMYB 306 interference fragment sense strand amplification downstream primer Y5289): 5'-TCATCGATTGGGCGCGCCTGCTTTGTGCTCCTTCACTACA-3';

SEQ ID NO.11 (upstream primer Y5290 of the cleavage site of the antisense strand of the MsMYB306 interfering fragment): 5'-CTTAATTAACTCTCTAGATGGCCAAACAAGCCTTATCTGA-3';

SEQ ID NO.12 (primer Y5291 downstream of the cleavage site of the antisense strand of the MsMYB306 interfering fragment): 5'-TTGCAGGTATTTGGATCCTGCTTTGTGCTCCTTCACTACA-3';

SEQ ID NO.13 (upstream quantitative primer Y4928 of MsMYB306 gene):

5’-GTTCGCAGCTTCCGAATAAAG-3’；

SEQ ID NO.14 (downstream quantitative primer Y4929 of MsMYB306 gene):

5’-ACCAAGGGTTGTAGGGTTATG-3’；

SEQ ID NO.15 (upstream quantitative primer of action gene Y358):

5’-CCCACTGGATGTCTGTAGGTT-3’；

SEQ ID NO.16 (downstream quantitative primer of action gene Y359):

5’-AGAATTAAGTAGCAGCGCAAA-3’；

SEQ ID NO.17(Y953)：

5’-ATGAGCCCAGAACGACGC-3’；

SEQ ID NO.18(Y954)：

5’-TCAAATCTCGGTGACGGG-3’。

Claims

1. the nucleotide sequence of the MYB transcription factor MYB306 gene of alfalfa MYB is as follows (1) or (2):

(1) A nucleotide sequence shown as SEQ ID NO. 1;

2. The protein encoded by the alfalfa MYB transcription factor MYB306 gene of claim 1, which has the amino acid sequence of (a) or (b) as follows:

(a) An amino acid sequence as shown in SEQ ID NO. 2;

3. A biological material related to the alfalfa MYB transcription factor MYB306 gene of claim 1, wherein the biological material is a biological material comprising the MYB transcription factor MYB306 gene, or a biological material for silencing, interfering or inhibiting the MYB transcription factor MYB306 gene;

(a) An expression cassette containing the MYB transcription factor MYB306 gene;

(b) A recombinant vector containing the MYB transcription factor MYB306 gene;

(c) A recombinant vector comprising the expression cassette of (a);

(e) A recombinant microorganism comprising the expression cassette of (a);

(f) A recombinant microorganism comprising the recombinant vector of (b);

(g) A recombinant microorganism comprising the recombinant vector of (c);

interference sequences of MYB transcription factor MYB306 genes;

(iii) an interfering vector for the MYB transcription factor MYB306 gene;

4. The biomaterial of claim 3, wherein the primer set for amplifying (i) the MYB transcription factor MYB306 gene interference sequence comprises an upstream and downstream primer for amplifying the sense strand of the MsMYB306 interference fragment and an upstream and downstream primer for amplifying the cleavage site of the antisense strand of the MsMYB306 interference fragment;

MsMYB306 interfering fragment sense strand amplification upstream primer Y5288:

5’-ACCATGGGGCGCGCCTGGCCAAACAAGCCTTATCTGA-3’；

5’-TCATCGATTGGGCGCGCCTGCTTTGTGCTCCTTCACTACA-3’；

5’-CTTAATTAACTCTCTAGATGGCCAAACAAGCCTTATCTGA-3’；

5’-TTGCAGGTATTTGGATCCTGCTTTGTGCTCCTTCACTACA-3’。

5. use of the alfalfa MYB transcription factor MYB306 gene of claim 1, the protein of claim 2, or the biological material of claim 3 or 4 in (1) or (2) below:

(1) Transgenic plants with increased cold tolerance are raised or cultivated;

6. The use according to claim 5, wherein silencing or interfering with the alfalfa MYB transcription factor MYB306 gene of claim 1 in the target plant increases the cold tolerance of the plant.

7. The use according to claim 5, wherein overexpression of the alfalfa MYB transcription factor MYB306 gene of claim 1 in the plant of interest reduces the cold tolerance of the plant.

8. A method of increasing cold tolerance in a plant, wherein silencing or interfering with the alfalfa MYB transcription factor MYB306 gene of claim 1 in a plant of interest increases cold tolerance in the plant.

9. The method of claim 7, wherein silencing or interfering with the alfalfa MYB transcription factor MYB306 gene of claim 1 in the target plant is: constructing an interference vector of the alfalfa MYB transcription factor MYB306 gene according to claim 1, and transferring the interference vector into a plant by an agrobacterium-mediated method to obtain a transgenic plant with improved cold resistance.

10. The use according to claim 5 or 6 or 7, the method according to claim 8 or 9, wherein the plant is alfalfa.