A kind of alfalfa MsEXPA4 gene and its application in regulating low temperature resistance

Abstract

The present invention relates to the field of biotechnology, and in particular, to a gene of alfalfa MsEXPA4 and its application in regulating low temperature resistance. The present invention uses BD-MsDREB1C as a bait protein to capture a candidate interacting protein MsEXPA4 through a Y2H yeast library, and verifies the interaction between MsDREB1C and MsEXPA4; the low temperature resistance analysis of the overexpressing MsEXPA4 strain found that whether it was a freezing treatment under plant cold acclimation or a freezing treatment under in vitro leaf cold acclimation, the overexpressing alfalfa strain showed better freezing resistance than the wild type, which was manifested in relieving the damage of low temperature to the permeability of plant cell membranes, relieving the damage caused by low temperature to chlorophyll in plant leaves, and enhancing the activity of antioxidant enzymes to alleviate the oxidative damage caused by low temperature. The present invention discloses the direct interaction between MsEXPA4 and MsDEEB1C, and shows that the physiological responses of the two to low temperature are basically the same, and the low temperature resistance response induced by cold acclimation of alfalfa is positively regulated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an alfalfa MsEXPA4 gene and an application thereof in regulating low temperature resistance. Background Art

[0002] DREB1 / CBFs are transcription factors of the DREB family and are considered to be the "master switch" of gene regulation in the cold tolerance process induced by cold acclimation. The low temperature regulatory network involved in DREB1 / CBFs is very complex and is directly or indirectly regulated by upstream regulatory factors to respond to cold. Currently, many researchers are committed to exploring the regulatory interactors of MsDREB1C. The MYC helix-loop-helix transcription factor ICE1 (CBF expression inducer 1) is a positive regulator of DREB1 / CBFs and plays an important role in regulating the constitutive expression of DREB1 / CBFs. There is increasing evidence that DREB1 / CBFs can be indirectly regulated by other proteins through ICE1, such as OPEN STOMATOSIS 1 (OST1), MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MITOGEN-ACTIVATED PROTEIN KINASE 6 (MPK6), BRASSINOSTEROID-INSENSITIVE2 BIN2, PLANT U-BOX E3 ligases PUB25 and PUB26, and the highly expressed osmotic response gene HOS1, and respond to low temperature by positive or negative regulation. At the same time, studies have shown that the expression of DREB1 / CBFs in cold stress is also directly regulated by MYB15, 14-3-3, calmodulin binding transcription activator (CAMTA), plant phytochrome-interacting factor 3 (PIF3), and B-box (BBX)37. The DREB1 / CBFs promoter has a MYB recognition sequence. Overexpression of MYB15 in Arabidopsis leads to a decrease in the expression of DREB1 / CBFs genes and reduced freezing tolerance; while the myb15 mutant leads to an increase in the expression of DREB1 / CBFs genes at low temperatures, showing increased tolerance to freezing stress. At the same time, the Arabidopsis calmodulin-binding transcription activator (CAMTA) family is a positive regulator of cold acclimation. It can bind to the CM2 motif in the CBF2 promoter, activate CBF2 expression, and enhance Arabidopsis freezing resistance, while the double camta1 camta3 mutant plants are impaired in freezing resistance. DREB1 / CBFs can also be regulated by 14-3-3 proteins to affect low temperature resistance. 14-3-3 proteins are phosphopeptide binding proteins that can be phosphorylated by the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1). The phosphorylated 14-3-3 proteins interact with CBF proteins in the nucleus and destabilize them, reducing their expression. In addition, crpk1 and 14-3-3 mutants in Arabidopsis thaliana exhibit cold tolerance, while transgenic plants overexpressing 14-3-3 exhibit cold sensitivity. Plant phytochrome-INTERACTING FACTOR 3 (PIF3) is a key transcription factor that inhibits photomorphogenesis and can negatively regulate Arabidopsis thaliana freezing resistance by directly binding to the promoters of CBF genes to downregulate their expression.At the same time, the B-box protein MdBBX37 in apple (Maluspumila) has also been reported to bind to the promoters of MdCBF1 and MdCBF4 to activate their transcription, thereby promoting plant cold tolerance. However, research on the function and mechanism of DREB1 / CBFs under low temperature has mainly focused on the model plant Arabidopsis, and research in alfalfa is almost blank.

[0003] Previous studies of the present invention have found that overexpression of MsDREB1C can improve the low temperature resistance of alfalfa under cold acclimation conditions, but its molecular regulatory mechanism has not yet been resolved. In order to further analyze the cold tolerance mechanism of MsDREB1C, MsDREB1C interacting proteins were screened through the Y2H yeast library, and the MsDREB1C protein interaction network was analyzed. The proteins interacting with MsDREB1C were explored and their functions were verified. Summary of the invention

[0004] In order to achieve the above object, the present invention first provides an alfalfa MsEXPA4 gene, the nucleotide sequence of the MsEXPA4 gene is shown in SEQ ID No.3.

[0005] The present invention also provides an alfalfa MsEXPA4 gene, and the encoding amino acid sequence of the MsEXPA4 gene is shown as SEQ ID No.4.

[0006] The present invention also provides a biological material related to the above-mentioned MsEXPA4 gene, which is any one of the following B1) to B5):

[0007] B1) an expression cassette containing the above-mentioned MsEXPA4 gene;

[0008] B2) a recombinant vector containing the above-mentioned MsEXPA4 gene,

[0009] B3) a recombinant microorganism containing the above-mentioned MsEXPA4 gene;

[0010] B4) a recombinant vector containing the expression cassette described in B1);

[0011] B5) A recombinant microorganism containing the recombinant vector described in B2).

[0012] The present invention also provides an application of the MsEXPA4 gene or the biological material in alfalfa breeding.

[0013] In certain embodiments, the application is to construct alfalfa with good low temperature resistance;

[0014] Optionally, the low temperature resistance measurement index comprises one or more of survival rate, electrical conductivity, chlorophyll content, malondialdehyde (MDA) content, hydrogen peroxide (H2O2) content, superoxide dismutase (SOD) content, catalase (CAT) content, and plant protein content.

[0015] In certain embodiments, the use is achieved by regulating the expression of the MsEXPA4 gene.

[0016] In certain embodiments, regulating the expression of the MsEXPA4 gene is up-regulating the expression of the MsEXPA4 gene or down-regulating the expression of the MsEXPA4 gene.

[0017] In certain embodiments, the regulating the expression of the MsEXPA4 gene is silencing the expression of the MsEXPA4 gene or overexpressing the MsEXPA4 gene.

[0018] The present invention also provides a method for detecting the low temperature resistance of alfalfa, which comprises the step of quantitatively detecting the expression amount of the MsEXPA4 gene.

