Application of alfalfa gene MsCBF4 in improving cold resistance of plants

Abstract

The invention discloses an application of an alfalfa gene MsCBF4 in improving the cold resistance of plants, and belongs to the technical field of gene engineering. In order to excavate a gene with a clear anti-freezing function from medicago sativa, the medicago sativa gene MsCBF4 is successfully cloned, and the expression of the MsCBF4 gene is found to be induced by cold stress and freezing stress through the expression specificity and space-time expression analysis of the gene in the medicago sativa. Besides, a plant overexpression vector containing the alfalfa MsCBF4 gene and recombinant agrobacterium are constructed, transgenic arabidopsis thaliana overexpressing the MsCBF4 gene is obtained through a genetic transformation experiment, freezing tolerance analysis on the transgenic arabidopsis thaliana overexpressing the MsCBF4 gene shows that the overexpression of the MsCBF4 gene enhances the tolerance of the arabidopsis thaliana to freezing stress, and the transgenic arabidopsis thaliana overexpressing the MsCBF4 gene can be used for preventing freezing stress. Therefore, the MsCBF4 gene can be used for improving the cold resistance of the plant.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a Medicago sativa gene MsCBF4 for use in improving the cold tolerance of plants. BACKGROUND

[0002] Medicago sativa (alfalfa) is the second largest cultivated legume forage grass after Glycine max (soybean) in the world, and is a perennial plant with wide ecological adaptability. Its planting area is in major climate zones around the world, especially in temperate regions. This species plays an irreplaceable role in forage production and ecological restoration due to its excellent cold and drought tolerance. Medicago sativa Glycine max However, in the cold northern regions and variable climate growth environments, alfalfa still faces severe freezing stress challenges. Extreme low temperatures, especially persistent cold in winter and frequent late spring cold, and other abnormal temperature mutations not only significantly inhibit the normal growth and development of the plant, but also cause a significant decline in productivity, hinder biomass accumulation, and degrade community structure, resulting in significant economic losses. Therefore, improving the cold resistance of alfalfa has become a key breeding goal to ensure stable and high yields and expand the range of suitable species.

[0003] At present, the main way to improve the cold resistance of alfalfa still relies on traditional genetic breeding methods. This strategy gradually accumulates cold tolerance-related genetic loci through systematic phenotype screening and multi-generation stress domestication to breed cold-resistant germplasm resources. Although this method has achieved certain results in some contexts, it essentially relies on natural genetic recombination, and the process has obvious limitations: on the one hand, while introducing target cold-resistant genes, a large number of non-target genes are often linked and introduced, making it difficult to balance ideal plant type and other agronomic traits; on the other hand, to stabilize the target cold-resistant traits, multiple rounds of tedious phenotype screening and backcrossing processes are usually required, resulting in a long breeding cycle and low efficiency, which cannot meet the demand for rapid updating of modern forage varieties.

[0004] With the development of molecular biology technology, transgenic strategies provide a new technical direction for alfalfa cold resistance breeding. This technology can achieve precise integration of cold tolerance traits by introducing specific functional genes, effectively overcoming the linkage problem in traditional breeding, and significantly shortening the breeding period, laying a scientific foundation for creating new alfalfa germplasm with high self-owned intellectual property rights. However, the successful application of this technology is highly dependent on the mining and verification of key cold-resistant functional genes. At present, the key genes suitable for alfalfa and capable of significantly improving its cold resistance are still relatively limited, which restricts the systematic development and widespread application of this technology in alfalfa breeding.

[0005] With the development of molecular biology technology, transgenic strategies provide a new technical direction for alfalfa cold resistance breeding. This technology can achieve precise integration of cold tolerance traits by introducing specific functional genes, effectively overcoming the linkage problem in traditional breeding, and significantly shortening the breeding period, laying a scientific foundation for creating new alfalfa germplasm with high self-owned intellectual property rights. However, the successful application of this technology is highly dependent on the mining and verification of key cold-resistant functional genes. At present, the key genes suitable for alfalfa and capable of significantly improving its cold resistance are still relatively limited, which restricts the systematic development and widespread application of this technology in alfalfa breeding. ​

[0006] Therefore, identifying, isolating and verifying a new gene with clear anti-freezing function from alfalfa or its close species, and then developing its application in improving the anti-freezing ability of alfalfa and even other crops, has important theoretical and practical significance for breaking through the existing breeding bottleneck and accelerating the breeding process of cold-resistant high-quality alfalfa new varieties. SUMMARY

[0007] In order to mine a gene with clear anti-freezing function from alfalfa, and then develop its application in improving the cold resistance of alfalfa and even other crops, the present application provides the following technical solutions: The first object of the present application is to provide an alfalfa gene MsCBF4 application of the alfalfa gene MsCBF4 for improving the cold resistance of plants, the nucleotide sequence of the alfalfa gene MsCBF4 is shown as SEQ ID NO. 1.