[0019] Finally, the present invention provides a method for preparing high and low temperature resistant alfalfa, which comprises the step of overexpressing the MsEXPA4 gene.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] The present invention uses BD-MsDREB1C as a bait protein to capture a candidate interacting protein MsEXPA4 (having 98.84% homology with Medicago truncatula MtEXPA4) through a Y2H yeast library; through yeast two-hybrid experiments, GST-pull down experiments and BiFC experiments, the interaction between MsDREB1C and MsEXPA4 is verified; through gene cloning, construction of expression vectors and genetic transformation of alfalfa, an alfalfa strain overexpressing MsEXPA4 is obtained; and through low temperature resistance analysis of the strain overexpressing MsEXPA4, it is found that whether it is a freezing treatment under cold acclimation of plants or a freezing treatment under cold acclimation of detached leaves, the alfalfa strain overexpressing shows better freezing resistance than the wild type, which is manifested in that the damage to the permeability of plant cell membranes caused by low temperature is alleviated, the damage to chlorophyll in plant leaves caused by low temperature is alleviated, and the activity of antioxidant enzymes is enhanced to alleviate the oxidative damage caused by low temperature. In summary, the present invention reveals the direct interaction between MsEXPA4 and MsDEEB1C, and shows that the physiological responses of the two to low temperature are basically consistent, and they positively regulate the cold acclimation-induced low temperature tolerance response of alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Using different 3AT concentration gradients to inhibit MsDREB1C autoactivation;

[0023] Figure 2 "Mickey" binders observed under a 40° objective during hybridization;

[0024] Figure 3 MsDREB1C candidate interacting proteins screened by Y2H mating;

[0025] Figure 4 DNA level identification of candidate interacting proteins of MsDREB1C;

[0026] Figure 5 Multiple sequence alignment of candidate interacting protein MsEXPA4; Note: MsEXPA4 was aligned with Arabidopsis EXPA4 (AtEXPA4, NP_181500.1), Medicago truncatula EXPA4 (MtEXPA4, XP_003616412.1), Glycine max EXPA4 (GmEXPA4, XP_003519349.1), and Rice EXPA4 (OsEXPA4, NP_001407476.1). The percentage at the bottom represents the sequence homology between MsEXPA4 and its orthologous proteins;

[0027] Figure 6 The interaction between MsDREB1C and MsEXPA4 in yeast two-hybrid “one-to-one” validation;

[0028] Figure 7 Protein expression and purification of MsDREB1C and MsEXPA4; (a): SDS-PAGE detection of MsDREB1C protein expression; (b)-(c): SDS-PAGE detection of MsDREB1C protein purification; (d): SDS-PAGE detection of MsEXPA4 protein expression; (e)-(f): SDS-PAGE detection of MsEXPA4 protein purification;

[0029] Figure 8 GST pull-down experiments showed that MsEXPA4 interacted with MsDREB1C;

[0030] Fig. 9 Bimolecular fluorescence complementation (BiFC) experiments showed that MsEXPA4 interacted with MsDREB1C in vivo;

[0031] Fig.10 Basic information of MsEXPA4 gene;

[0032] (a)-(b): Gel electrophoresis of MsEXPA4 amplification from alfalfa; (c): MsEXPA4 gene sequence; (d): MsEXPA4 protein sequence;

[0033] Fig.11 Analysis of the expression level of MsEXPA4 in transgenic alfalfa;

[0034] Fig.12 Phenotype of alfalfa overexpressing MsEXPA4 under low temperature stress; (a): Phenotypic status of WT and transgenic alfalfa lines (MsEXPA4-7, MsEXPA4-14, MsEXPA4-8) grown in the greenhouse; (b): Phenotypic status of WT and transgenic alfalfa lines after low temperature treatment. Before freezing treatment, alfalfa was pretreated at 4°C for 1 week for cold acclimation. The freezing treatment was carried out using a gradient cooling method of 1°C per hour until it dropped to -9°C and maintained for 1 hour. After freezing treatment, the plants were transferred to 4°C overnight and then placed in the greenhouse to recover for 7 days. (c) Figure is an enlarged view of the frame in (b);

[0035] Fig.13 Overexpression of MsEXPA4 reduced cell damage and oxidative damage in alfalfa after low temperature; (a): damage rate; (b): electrical conductivity; (c): MDA content; (d): H2O2 content; (e): SOD activity; (f): CAT activity;

[0036] Fig.14 Overexpression of MsEXPA4 in alfalfa reduced chlorophyll damage after low temperature; (a): Chlorophyll a; (b): Chlorophyll b; (c): Total chlorophyll content;

[0037] Fig.15 Effect of overexpression of MsEXPA4 on the freezing resistance of detached alfalfa leaves; (a): Phenotype of detached alfalfa leaves overexpressing MsEXPA4 before low-temperature treatment; (b): Phenotype of detached alfalfa leaves overexpressing MsEXPA4 after low-temperature treatment; Before freezing treatment, the leaves were pretreated at 4°C for 2 days for cold acclimation, and then frozen using a gradient cooling method of 1°C per hour until the temperature dropped to -10°C and maintained for 1 hour. After freezing treatment, the leaves were transferred to 4°C overnight and placed in a growth chamber for recovery for 7 days; (c): damage rate; (d): chlorophyll a; (e): chlorophyll b; (f): total chlorophyll content. DETAILED DESCRIPTION

[0038] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0039] The materials involved in the present invention are:

[0040] The wild type of plant material used in this study was alfalfa "Zhongmu No. 1", which was bred and preserved in our laboratory. It was planted in a 9cm×9cm×12cm pot containing equal proportions of sterilized nutrient soil and vermiculite using the greenhouse soil culture method at 22℃ / 20℃ and 14h / 10h day / night cycle.

[0041] Freezing treatment of whole plants: After the overexpression and wild-type alfalfa were grown in the greenhouse for 4 weeks, the plants were transferred to a 4°C low-temperature incubator for cold acclimation. After 7 days of cold acclimation, they were frozen by a gradient cooling method of 1°C per hour until they dropped to -9°C and maintained for 1 hour. After freezing treatment, the plants were transferred to 4°C overnight and then placed in the greenhouse to recover for 7 days.

[0042] Freezing treatment of detached leaves: Take detached leaves of the same batch of disease-free wild-type and overexpression strains and place them in a culture dish filled with 25 mL of ultrapure water for low-temperature treatment. Before freezing treatment, detached alfalfa leaves were cold-acclimated at 4°C for 2 days, and then frozen by a gradient cooling method of 1°C per hour until it dropped to -10°C and maintained for 1 hour. After freezing treatment, the plants were transferred to 4°C overnight and placed in a light incubator to recover for 7 days.

[0043] The nucleotide sequence of MsDREB1C is shown as SEQ ID No.1, and the amino acid sequence encoded by the gene is shown as SEQ ID No.2.

[0044] SEQ ID No.1:

[0045] ATGGATATGTTAACAACTAACAACTCTTCTTATCCACACCAGTTTTCTCCAAATTCCACCGAGAGTTCATTTGCGAATTCAGAGGGCTCTCAGGGGATGTCAATTTCCAACGAGGAAGTGAGGTTAGCTGCGACGACTCCAAAGAAGCGTGCTGGGAGGAAGAAGTTCCAGGAGACTCGCCACCCGGTATATAGGGGTGTGAGGAAGAGGAACTTAGATAAATGGGTGTGTGAAATGAGGGAGCCAAACAAGAAGACTAAGATTTGGCTAGGCACTTTTCCGACAGCAGAGATGGCAGCCCGAGCACATGATGTTGCTGCGATGGCATTGAGGGGCCACTACGCCTGTCTCAACTTTGCAGACTCTGCGTGGCGGCTCCCTGTACCTGCCACCACTCAGGCAAAAGATATACAAAAAGCGGCTGCACAGGCCGCAGAGGCTTTCAGACCGGACAAGACTTCGATGACTAATGACATTGACACGGCTATTGCCACCTCTTCTACGGAGGAGCAGAGTAGGACTTGTATGGAAGAGGAAGAAGAGGGGACGATGAACATGCCAGAGTTGTTGAGGAATATGGCGCTAATGTCTCCTACACATAGCTTTGGGTATCATGAGTATGAAGATATTCATGTTCAAGACTTTCCAGATTTTCAAGATGAAGAGGTATCACTATGGAATTATTAA;

[0046] SEQ ID No.2:

[0047] MDMLTTNNSSYPHQFSPNSTESSFANSEGSQGMSISNEEVRLAATTPKKRAGRKKFQETRHPVYRGVRKRNLDKWVCEMREPNKKTKIWLGTFPTAEMAARAHDVAAMALRGHYACLNFADSAWRLPVPATTQAKDIQKAAAQAAEAFRPDKTSMTNDIDTAIATSSTEEQSRTCMEEEEEGTMNMPELLRNMALMSPTHSFGYHEYEDIHVQDFPDFQDEEVSLWNY;

[0048] The nucleotide sequence of MsEXPA4 is shown in SEQ ID No.3, and the amino acid sequence encoded by the gene is shown in SEQ ID No.4.

[0049] SEQ ID No.3:

[0050] ATGACTAAGCTCAACATCACCACCATTTTCACCTTCTTAGCCCTTCTCCTCACTTCAACCGAAGCCAGACTCCCCGGTGTCTACACCGGCGGAGCATGGCAAACCGCTCACGCCACCTTCTACGGCGGCAGCGACGCCTCCGGAACAATGGGAGGAGCATGTGGATATGGAAATTTGTATAGTCAAGGTTATGGTGTGAACACAGCAGCATTAAGCACAGCACTATTCAACAACGGTTTAAGCTGTGGCTCGTGTTTTGAGTTAAAGTGTTCCAATGATCCTAGTTGGTGCCACCCTGGAAGTCCTTCAATATTGGTAACTGCTACAAATTTTTGTCCACCAAATTTTGCTCAACCAAGTGATAATGGAGGATGGTGTAACCCTCCACGACCACACTTTGATCTTGCTATGCCTATGTTCCTCAAAATTGCTCAATATCGTGCTGGTATTGTTCCTGTTTCTTACCGCAGGGTACCATGCAGAAAACAAGGAGGAATAAGATACACAATCAACGGCTTCCGTTACTTCAACTTGGTTCTAATCACCAACGTTGCAGGTGCAGGTGATATAGTGAAGACTAGTGTTAAAGGCTCAAAAACTGGTTGGATGAGTATGAGTCGTAACTGGGGGCAAAATTGGCAATCAAATTCTGTTTTGGTTGGTCAATCACTTTCTTTTAGGGTTACATCCAGTGATAAACGTACTTCAACTTCTTGGAATATTGTTCCCTCTAATTGGCAATTTGGTCAAACTTTCACTGGCAAGAATTTTCGAGTCTAA;

[0051] SEQ ID No.4:

[0052] MTKLNITTIFTFLALLLTSTEARLPGVYTGGAWQTAHATFYGGSDASGTMGGACGYGNLYSQGYGVNTAALSTALFNNGLSCGSCFELKCSNDPSWCHPGSPSILVTATNFCPPNFAQPSDNGGWCNPPR PHFDLAMPMFLKIAQYRAGIVPVSYRRVPCRKQGGIRYTINGFRYFNLVLITNVAGAGDIVKTSVKGSKTGWMSMSRNWGQNWQSNSVLVGQSLSFRVTSSDKRTSTSWNIVPSNWQFGQTFTGKNFRV.

[0053] Agrobacterium GV3101 and EHA105 competent cells were purchased from Beijing Huayueyang Biotechnology Co., Ltd.;

[0054] Hygromycin B and phytogel (Biotopped) were purchased from Beijing Deyi Hengda Biotechnology Co., Ltd.;

[0055] MS Basal Medium (Phytotechnology), KINETIN SOLUTION (Phytotechnology), 2,4-D ACID SOLUTION (10 mg / mL) 2,4-D solution (Phytotechnology), 6-BASOLUTION (1.0 mg / mL) (Phytotechnology), AGAR MICROPROPAGATION agar (Phytotechnology), and SH medium powder (Phytotechnology) were purchased from Beijing Ximeijie Technology Co., Ltd.;

[0056] Hoagland nutrient solution was purchased from Qingdao Green Life Biotechnology Co., Ltd.

[0057] The preparation of SH3a solid culture medium (1 L) is shown in Table 1:

[0058] Table 1 Preparation of SH3a solid culture medium (1L)

[0059] Reagents Dosage SH medium powder 13.2g sucrose 20g Vitamins (1000×) 1mL Iron salt (500×) 2mL Inositol (50mg / mL) 2mL 2,4-D (10 mg / mL) 500μL 6-BA (1 mg / mL) 250μL Agar powder 7g Ultrapure water Make up to 1L

[0060] After adding various solutions, the pH is finally adjusted to 5.95. After the culture medium is prepared, it needs to be sterilized by high-pressure steam at 121°C for 15 minutes.

[0061] The preparation of MSBK solid medium (1L) is shown in Table 2:

[0062] Table 2 Preparation of MSBK solid medium (1L)

[0063] Reagents Dosage MS Basal Medium Powder 4.43g sucrose 30g KINETIN (1 mg / mL) 1mL 6-BA (1 mg / mL) 500μL Agar powder 7g Ultrapure water Make up to 1L

[0064] After adding various solutions, the pH is finally adjusted to 5.95. After the culture medium is prepared, it needs to be sterilized by high-pressure steam at 121°C for 15 minutes.

[0065] The preparation of SH9a solid culture medium (1 L) is shown in Table 3:

[0066] Table 3 Preparation of SH9a solid culture medium (1L)

[0067] Reagents Dosage SH medium powder 13.2g sucrose 10g Vitamins (1000×) 1mL Iron salt (500×) 2mL Inositol (50mg / mL) 2mL Agar powder 7g Ultrapure water Make up to 1L

[0068] After adding various solutions, the pH is finally adjusted to 5.95. After the culture medium is prepared, it needs to be sterilized by high-pressure steam at 121°C for 15 minutes.

[0069] The yeast defective medium, 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-α-gal), and 3-amino-1,2,4-triazole (3AT) used were purchased from Beijing Coolbo Technology Co., Ltd.;

[0070] Hygromycin B and phytogel (Biotopped) were purchased from Beijing Deyi Hengda Biotechnology Co., Ltd.;

[0071] Inositol and sucrose were purchased from Beijing Guangda Hengyi Technology Co., Ltd.;

[0072] The RNA extraction kit (Promega) and plasmid extraction kit (Omega) were purchased from Beijing Deyi Hengda Biotechnology Co., Ltd.; reverse transcriptase, quantitative enzyme, gene cloning, T-vector ligation, Escherichia coli DH5α competent cells were provided by Nanjing Novizan Biotechnology Co., Ltd.; DNA gel recovery kit, seamless cloning and recombinase were provided by Beijing Quanshijin Biotechnology Co., Ltd.;

[0073] The kits used for physiological index determination, such as MDA, H2O2, SOD, CAT, and protein content detection kits, were purchased from Nanjing Jiancheng Bioengineering Institute; Agrobacterium EHA105 competent cells were purchased from Beijing Huayueyang Biotechnology Co., Ltd.; yeast Y2H and Y187 strains were preserved by our laboratory; the vectors used, such as Super 1300, pGBKT7 (BD), and pGADT7 (AD), were all preserved by our laboratory.