[0008] The second object of the present application is to provide an application of an alfalfa gene MsCBF4 for breeding cold-resistant plants using the alfalfa gene MsCBF4 , the nucleotide sequence of the alfalfa gene MsCBF4 is shown as SEQ ID NO. 1.

[0009] The third object of the present application is to provide an application of a recombinant vector containing the above-mentioned alfalfa gene MsCBF4 , the application is to use the recombinant vector to improve the cold resistance of plants.

[0010] The fourth object of the present application is to provide an application of a recombinant vector containing the above-mentioned alfalfa gene MsCBF4 , the application is to use the recombinant vector to breed cold-resistant plants.

[0011] In an embodiment of the present application, the recombinant vector is constructed by recombining the alfalfa gene MsCBF4 onto pCAMBIA1302-GFP plasmid.

[0012] The fifth object of the present application is to provide an application of a recombinant bacteria containing the above-mentioned alfalfa gene MsCBF4 or the above-mentioned recombinant vector, the application is to use the recombinant bacteria to improve the cold resistance of plants.

[0013] The fifth object of the present application is to provide an application of a recombinant bacteria containing the above-mentioned alfalfa gene MsCBF4 or the above-mentioned recombinant vector, the application is to use the recombinant bacteria to breed cold-resistant plants.

[0014] In one embodiment of the present invention, the host of the recombinant bacteria is Agrobacterium.

[0015] In one embodiment of the present invention, the plant is Arabidopsis thaliana.

[0016] The seventh objective of this invention is to provide a method for improving the cold resistance of Arabidopsis thaliana, the method comprising the following steps: (1) Incorporate alfalfa genes MsCBF4 The recombinant gene was inserted into the pCAMBIA1302-GFP plasmid to obtain the recombinant vector pCAMBIA1302-MsCBF4-GFP, containing the alfalfa gene. MsCBF4 The nucleotide sequence is shown in SEQ ID NO.1; (2) The recombinant vector pCAMBIA1302-MsCBF4-GFP obtained in step (1) was transformed into Agrobacterium competent cells and cultured to obtain the alfalfa gene-carrying cells. MsCBF4 Agrobacterium; (3) Cultivate Arabidopsis seedlings to the initial flowering stage, place the Arabidopsis flower stamens into the Agrobacterium bacterial solution obtained in step (2) for 20-30 s, keep them in the dark for 24 h, and then carry out normal light culture to complete the overexpression transformation of Arabidopsis plants.

[0017] The beneficial effects of this invention are: This invention successfully cloned the alfalfa gene. MsCBF4 Through expression-specific and spatiotemporal expression analysis of this gene in alfalfa, it was found that... MsCBF4 Gene expression is induced by cold stress and freezing stress. Furthermore, this invention constructs a gene containing alfalfa. MsCBF4 The plant overexpression vector of the gene and recombinant Agrobacterium were obtained, and the overexpression was obtained through genetic transformation experiments. MsCBF4 Transgenic Arabidopsis thaliana with overexpressed genes, after... MsCBF4 Analysis of the cryogenic tolerance of transgenic Arabidopsis thaliana revealed that freezing stress exacerbated the membrane response in Arabidopsis thaliana, while overexpression of the gene... MsCBF4 The degree of gene damage in Arabidopsis lines was significantly less than that in WT lines, leading to the conclusion that... MsCBF4 The conclusion is that overexpression of the gene enhances the tolerance of Arabidopsis to freezing stress. Attached Figure Description