[0074] The preparation of LB solid medium (1 L) is shown in Table 4:

[0075] Table 4 Preparation of LB solid medium (1L)

[0076] Reagents Dosage Yeast Extract 5g Tryptone 10g NaCl 10g Agar powder 15g Ultrapure water Make up to 1L

[0077] LB liquid medium does not need to add agar powder. After the medium is prepared, it needs to be sterilized by high pressure steam at 121℃ for 15 minutes.

[0078] The preparation of YEB solid medium (1 L) is shown in Table 5:

[0079] Table 5 Preparation of YEB solid medium (1L)

[0080] Reagents Dosage Yeast Extract 1g Tryptone 10g MgSO4·7H2O 1g sucrose 5g Agar powder 15g Ultrapure water Make up to 1L

[0081] YEB liquid medium does not need to add agar powder. After the medium is prepared, it needs to be sterilized by high pressure steam at 121℃ for 15 minutes.

[0082] The preparation of YPDA solid medium (1 L) is shown in Table 6:

[0083] Table 6 Preparation of YPDA solid medium (1L)

[0084]

[0085]

[0086] YPDA liquid medium does not need to be added with agar powder. 20% glucose and 0.4% adenine sulfate need to be prepared separately and sterilized before mixing with other sterilized ingredients.

[0087] The preparation of SD / -Leu or SD / -Trp or SD / -Leu, -Trp or SD / -Leu, -Trp, -His, -Ade yeast defective medium solid medium (1L) is shown in Table 7:

[0088] Table 7 Preparation of solid medium for yeast defective medium (1L)

[0089] Reagents Dosage SC / yeast deficient medium 8g Agar powder 15g Ultrapure water 900mL Glucose (20%) 100mL

[0090] Note: Agar powder is not required when preparing SD / yeast defect liquid medium. Adjust the pH of the mixture to 5.8 and sterilize it at 121℃ for 15 minutes. 20% glucose needs to be prepared separately and sterilized before mixing with other ingredients.

[0091] The preparation of YPDA solid medium (1 L) is shown in Table 8:

[0092] Table 8 Preparation of YPDA solid medium (1L)

[0093] Reagents Dosage Yeast Extract 10g Tryptone 20g Agar powder 15g Ultrapure water 885mL Adenine sulfate 15mL Glucose (20%) 100mL

[0094] YPDA liquid medium does not need to be added with agar powder. 20% glucose and 0.4% adenine sulfate need to be prepared separately and sterilized before mixing with other sterilized ingredients.

[0095] The preparation method of 2×YPDA medium (1L) is shown in Table 9:

[0096] Table 9 Preparation of YPDA solid medium (1L)

[0097] Reagents Dosage Yeast Extract 20g Tryptone 40g Agar powder 15g Ultrapure water 770mL Adenine sulfate (0.4%) 30mL Glucose (20%) 200mL

[0098] The preparation method of 0.5×YPDA medium (1L) is shown in Table 10:

[0099] Table 10 Preparation of YPDA solid culture medium (1L)

[0100] Reagents Dosage Yeast Extract 5g Tryptone 10g Agar powder 15g Ultrapure water 942.5mL Adenine sulfate (0.4%) 7.5mL Glucose (20%) 50mL

[0101] Example 1 Different concentration gradients of 3AT inhibit the transcriptional autoactivation activity of MsDREB1C

[0102] Transcriptional autoactivation inhibition assay: Construction of BD-MsDREB1C vector: Use -Basic Seamless Cloning and Assembly Kit (Novizan) was used for homologous recombination, and the reaction system is shown in Table 11:

[0103] Table 11 Reaction system

[0104] Reagents Dosage 2×Basic Assembly Mix 3μL Linearized vector 2μL Inserted target fragment 1μL

[0105] The above reactants were placed on ice for transformation into competent E. coli DH5α after being reacted at 50℃ in a PCR instrument for 30 minutes. After transformation, the bacterial solution was spread on a solid culture medium containing the corresponding antibiotics (Super 1300 and BD vectors are both Kan-resistant) and cultured at 37℃ overnight. After monoclonal identification, the bacterial solution with the correct band was selected and sent to Qingke Bio for sequencing. After sequence alignment, the bacterial solution with the correct sequencing was selected and mixed with 50% glycerol in a ratio of 1:1, and then stored at -80℃ for future use. Before transforming Agrobacterium, the bacterial solution was activated and the plasmid was extracted.

[0106] (2) Transform yeast Y2H competent cells and complete BD-MsDREB1C+AD, PGBKT7-53+PGADT7-T, PGBKT7-Lam+PGADT7-T, and BD+AD co-transformation.

[0107] (3) 4 μl of the above cotransfection products were titrated on SD / -Trp, -Leu (DDO) and SD / -Trp, -Leu, -His, -Ade (QDO) solid culture media and QDO (10 mM, 20 mM, 30 mM, 40 mM) supplemented with different concentration gradients of 3AT. Note that all bacterial cultures were adjusted to a uniform OD600 value of 0.5 (1×) before titration, and each cotransfection product was diluted 10×, 100×, and 1000×, respectively.

[0108] (4) After titration, the culture medium plate was inverted and cultured in a 30°C incubator for 5 days.

[0109] (5) After 5 days, observe and photograph the colony status to determine the final concentration gradient of 3AT for the subsequent Y2H mating screening library.

[0110] The nuclear transcription factor MsDREB1C has transcriptional self-activation activity. In order to avoid the high false positive rate during Y2H screening, the self-activation of MsDREB1C was inhibited by 3AT (3-amino-1,2,4-triazole) with different concentration gradients. The results showed that 3AT can effectively change the self-activation activity of MsDREB1C, that is, as the concentration of added 3AT increases, its self-activation gradually weakens; when the 3AT concentration in the QDO four-deficient medium (SD / -Trp, -Leu, -His, -Ade) increases to 30mM, MsDREB1C self-activation is less; when it increases to 40mM concentration, the self-activation activity of MsDREB1C is almost inhibited. In order to reduce the self-activation of MsDREB1C while ensuring the capture rate, QDO solid medium containing 30mM 3AT was finally selected for the Y2H mating screening library of the bait protein MsDREB1C (see Figure 1 ).

[0111] Example 2 Results of Y2H mating screening of bait protein MsDREB1C

[0112] Y2H mating "one pair library" screening:

[0113] (1) Take out the BD-MsDREB1C-Y2H bacterial solution from -80°C, streak it on a YPDA solid plate, and culture it upside down in a 30°C incubator for 2 days.

[0114] (2) Pick up fresh BD-MsDREB1C-Y2H colonies of 2-3 mm in size in a clean bench and place them in a 250 mL conical flask containing 50 mL SD / -Trp liquid culture medium.

[0115] (3) The packaged culture medium was cultured in a 30°C constant temperature shaker at 250 rpm for 16-20 h until the OD600 reached 0.8.

[0116] (4) Transfer the turbid bacteria that have reached the target OD value into a 50 mL centrifuge tube, centrifuge at 1000 g for 5 min, and discard the supernatant to collect the bacteria.

[0117] (5) Resuspend the collected BD-MsDREB1C-Y2H cells in 4 mL of SD / -Trp liquid culture medium in a clean bench.

[0118] (6) Take out 1 mL of the AD bacterial solution from -80°C and thaw it in a water bath at room temperature.