[0018] Figure 1 For genes MsCBF4 The results of expression-specific and spatiotemporal expression analysis in alfalfa are shown in the figure; where A represents gene. MsCBF4 The image shows the expression levels of genes in different plant tissues of alfalfa, where B represents gene B. MsCBF4 The image shows the expression levels of alfalfa in the roots and leaves after different durations of freezing stress treatment. C represents the gene expression level.MsCBF4 The results of expression analysis of alfalfa roots and leaves after different durations of cold stress treatment are shown in the figure. Figure 2 The construction map of the recombinant vector pCAMBIA1302-MsCBF4-GFP; Figure 3 The image shows the PCR identification results of the recombinant plasmid pCAMBIA1302-MsCBF4-GFP; where M is the marker, + is the positive control, - is the negative control (using ddH2O as a template), and 1, 2, 3, 4, and 5 are all positive clone plasmids of E. coli transformed with the recombinant vector. Figure 4 To overexpress alfalfa MsCBF4 Figure showing the results of screening and identification analysis of transgenic Arabidopsis thaliana lines expressing the gene; where A represents alfalfa overexpression. MsCBF4 The results of screening transgenic Arabidopsis thaliana lines for the gene are shown in Figure B, where B represents the identification of the exogenous gene using PCR. MsCBF4 The results of successful importation into Arabidopsis thaliana genomic DNA are shown in the figure. + indicates a positive control: recombinant plasmid pTOPO001-MsCBF4; - indicates a negative control (using ddH2O as a template); WT represents wild-type Arabidopsis thaliana lines; C indicates the detection of exogenous genes using qRT-PCR. MsCBF4 Figure showing the results of relative expression levels in transgenic Arabidopsis thaliana lines; Figure 5 To overexpress alfalfa MsCBF4 Figure 1 shows the results of the freeze tolerance analysis of transgenic Arabidopsis thaliana. In this figure, A shows the phenotypic results of Arabidopsis thaliana WT and MsCBF4-OE lines before freezing and after freezing stress and recovery at room temperature; B shows the statistical results of the relative electrical conductivity of leaves of Arabidopsis thaliana WT and MsCBF4-OE lines before and after freezing treatment; and C shows the statistical results of the malondialdehyde content of leaves of Arabidopsis thaliana WT and MsCBF4-OE lines before and after freezing treatment. Detailed Implementation

[0019] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be noted that the following examples are only used to explain the present application, but not to limit the scope of the present application. The following examples are only a part of the embodiments of the present application, not all. Those skilled in the art can refer to the content herein to appropriately improve the process parameters to achieve the purpose of the present application. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content and scope of the present application, to realize and apply the present application technology. In the art, other technicians will not make creative efforts, and the examples they obtain are protected by the present application.

[0020] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art unless otherwise specified, which can be obtained by commercial channels by those skilled in the art.

[0021] The alfalfa used in the following examples is Dongnong No. 1, which is recorded in the following literature: Yin X J, Cui G W, Hu G F, et al. Breeding report of new alfalfa variety Dongnong No. 1 [J]. Chinese Journal of Grassland Science, 2018, 40(01): 29-34. DOI: 10.16742 / j.zgcdxb.2018-01-05. The vector pCAMBIA1302-GFP used in the following examples is recorded in the following literature: Yao, X., Fang, K., Qiao, K., et al. Cooperative transcriptional regulation by ATAF1 and HY5 promotes light-induced cotyledon opening in Arabidopsis thaliana. Sci. Signaling, 2024, 17(817), eadf7318. Agrobacterium GV3101 used in the following examples is described in the following reference: Xia, M.S., Zhang, W.B., Chang, Y.T., et al. A preliminary investigation on the functional validation and interactions of PoWOX Genes in Peony (Paeonia ostii). Horticulturae, 2022, 8(3), 266. Example 1: Medicago truncatula MsCBF4 Cloning of the gene 1. Medicago truncatula seedling cultivation Full and healthy seeds of Medicago truncatula of Dongnong No. 1 were selected, soaked in warm water at 45 °C for 2 h to break dormancy, then surface sterilized with 75% ethanol for 30 s, and then placed in a culture dish with filter paper and cultured in the dark at 25 °C. When the radicle length was >1 cm, the seedlings were transplanted to sterile nutrient soil and cultivated in a phytotron (temperature 25 °C, light period 150 μmol·m -2 ·s -1 , photoperiod 16 h / 8 h) to the three-leaf stage to obtain uniform Medicago truncatula seedlings.

[0022] 2. RNA extraction and reverse transcription Total RNA was extracted from the leaves of Medicago truncatula seedlings obtained in step 1 using the RNA Easy Fast Plant Tissue RNA Fast Extraction Kit (Beijing Tiangen Bioscience Co., Ltd.) according to the manufacturer's instructions. The HiScript IIIRT SuperMix for qPCR (+gDNA wiper) kit (Nanjing Novogene Bioinformatics Technology Co., Ltd.) was used to reverse transcribe the total RNA according to the manufacturer's instructions to obtain cDNA. ®

[0023] 3. PCR reaction According to the CDS region sequence of Medicago truncatula MsCBF4 gene (nucleotide sequence as shown in SEQ ID NO. 1, amino acid sequence as shown in SEQ ID NO. 2), the CDS region specific PCR amplification primers were designed using Primer Premier 5.0. The designed primers KzMsCBF4-F (nucleotide sequence as shown in SEQ ID NO. 3) and KzMsCBF4-R (nucleotide sequence as shown in SEQ ID NO. 4) were synthesized by Sheng Wu Bioengineering Shanghai Co., Ltd. MsCBF4

[0024] To ensure the gene​​MsCBF4 CDS full-length clone accuracy, with high-fidelity polymerase Tks Gflex TM DNAPolymeras performed PCR reaction on the cDNA obtained in step 2, to obtain PCR amplification product.

[0025] SEQ ID NO. 1: ATGCCGAAGAAGCGTGCAGGGAGGAAGAAGTTCAGAGAAACACGTCACCCTGTGTACCGTGGTGTAAGAAAGAGGGATTCCGGGAAGTGGGTTTGTGAAGTAAGAGAACCTAACAAGAAAACCAGAATTTGGCTAGGAACATTTCCTACACCGGAGATGGCGGCGCGTGCACACGATGTTGCGGCGATTGCGTTGAGGGGAAGGTCTGCTTGTCTTAACTTTGCTGATTCTGCATGGAGGTTGCCGGTCCCGGCTACGGCGGATGCAAGGGATATTCAAAAGGCAGCGGCTGAAGCAGCGGAGGCTTTCAGGCCGGAGAGTGTGTTTGAGAATAGTGAGGAGAGGAAGGATAGTGAATCTTCATCAACGGTGGCGGTGGCAGAGACGGTGATGGAGCAACGAGAGGAAGAAGAGGATACAGTGCCGGAGTATTTGAGAAACATGGTGCTAATGTCACCGGCGCATTACTGGGGAAGTGATTGTGGTGTTGCTGACGTGGAATTTGATGAAACTGAAATTTCATTGTGGAGTTATTCATTTTAG; SEQ ID NO. 2: MPKKRAGRKKFRETRHPVYRGVRKRDSGKWVCEVREPNKKTRIWLGTFPTPEMAARAHDVAAIALRGRSACLNFADSAWRLPVPATADARDIQKAAAEAAEAFRPESVFENSEERKDSESSSTVAVAETVMEQREEEEDTVPEYLRNMVLMSPAHYWGSDCGVADVEFDETEISLWSYSF; SEQ ID NO. 3: 5'-ATGCCGAAGAAGCGTGCAGG-3'; SEQ ID NO. 4: 5'-CTAAAATGAATAACTCCACAATGAAAT-3'.

[0026] 4. Connection transformation and sequencing of pTOPO001 vector The PCR product obtained in step 3 was connected with the entry vector pTOPO001 (purchased from Beijing Jinsha Biotechnology Co., Ltd., item number: TC602), and the connection product was transformed into E. coli DH5a competent cells, coated on LB solid medium containing 100 mg / L Amp, and cultured overnight. The positive clone colonies were extracted for plasmid, and sent to Shenguo Biotechnology (Shanghai) Co., Ltd. for sequencing, and the plasmid with correct sequencing was named pTOPO001-MsCBF4.

[0027] The sequencing results show that the size of the PCR amplified fragment is 543 bp, and the nucleotide sequence is shown as SEQ ID NO. 1. The fragment contains MsCBF4 CDS region sequence of the gene.

[0028] Example 2, gene MsCBF4 Expression specificity and temporal and spatial expression analysis in Medicago sativa 1. Plant material acquisition and treatment According to the method in Example 1, the Medicago sativa seedlings were cultivated to 4 weeks old, then moved to a low temperature light incubator for low temperature stress treatment (temperature 4℃, humidity 80%, light intensity 12000 lx, light cycle 16 / 8 h), and the leaf and root tissues of Medicago sativa were sampled at 0, 1, 3, 6, 12, 24 h of low temperature stress treatment, frozen in liquid nitrogen and stored in-80℃ environment, for verifying the expression of the gene MsCBF4 Tissue expression under cold stress in Medicago sativa.