[0119] (7) Mix 1 mL of AD solution and 4 mL of BD solution and add them into two 1 L conical flasks.

[0120] (8) Rinse tubes AD and BD twice each with 45 mL of 2×YPDA liquid culture medium and divide the mixture evenly into two 1 L conical flasks.

[0121] (9) Place two 1 L conical flasks containing the mixed solution in a 30°C constant temperature shaker at 30-50 rpm for 20-24 h.

[0122] (10) After culturing for 20 h, observe the hybridization solution under a 40× microscope to see whether there are “Mickey Mouse Head” or “Shamrock” shaped conjugates. If so, proceed to the next step. If no conjugates are seen, continue culturing for 4 h.

[0123] (11) Transfer the hybridized bacterial solution to a 50 mL centrifuge tube in a clean bench and centrifuge at 1000 g for 10 min. Discard the supernatant and collect the bacterial cells.

[0124] (12) Resuspend the cells in 50 mL of 0.5× YPDA liquid medium in a clean bench and use it to rinse the conical flask used for hybridization twice.

[0125] (13) Centrifuge the mixture at 1000 g for 10 min at room temperature.

[0126] (14) After centrifugation, remove the supernatant and resuspend the cells in 10 mL of 0.5× YPDA liquid culture medium.

[0127] (15) The resuspended cells were evenly spread onto 150 mm diameter QDO (containing 3AT) plates using glass beads, with approximately 200–300 μL per plate, for a total of about 40 plates.

[0128] (16) Incubate the cells in an inverted position at 30°C for 5-7 days. Observe the growth of the colonies every day and mark the colonies according to their growth rate. Prepare an additional QDO medium for positive control.

[0129] (17) The fast-growing and full colonies were dissolved in 0.9% NaCl and then dropped onto QDO (containing 3AT) + X-α-gal for verification again.

[0130] (18) Incubate the plate upside down at 30°C in an incubator for 5–7 days and observe the status of the colonies.

[0131] When the BD bacterial solution containing the bait protein MsDREB1C was hybridized with the AD bacterial solution, the "Mickey" binder ( Figure 2 ).

[0132] Later, the collected bacteria were cultured and grown on 40 QDO (and 30mM 3AT) solid medium. After 7 days of growth, plump and fast-growing single colonies were selected for further screening on QDO plates containing 30mM 3AT and X-α-gal and QDO plates containing 40mM 3AT and X-α-gal to screen single colonies that grew plump and turned blue on both media ( Figure 3 After the selected colonies were expanded and yeast plasmids were extracted, they were further screened at the DNA level using primers AD-F and AD-R through amplification ( Figure 4 ), and finally the PCR products with bands on 1% agarose gel were selected for sequencing.

[0133] Example 3 Obtaining candidate interacting protein MsEXPA4

[0134] Identification of Screening Candidate Proteins: DNA Level Identification

[0135] (1) The plump and blue colonies grown in QDO (containing 3AT) + X-α-gal medium were picked and placed in QDO liquid medium and cultured at 30°C and 250 rpm for 24 h.

[0136] (2) After the bacterial solution becomes turbid, use the yeast plasmid extraction kit of Solebo to extract the yeast plasmid.

[0137] (3) After PCR, the DNA was sent to Qingke Company for sequencing.

[0138] Sequencing, alignment and functional annotation: The sequenced fragments were aligned with the ZM-4 reference genome (Long et al. 2022), and the protein sequences were used for protein sequence alignment and annotation in Ensembl plants. Functional annotation was performed through literature review and used as candidate interacting proteins for later verification.

[0139] Multiple sequence alignment of MsEXPA4 with EXPA4 protein sequences of other species: The protein sequence of MsEXPA4 was aligned with that of Arabidopsis thaliana (AtEXPA4, NP_181500.1), Medicago truncatula (MtEXPA4, XP_003616412.1), soybean (GmEXPA4, XP_003519349.1), and rice (OsEXPA4, NP_001407476.1) using DNAMAN.

[0140] Sequence alignment and literature review of the sequencing results revealed that a capture protein had a 98.8% sequence similarity with the Medicago truncatula expansin-A4 protein (MtEXPA4, XP_003616412.1), so the protein was named Medicago truncatula expansin-A4 (MsEXPA4). MsEXPA4 was selected as a candidate interacting protein of MsDREB1C. Multiple sequence alignment with EXPA4 from other species revealed that MsEXPA4 was highly conserved with EXPA4 from other species, with signal peptide, HFD, domain1 and domain2. The highest protein sequence homology between MsEXPA4 and Medicago truncatula MtEXPA4 was 98.84%; the protein sequence homology with soybean GmEXPA4, Arabidopsis AtEXPA4, corn ZmEXPA4, and rice OsEXPA4 was 88.63%, 83.59%, 76.54%, and 70.28%, respectively. Figure 5 ). Although some genes of the expansin family in Arabidopsis have been reported to be associated with low temperature, their interaction with DREB in plants and their functions in alfalfa have not yet been identified and revealed. Therefore, the captured MsEXPA4 protein was selected as the target protein for subsequent studies.

[0141] Example 4 Yeast two-hybrid verification of interaction between MsDREB1C and MsEXPA4 in vitro

[0142] Y2H yeast two-hybrid assay: According to the method of Example 1, MsDREB1C, 53, and lam were respectively constructed into the pGBKT7 (BD) vector to obtain BD-DREB1C, BD-53, and BD-lam fusion vectors; T and MsEXPA4 were constructed into the pGBADT7 (AD) vector to obtain AD-T and AD-MsEXPA4 fusion vectors, respectively. These plasmids were co-transformed into Y2H yeast cells. The co-transformation products of BD-MsDREB1C and AD-MsEXPA4 were used for Y2H one-to-one verification, the co-transformation products of BD-53 and AD-T were used as positive controls, the co-transformation products of BD-lam and AD-T were used as negative controls, and the co-transformation products of BD and AD were used as double negative controls. Using DDO medium as a control, the growth of the co-transformation products on QDO medium containing X-α-gal was detected. In addition, in order to inhibit the self-activation of MsDREB1C, a high concentration of 100mM 3AT was added to the QDO medium.

[0143] To further explore whether MsDREB1C interacts with MsEXPA4, a yeast two-hybrid "one-to-one" experiment was used for verification. The results showed that the yeast strain Y2H Gold carrying the co-transformation product of pGBKT7(BD)-MsDREB1C and pGADT7(AD)-MsEXPA4 grew well and turned blue on the X-α-gal medium of SD / -Trp, -Leu, -His, -Ade(QDO), and the addition of 100mM 3AT effectively inhibited the self-activation of MsDREB1C ( Figure 6 ). Therefore, yeast two-hybrid assay demonstrated that MsDREB1C interacts with MsEXPA4.

[0144] Example 5 GST-pull down validation of direct interaction between MsDREB1C and MsEXPA4 in vitro

[0145] GST-pull down test: According to the method of Example 1, MsDREB1C and MsEXPA4 were recombined into pET-32a with His tag sequence and PGEX-4T-1 vector with GST tag sequence, respectively. The recombinant fusion protein was expressed by IPTG induction and analyzed by 12% SDS-PAGE. The precipitate was resuspended in 20 mL lysis buffer (20 mM Tris-HCl, containing 1 mM PMSF and bacterial protease inhibitor cocktail, pH 8.0) and ultrasonically disrupted (400 W, 20 min). After washing and dissolving the precipitate, the supernatant was taken for gradient dilution (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0 buffer, purified by Ni-IDA column and GST column, respectively, and analyzed again by 12% SDS-PAGE. For the pull-down experiment, GST protein was used as a negative control. According to the manufacturer's instructions, the protein GST-MsEXPA4 and protein His-MsDREB1C were subjected to pull-down experiments using the GST Protein Interaction Pull-down Kit (21516; Thermo Fisher Scientific, Shanghai, China) to verify whether there is an interaction between the two proteins. Finally, the eluted samples were detected by Western Blotting.

[0146] To further verify the direct interaction between MsDREB1C and MsEXPA4, MsDREB1C and MsEXPA4 were recombined into pET-32a with a His tag sequence and PGEX-4T-1 vector with a GST tag sequence, respectively. After SDS-PAGE detection of the protein expression of MsDREB1C and MsEXPA4, protein purification was performed on them respectively ( Figure 7 Subsequently, GST-MsEXPA4 and His-MsDREB1C protein pull-down experiments were used to further reveal the direct interaction between MsEXPA4 and MsDREB1C in vitro ( Figure 8 ).

[0147] Example 6 BiFC Verification of the Interaction between MsDREB1C and MsEXPA4 in Vivo

[0148] Bimolecular fluorescence complementation (BiFC) test: MsDREB1C and MsEXPA4 were connected to pSPYCE and pSPYNE vectors respectively according to the method of Example 1. The fusion Agrobacterium GV3101 strain containing cYFP-MsDREB1C and nYFP-MsEXPA4 was co-transformed into tobacco leaves. The fusion Agrobacterium GV3101 containing an empty vector was used as a control. After 48 h of cultivation, the epidermal cells of the transformed tobacco were imaged and observed under a laser confocal microscope (Zeiss, Germany).

[0149] To verify the interaction between MsDREB1C and MsEXPA4 in vivo, the CDS sequences of MsDREB1C and MsEXPA4 were constructed into pSPYCE and pSPYNE vectors, respectively, and the fusion Agrobacterium GV3101 strain containing cYFP-MsDREB1C and nYFP-MsEXPA4 was co-transformed into tobacco leaves. After 48 hours of culture, the epidermal cells of the transformed tobacco were imaged and observed under a laser confocal microscope (Zeiss, Germany) by making slides. The results showed ( Fig. 9 ), a yellow fluorescence signal was observed in cells co-expressing cYFP-MsDREB1C and nYFP-MsEXPA4, while no yellow fluorescence signal was observed in the co-transfected fusion Agrobacterium GV3101 containing an empty vector (such as cYFP-MsDREB1C+nYFP; cYFP+nYFP-MsEXPA4; cYFP+nYFP), indicating that MsDREB1C and MsEXPA4 interact with each other in vivo.

[0150] Example 7 Cloning of alfalfa MsEXPA4

[0151] Gene cloning of MsEXPA4:

[0152] The cDNA of alfalfa "Zhongmu 1" was used as a template and the gene of MsEXPA4 was cloned using the 2×Phanta Max Master Mix (Novizan) kit. The DNA gene fragments were recovered from the gel using the Quick Gel Extraction Kit (Quanshijin); T-linking was performed using the TA / Blunt-Zero Cloning Kit (Novazan); E. coli was transformed using the DH5α competent cell. The plasmid was extracted using the EZNAPlasmid Miniprep Protocol (Omega) kit and the primers were used for single clone identification;

[0153] Linearized vector construction

[0154] (1) Single enzyme digestion of Super1300 vector was performed using restriction endonuclease QuickCutTM SmaⅠ (Takara) according to the following reaction system as shown in Table 12:

[0155] Table 12 Reaction system

[0156] Reagents Dosage SmaⅠ 1μL 10×Quickcut Buffer 5μL Super 1300 vector plasmid 1ng Ultrapure water to 50μL

[0157] The above reactants were reacted in a PCR instrument at 37°C for 1 hour and then detected by 1% agarose gel electrophoresis. The linearized vector was cut and gel recovered, and the concentration of the recovered product was detected by an ultra-micro spectrophotometer.

[0158] (2) Single enzyme digestion of the BD vector was performed using restriction endonuclease QuickCutTM EcoRI (Takara) according to the following reaction system as shown in Table 13:

[0159] Table 13 Reaction system

[0160] Reagents Dosage EcoRⅠ 1μL 10×Quickcut Buffer 5μL BD vector plasmid 1ng Ultrapure water to 50μL

[0161] The above reactants were reacted in a PCR instrument at 37°C for 1 hour and then detected by 1% agarose gel electrophoresis. The linearized vector was cut and gel recovered, and the concentration of the recovered product was detected by an ultra-micro spectrophotometer.

[0162] Homologous recombination of target gene and linearized vector: use -Basic Seamless Cloning and Assembly Kit (Novizan) was used for homologous recombination, and the reaction system is shown in Table 14:

[0163] Table 14 Reaction system

[0164] Reagents Dosage 2×Basic Assembly Mix 3μL Linearized vector 2μL Inserted target fragment 1μL

[0165] The above reactants were placed on ice for transformation into competent E. coli DH5α after being reacted at 50℃ in a PCR instrument for 30 minutes. After transformation, the bacterial solution was spread on a solid culture medium containing the corresponding antibiotics (Super 1300 and BD vectors are both Kan-resistant) and cultured at 37℃ overnight. After monoclonal identification, the bacterial solution with the correct band was selected and sent to Qingke Bio for sequencing. After sequence alignment, the bacterial solution with the correct sequencing was selected and mixed with 50% glycerol in a ratio of 1:1, and then stored at -80℃ for future use. Before transforming Agrobacterium, the bacterial solution was activated and the plasmid was extracted.

[0166] Alfalfa genetic transformation:

[0167] Transformation of Agrobacterium competent cells:

[0168] (1) Take out the competent Agrobacterium cells (GV3101 or EHA105) from the -80°C ultra-low temperature freezer and quickly place them on ice to thaw for about 3-5 minutes.

[0169] (2) Add 1 μg (volume calculated based on concentration) of the homologous recombination plasmid containing the target gene to be transformed into 100 μL of Agrobacterium competent cells and mix gently.

[0170] (3) Place the centrifuge tube containing the mixture on ice for 30 min.

[0171] (4) After the ice bath, place it in liquid nitrogen for quick freezing for 5 minutes.

[0172] (5) After quick freezing, place the centrifuge tube containing the mixture in a water bath at 37°C for 5 min.

[0173] (6) After the constant temperature water bath, place it immediately on ice and keep it in an ice bath for another 5 minutes.

[0174] (7) After the ice bath, add 700 μL of YEB solution without antibiotics in a clean bench and shake at 220 rpm at 28°C for 2-3 h to recover the resistance.

[0175] (8) The revived product was centrifuged at 4000 rpm for 1 min to collect the bacterial cells.

[0176] (9) Discard part of the supernatant in a clean bench and use the remaining 200 μL of culture medium to resuspend the bacteria at the bottom of the centrifuge tube. Use a bacterial spreader to evenly spread the resuspended bacteria on a YEB solid culture medium plate containing 50 mg / L kanamycin (Kan) and 25 mg / L rifampicin (Rif) antibiotics. Wrap the plate with tin foil and invert it in a 28°C constant temperature incubator for 2 days.