[0029] According to the method in Example 1, the Medicago sativa seedlings were cultivated to 4 weeks old, then moved to a low temperature light incubator for low temperature stress treatment (temperature 4℃, humidity 80%, light intensity 12000 lx, light cycle 16 / 8 h), and the leaf and root tissues of Medicago sativa were sampled at 0, 1, 3, 6, 12, 24 h of low temperature stress treatment, frozen in liquid nitrogen and stored in-80℃ environment, for verifying the expression of the gene MsCBF4 Tissue expression under cold stress in Medicago sativa.

[0030] According to the method in Example 1, the Medicago sativa seedlings were cultivated to 4 weeks old, then moved to a low temperature light incubator for low temperature stress treatment (temperature 4℃, humidity 80%, light intensity 12000 lx, light cycle 16 / 8 h), and the leaf and root tissues of Medicago sativa were sampled at 0, 1, 3, 6, 12, 24 h of low temperature stress treatment, frozen in liquid nitrogen and stored in-80℃ environment, for verifying the expression of the gene MsCBF4 Expression in different plant tissues of Medicago sativa.

[0031] 2. qRT-PCR expression detection Following the method described in Example 1, cDNA from different alfalfa samples obtained in step 1 was obtained. The cDNA was diluted 10-fold and used as a template, with QMsCBF4-F (SEQ ID NO. 5) and QMsCBF4-R (SEQ ID NO. 6) as gene sequences. MsCBF4 The amplification primers were used, with MsActin-F (SEQ ID NO.7) and MsActin-R (SEQ ID NO.8) as internal reference genes for amplification primers to perform qRT-PCR.

[0032] SEQ ID NO.5: 5'-GGGAAGTGGGTTTGTGAAGTAAGAG-3'; SEQ ID NO.6: 5'-ACGCAATCGCCGCAACATC-3'; SEQ ID NO.7: 5'-CAAAAGATGGCAGATGCTGAGGAT-3'; SEQ ID NO. 8: 5'-CATGACACCAGTATGACGAGGTCG-3'.

[0033] The results show that: MsCBF4 The gene is expressed at the highest level in the root and at the lowest level in the flower. Figure 1 (A) As the duration of cold stress and freezing stress treatments increased, the levels of [unclear text - possibly related to alfalfa leaf and root tissues] decreased. MsCBF4 Gene expression levels showed an increasing trend; under cold stress conditions MsCBF4 Gene expression levels were higher in roots than in leaves under freezing stress conditions. MsCBF4 Gene expression levels are higher in leaves than in roots. Figure 1 B and Figure 1 (C in the text). The above results indicate that... MsCBF4 Gene expression is induced by cold stress and freezing stress.

[0034] Example 3: Containing alfalfa MsCBF4 Construction of plant overexpression vectors for genes 1. Using the plasmid pTOPO001-MsCBF4 obtained in Example 1 as a template, primers 1302MsCBF4-F (SEQ ID NO. 9) and 1302MsCBF4-R (SEQ ID NO. 10) containing the homologous arm sequence of the vector pCAMBIA1302-GFP were designed. High-fidelity DNA polymerase Tks Gfle was used. TM DNA polymerase was used for PCR amplification. The PCR product was recovered again by 0.1% agarose gel electrophoresis and stored at -20°C for later use.

[0035] SEQ ID NO.9: 5'- GGACTCTTGACCATGGTA ATGCCGAAGAAGCGTGC-3' (underlined at the homologous arm and restriction site sequence); SEQ ID NO.10: 5'- CTTCTCCTTTACTAGT AAATGAATAACTCCACAATGAAATTTCAGTTTCATCA-3' (underlined at the homologous arm and restriction site sequence).

[0036] 2. The plant expression vector pCAMBIA1302-GFP was double-digested with Nco I and Spe I. The target band of pCAMBIA1302-GFP was recovered by agarose gel electrophoresis and stored at -20℃ for later use.

[0037] 3. The PCR product recovered in step 1 was ligated into the enzyme-digested pCAMBIA1302-GFP vector recovered in step 2 and incubated at 50°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated on LB agar containing 100 mg / L Kan, and incubated overnight. Plasmids were extracted from positive clones, and after PCR identification, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced plasmid was named pCAMBIA1302-MsCBF4-GFP.

[0038] The map of the recombinant vector pCAMBIA1302-MsCBF4-GFP includes the selection marker Hyg, the reporter gene, and the target gene. MsCBF4 Dual expression box ( Figure 2 PCR identification of the recombinant plasmid pCAMBIA1302-MsCBF4-GFP showed that the target gene... MsCBF4 Successfully integrated into the carrier ( Figure 3 ).

[0039] Example 4: Overexpression of alfalfa MsCBF4 Construction of transgenic Arabidopsis thaliana 1. Arabidopsis seedling cultivation After sterilization, Arabidopsis seeds were laid flat on MS solid medium (containing 3% sucrose, pH 5.8) and vernalized at 4℃ for 2 days. They were then cultured in an incubator with a light / dark cycle of 16 / 8 h, a relative temperature of 23℃, and a relative humidity of 70%. After 7 days of culture, Arabidopsis seedlings were transplanted into pots containing peat moss, vermiculite, and perlite in a ratio of 1:1:1 and cultured until the initial flowering stage (approximately 6 weeks old).

[0040] 2. Culture of Agrobacterium The recombinant plasmid pCAMBIA1302-MsCBF4-GFP obtained in Example 3 was transformed into Agrobacterium GV3101 competent cells. The cells were plated on YEP plates (containing 100 mg / L Kan and 50 mg / L Rif) and incubated upside down at 28°C for 2 days. Single colonies were picked and added to YEP liquid medium (containing 100 mg / L Kan and 50 mg / L Rif), and cultured on a shaker at 37°C and 200 rpm for 12 h to obtain bacterial culture. Simultaneously, single colonies were identified by PCR. The correctly identified Agrobacterium culture was stored at -80°C in 50% glycerol for later use.

[0041] 3. Overexpression transformation and identification in Arabidopsis thaliana After reviving the preserved Agrobacterium culture, transfer the culture from the shake flask to YEP liquid medium and incubate until OD500. 600 Cells were collected by centrifugation at 8000 rpm for 10 min at a concentration of 1.0, and then resuspended in Arabidopsis thaliana transformation osmotic buffer to allow OD to rise. 600 =0.8. The Arabidopsis thaliana flower stamens from step 1 were placed in a container filled with bacterial solution for 20-30 seconds. The flowerpot was then placed sideways on a tray and kept in the dark for 24 hours. Afterward, normal light cultivation was performed to complete the overexpression transformation of the Arabidopsis thaliana plants. The first batch of seeds was harvested as the T0 generation. After sterilization, the harvested seeds were sown into the culture medium. T1 generation positive plants were screened using 0.002% hygromycin. The obtained positive plants were transplanted into soil, and the same method was used until the T3 generation of completely homozygous plants was screened. Three transgenic Arabidopsis thaliana homozygous lines MsCBF4-OE with a hygromycin positivity rate were successfully obtained, named #27, #33, and #41, respectively. The homozygous lines were then identified by PCR and qRT-PCR.

[0042] The results showed that Arabidopsis thaliana lines #27, #33, and #41 were completely homozygous in the T3 generation, with a homozygosity rate of 100%. Figure 4 (A) DNA identification results of homozygous lines indicate the presence of exogenous genes. MsCBF4 Successfully introduced into Arabidopsis thaliana genomic DNA ( Figure 4 (B in the text). The foreign gene was detected by qRT-PCR. MsCBF4 It can be overexpressed in transgenic Arabidopsis thaliana, among which the #27 strain has the highest expression level. Figure 4 (C in the middle).

[0043] Example 5: Overexpression of alfalfa MsCBF4 Analysis of the freeze resistance of transgenic Arabidopsis thaliana. 1. Plant material treatment The method in example 4 was used to sterilize the seeds of Arabidopsis thaliana wild type (WT) and MsCBF4-OE T3 generation. After vernalization, the seedlings were moved to a low-temperature light incubator for 8 h of cold acclimation (temperature 4℃, humidity about 80%, light intensity 12000 lx, light cycle 16 / 8 h). Then the cold-acclimated seedlings were cooled from 4℃ to -5℃ in the low-temperature light incubator, followed by 2 h of -5℃ cold dark treatment. The leaves of Arabidopsis were sampled, frozen in liquid nitrogen and stored at -80℃ for detection of relative conductivity (REL) and malondialdehyde (MDA) content. The treated Arabidopsis was recovered at room temperature for 7 d and photographed.