[0177] (10) Pick several monoclonal colonies from the overnight culture medium plate and add them to 1 mL of YEB liquid culture medium (containing antibiotics corresponding to the culture medium plate). After culturing in a shaker at 28°C and 220 rpm for 24-48 h, use the corresponding vector and the primer at the other end of the gene to perform monoclonal identification. After confirming that the band is correct, mix the bacterial solution containing the target gene expression vector with 50% glycerol in a 1:1 ratio and store it in a -80°C low-temperature incubator.

[0178] Leaf genetic transformation:

[0179] (1) One living cell: Take the bacterial liquid stored in a -80°C low-temperature incubator and add 20 mL of fresh YEB (containing Kan and Rif) liquid culture medium at a ratio of 1:200, and culture it in a shaker at 28°C and 220 rpm for about 16 h;

[0180] (2) Two live cells: After one live cell becomes turbid, take one live cell and add it to 20 mL of fresh YEB (containing Kan and Rif) liquid culture medium at a ratio of 1:200, and culture it in a shaker at 28°C and 220 rpm for about 8-10 h until the OD600 reaches about 0.6;

[0181] (3) Prepare materials: Use scissors disinfected with 75% ethanol to cut several leaves of alfalfa that are in good growth condition and free of disease, and wash them in ultrapure water containing 10% sodium hypochlorite for 10 minutes;

[0182] (4) Transfer the disinfected leaves to a new sterilized conical flask in a clean bench and wash the leaves with sterile ultrapure water 5-6 times until the residual sodium hypochlorite on the leaves is washed away;

[0183] (5) Add about 30 mL of sterile SH3a liquid culture medium (about 100 mL) to the cleaned leaves, then add the bacteria collected in step (2), fully resuspend, wrap and remove from the clean bench;

[0184] (6) Place the conical flask with leaves in an ultrasonic device to injure the leaves, and check the wound status of the leaves every 4 seconds until most leaves have wounds evenly distributed (be careful not to kill the leaves);

[0185] (7) Place it in a vacuum apparatus and evacuate for about 7 minutes. At this time, most of the leaves will be basically sunk under the liquid, allowing the bacterial liquid to fully contact the wounded leaves;

[0186] (8) Place the conical flask in a low-speed shaker in the dark for 30 min, then wash it 5-6 times in a clean bench until the liquid is no longer turbid.

[0187] (9) Remove the leaves, dry the surface water with sterile filter paper, and then spread them on SH3a antibiotic-free solid medium for co-culture for 24 h;

[0188] (10) After co-cultivation, the leaves were washed again, dried with filter paper, and then transferred to SH3a solid medium (containing 5 mg / L hygromycin B selection antibiotic and 400 mg / L cephalosporin antibiotic) for culture for about 2 months, during which the medium was changed every 15 days;

[0189] (11) After 2 months of callus culture, the soft calli were transferred to MSBK solid medium (containing 5 mg / L hygromycin B selection antibiotic and 400 mg / L cephalosporin antibiotic) for bud culture;

[0190] (12) After 15 days of bud culture, the buds were transferred to SH9a solid medium (containing 5 mg / L hygromycin B selection antibiotics and 400 mg / L cephalosporin antibiotics) to continue rooting culture for about 4 months. After the alfalfa regenerated plants were complete and the roots were fully developed, soil culture was carried out;

[0191] (13) After the strains have grown strong, the identification of transgenic positive strains can be carried out.

[0192] Identification of MsEXPA4 transgenic alfalfa:

[0193] First, DNA level identification is performed. The identification steps are as follows:

[0194] (1) Prepare DNA extraction buffer (100 mL): 20 mL 1 M Tris (pH 9.0) + 20 mL 2 M LiCl + 5 mL 0.5 M EDTA (pH 8.0) + 5 mL 20% SDS solution + 50 mL ultrapure water

[0195] (2) Take a small portion of plant leaf tissue and place it in a 1.5 mL centrifuge tube;

[0196] (3) Add 200 μL DNA extraction buffer and grind the leaves thoroughly with a grinding rod;

[0197] (4) Centrifugation at 13,000 rpm for 5 min;

[0198] (5) Carefully transfer 160 μL of supernatant to a new centrifuge tube, add 160 μL of isopropanol, and immediately pipette to mix;

[0199] (6) Centrifugation at 13,000 rpm for 5 min;

[0200] (7) Discard the supernatant and invert the centrifuge tube to dry for 30 min;

[0201] (8) Add 20 μL ultrapure water to resuspend the eluted DNA;

[0202] (9) Take 2 μL of DNA as a template and use the primers MsEXPA4-1300-F and MsEXPA4-1300-R for positive identification (see Table 15).

[0203] (4) The PCR products were run on 1% agarose gel electrophoresis, and the positive strains identified by DNA level were cut out according to the size of the target band. TM Total RNA was extracted from alfalfa using the Total RNA Extraction Kit (Promega), and cDNA was synthesized from alfalfa using the HiScript III All-in-one RT SuperMix Perfect for qPCR (NoviZan) kit, followed by identification at the qRT-PCR level.

[0204] Table 15 Primer sequences

[0205]

[0206] In order to verify the function and mechanism of MsEXPA4 interacting with MsDREB1C under low temperature stress, the cDNA of RNA extracted from leaves of alfalfa "Zhongmu 1" was used as a template and MsEXPA4-F and MsEXPA4-R were used as primers to amplify MsEXPA4 ( Fig.10 , ab). The CDS is 780 bp in length ( Fig.10 , c), encoding 260 amino acids ( Fig.10 , d).

[0207] Example 8 Identification of alfalfa strains overexpressing MsEXPA4

[0208] Through genetic transformation, alfalfa regeneration lines with the MsEXPA4 gene were obtained. DNA level identification was performed on the obtained transgenic MsEXPA4 regeneration alfalfa lines, and a total of 15 positive lines were obtained ( Fig.11 ). Subsequently, the transcription level of MsEXPA4 in 15 positive strains and WT was analyzed by qRT-PCR. The results showed that the expression level of MsEXPA4 in WT was almost zero, so the expression level of MsEXPA4 among positive strains was compared ( Fig.11 ), and finally selected three strains, OE7, OE14, and OE8, for subsequent experiments.

[0209] Example 9 Analysis of frost resistance of alfalfa overexpressing MsEXPA4

[0210] Determination of survival rate: After the alfalfa was recovered from freezing for 7 days, the survival rate was calculated.

[0211] Determination of conductivity:

[0212] (1) Prepare a 15 mL centrifuge tube, add 14 mL of ultrapure water, and prepare a blank control P0.

[0213] (2) Weigh 0.1 g of the leaf to be tested and cut it into pieces in an EP tube.

[0214] (3) After shaking on a low-speed shaker for 24 hours, the conductivity was measured as P1.

[0215] (4) Boil it in a 100°C water bath for 30 min.

[0216] (5) After cooling to room temperature, the conductivity is measured as P1' and the blank control is P0'.

[0217] (6) Calculate according to the formula: Conductivity (%) = (P1-P0) / (P1'-R0') × 100%

[0218] Determination of chlorophyll content:

[0219] (1) Prepare a 15 mL centrifuge tube and add 10 mL of 95% ethanol. Also prepare a blank control.

[0220] (2) Weigh 0.1 g of the leaf to be tested and cut it into pieces in an EP tube.

[0221] (3) Wrap the centrifuge tube containing the leaves to be tested with tin foil and leave it in the dark for 48 hours until the leaves are completely discolored.