[0044] 2. REL detection 0.1 g of Arabidopsis leaves were weighed and placed in 10 mL of deionized water for vacuum infiltration at room temperature for 12 h. The initial conductivity (L1) was measured using a conductivity meter. Then the leaves were boiled for 30 min, cooled to 25℃, and the conductivity was measured again (L2). REL (%) = L1 / L2 x 100.

[0045] 3. MDA content detection 0.1 g of Arabidopsis leaves were homogenized in phosphate buffer (50 mM, pH 7.0) and centrifuged at 12000 rpm for 20 min at 4℃ to obtain the supernatant. The MDA content was determined by the thiobarbituric acid method.

[0046] The results showed that there was no significant difference in phenotype between WT and MsCBF4-OE lines at room temperature before freezing treatment. After freezing stress and 7 d of recovery at room temperature, the MsCBF4-OE lines showed obvious frost resistance and high survival state, among which the #27 line had the best survival state (Fig. 1A). Figure 5 REL detection found that there was no significant difference in REL between lines at room temperature before freezing treatment. After freezing stress, the REL of each line increased, and the REL of the three overexpression lines was significantly lower (p<0.05) than that of the WT line, among which the #41 line had the lowest REL (Fig. 1B). Figure 5 MDA content detection found that there was no significant difference in MDA content between lines at room temperature before freezing treatment. After freezing treatment, the MDA content of each line increased, and the MDA content of the three overexpression lines was significantly lower (p<0.05) than that of the WT line, among which the #27 line had the lowest MDA (Fig. 1C). Figure 5 The above results showed that freezing stress would exacerbate the membrane response of Arabidopsis, and the damage of Arabidopsis lines overexpressing MsCBF4 gene was significantly lighter than that of the WT line. MsCBF4

[0047] In summary, MsCBF4 ​Overexpression of the gene enhances the tolerance of Arabidopsis to freeze stress.

[0048] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. An alfalfa gene MsCBF4 Its application in improving the cold resistance of plants is characterized by... The alfalfa gene MsCBF4 The nucleotide sequence is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

2. An alfalfa gene MsCBF4 The application is characterized by, The application utilizes alfalfa genes. MsCBF4 Breeding cold-resistant Arabidopsis thaliana, the alfalfa gene MsCBF4 The nucleotide sequence is shown in SEQ ID NO.

1.

3. A type of alfalfa containing the gene described in claim 1 MsCBF4 The application of recombinant vectors is characterized by, The application involves using the recombinant vector to improve the cold resistance of Arabidopsis thaliana.

4. A type of alfalfa containing the gene described in claim 1 MsCBF4 The application of recombinant vectors is characterized by, The application involves using a recombinant vector to cultivate cold-resistant Arabidopsis thaliana; the recombinant vector is a vector containing the alfalfa gene. MsCBF4 The GFP plasmid was constructed by recombining the GFP plasmid with the pCAMBIA1302-GFP plasmid.

5. A type of alfalfa containing the gene described in claim 1 MsCBF4 Or the application of the recombinant bacteria of the recombinant vector as described in claim 3, characterized in that, The application involves using recombinant bacteria to improve the cold resistance of Arabidopsis thaliana; the host of the recombinant bacteria is Agrobacterium.

6. A type of alfalfa containing the gene described in claim 1 MsCBF4 Or the application of the recombinant bacteria of the recombinant vector as described in claim 3, characterized in that, The application involves using recombinant bacteria to cultivate cold-resistant Arabidopsis thaliana; the host of the recombinant bacteria is Agrobacterium.

7. A method for improving the cold resistance of Arabidopsis thaliana, characterized in that, Includes the following steps: (1) Incorporate alfalfa genes MsCBF4 The recombinant gene was inserted into the pCAMBIA1302-GFP plasmid to obtain the recombinant vector pCAMBIA1302-MsCBF4-GFP, containing the alfalfa gene. MsCBF4 The nucleotide sequence is shown in SEQ ID NO.1; (2) The recombinant vector pCAMBIA1302-MsCBF4-GFP obtained in step (1) was transformed into Agrobacterium competent cells and cultured to obtain the alfalfa gene-carrying cells. MsCBF4 Agrobacterium; (3) Cultivate Arabidopsis seedlings to the initial flowering stage, place the Arabidopsis flower stamens into the Agrobacterium bacterial solution obtained in step (2) for 20-30 s, keep them in the dark for 24 h, and then carry out normal light culture to complete the overexpression transformation of Arabidopsis plants.

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