[0222] (4) Measure the OD values ​​at 665nm and 649nm and record them as A 665 and A 649 , remember to mix the liquid before measuring.

[0223] (5) Calculate according to the formula:

[0224] Chlorophyll a: 13.95×A 665 -6.88×A 649

[0225] Chlorophyll b: 24.96×A 649 -7.32×A 665

[0226] Total chlorophyll content: chlorophyll a + chlorophyll b

[0227] Determination of malondialdehyde (MDA) content:

[0228] The malondialdehyde (MDA) test kit (A003-1) of Nanjing Jiancheng Bioengineering Institute was used to determine the MDA content using the thiobarbituric acid (TBA) method according to the instructions, that is, MDA in the peroxidation lipid degradation product is condensed with TBA to form a red product with a maximum absorption peak at 532nm.

[0229] Determination of hydrogen peroxide (H2O2) content: Use the hydrogen peroxide (H2O2) test kit (A064-1-1) of Nanjing Jiancheng Bioengineering Research Institute to determine the H2O2 content according to the instructions, that is, H2O2 can react with molybdic acid to generate a complex. The amount of H2O2 can be calculated by measuring the amount of complex at 405nm.

[0230] Determination of superoxide dismutase (SOD): The activity of SOD was determined using the superoxide dismutase (SOD) test kit (A001-1-2) of Nanjing Jiancheng Bioengineering Institute according to the instructions using the xanthine oxidase method.

[0231] Determination of Catalase (CAT):

[0232] The CAT content was determined using the Nanjing Jiancheng Bioengineering Research Institute Catalase (CAT) test kit (A007-1-1) according to the instructions. That is, the reaction of CAT decomposing H2O2 can be quickly terminated by adding ammonium molybdate. The remaining H2O2 reacts with ammonium molybdate to produce a light yellow complex. The change in the complex is measured at 405nm, and the activity of CAT can be calculated.

[0233] Determination of vegetable protein content:

[0234] The protein content was determined using the Nanjing Jiancheng Bioengineering Institute Protein Quantification (TP) Assay Kit (A045-4-2) according to the instructions. That is, under alkaline conditions, the protein converts Cu 2+ Reduction to Cu + , Cu + It forms a purple complex with the BCA reagent, with a maximum absorption peak at 562nm. Since absorbance is proportional to concentration, the concentration of the protein to be tested can be calculated by measuring absorbance.

[0235] Damage rate determination:

[0236] For the freezing treatment of plants: after 7 days of recovery from freezing treatment, if the damage to the plants is greater than 1 / 2, they are counted as injured plants, and the damage rate can be calculated by dividing the number of injured plants by the total number of plants.

[0237] For the freezing treatment of detached leaves: after 7 days of recovery from freezing treatment, the damage rate was obtained by dividing the number of leaves with yellowing area greater than 1 / 3 by the total number of leaves.

[0238] To detect the biological function of MsEXPA4 under low temperature stress, alfalfa lines overexpressing MsEXPA4 (MsEXPA4-7, MsEXPA4-14, and MsEXPA4-8) were selected for low temperature tolerance evaluation under one week of cold acclimation. The freezing treatment was carried out by a gradient cooling method of 1°C per hour until it dropped to -9°C and maintained for 1 hour. The phenotypic analysis of all lines after low temperature stress treatment showed that all transgenic lines grew well with the wild type under normal growth conditions ( Fig.12 , a). However, after low temperature treatment, WT was more seriously damaged. After 7 days of recovery, many leaves of the plants wilted and turned yellow. However, leaves of the strains overexpressing MsEXPA4-7, MsEXPA4-14, and MsEXPA4-8 were less damaged by low temperature. After 7 days of recovery, most leaves remained green ( Fig.12 The above results showed that alfalfa lines overexpressing MsEXPA4 had stronger low temperature tolerance.

[0239] After 7 days of recovery, the plant damage rates of MsEXPA4-7, MsEXPA4-14, and MsEXPA4-8 were reduced by 46%, 41%, and 35%, respectively, compared with WT. Fig.13 , a). In order to detect the physiological and biochemical response of low temperature stress to cell membrane permeability damage, the conductivity and MDA content were determined and it was found that under normal growth conditions, no significant difference was observed between MsEXPA4-OE and control alfalfa plants; after low temperature treatment, the conductivity and MDA content of the overexpression strain were significantly lower than those of WT, indicating that the cell membrane was less damaged ( Fig.13 , bc). The results of the determination of oxidation-antioxidation related indicators showed that under normal conditions, there were no significant differences in the physiological indicators of H2O2, SOD, and CAT. However, after low temperature treatment, the strain overexpressing MsEXPA4 had lower H2O2 content and higher SOD and CAT antioxidant enzyme activities ( Fig.13 , df), indicating that overexpression of MsEXPA4 alleviated the oxidative damage caused by low temperature.

[0240] In addition, the leaves of the wild-type plants wilted and turned yellow after low temperature treatment, and the contents of chlorophyll a, chlorophyll b, and total chlorophyll were significantly lower than those of the transgenic lines ( Fig.14 ), indicating that overexpression of MsEXPA4 reduced chlorophyll loss caused by low temperature ( Fig.14 ).

[0241] In addition, the detached leaves of overexpressing MsEXPA4 and wild-type alfalfa were treated with freezing ( Fig.15 ) found that overexpression of MsEXPA4 significantly reduced the damage rate of leaves after freezing treatment ( Fig.15 , ac), and has a higher green retention after low temperature treatment, which is reflected in the higher chlorophyll content ( Fig.15 , df).

[0242] In summary, overexpression of MsEXPA4 in alfalfa plays an important role in enhancing cold acclimation-induced freezing resistance. The present invention reveals the direct interaction between MsEXPA4 and MsDEEB1C, and shows that the physiological responses of the two to low temperature are basically the same, positively regulating the low temperature tolerance response induced by cold acclimation in alfalfa.

[0243] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of the MsEXPA4 gene in alfalfa breeding, wherein the application is to construct alfalfa with good low temperature resistance by overexpressing the MsEXPA4 gene in alfalfa; in, The nucleotide sequence of the MsEXPA4 gene is shown in SEQ ID No.

3.

2. The use according to claim 1, characterized in that The amino acid sequence encoded by the MsEXPA4 gene is shown in SEQ ID No.

4.

3. Application of a biological material of the MsEXPA4 gene in alfalfa breeding, wherein the application is to construct alfalfa with good low temperature resistance by overexpressing the MsEXPA4 gene; in, The nucleotide sequence of the MsEXPA4 gene is shown in SEQ ID No. 3; Wherein, the biological material is an expression cassette of the MsEXPA4 gene.

4. The use according to claim 3, characterized in that The biological material is a recombinant vector of the MsEXPA4 gene.

5. The use according to claim 3, characterized in that The biological material is a recombinant microorganism of the MsEXPA4 gene.

6. The use according to claim 3, characterized in that The biological material is a recombinant vector containing a MsEXPA4 gene expression box.

7. The use according to claim 3, characterized in that The biological material is a recombinant microorganism containing a MsEXPA4 gene recombinant vector.

8. A method for preparing high and low temperature resistant alfalfa, characterized in that: The method comprises the step of overexpressing the MsEXPA4 gene; Among them, the nucleotide sequence of the MsEXPA4 gene is shown in SEQ ID No. 3.

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