Alfalfa calmodulin-like protein cml50 gene and application thereof

Abstract

The application discloses alfalfa calmodulin CML50 genes and application thereof, and the nucleotide sequence of the calmodulin CML50 gene is as follows (1) or (2): (1) a nucleotide sequence as shown in SEQ ID NO. 1; (2) a nucleotide sequence with more than 90% homology with the nucleotide sequence and with equivalent functions. The application provides a method for cultivating cold-resistant plants by using the MsCML50 gene, and after overexpression of the gene in alfalfa, the cold resistance of the transgenic alfalfa is improved; meanwhile, after interference of the gene in alfalfa, the cold resistance of the transgenic alfalfa is reduced.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically involving the calmodulin CML50 gene of alfalfa and its application. Background Technology

[0002] Low temperatures, drought, and salinity negatively impact plant growth and development, leading to reduced crop and forage yields. They also affect the normal growth and quality of forest trees, fruit trees, flowers, and ornamental plants. Developing stress-resistant plant varieties is one of the main goals of agriculture. Therefore, isolating stress-resistant genes from plants with strong resilience is essential.

[0003] Calmodulin-like proteins (CMLs) are plant-specific calcium-binding proteins containing 2–6 EF-hand domains, acting as calcium signaling sensors (McCormack and Braam, 2003). CMLs bind calcium... 2+ After activation, it binds to its target proteins, thereby activating the latter and regulating physiological and biochemical responses. Studies have found that various types of proteins can become targets of CML, including protein kinases, transcription factors, metabolic enzymes, and transport proteins (Perochon et al., 2010). Recent studies have revealed that CML plays an important role in plant growth, development, and stress responses (Sun et al., 2021; Yu et al., 2022).

[0004] Numerous studies have reported on the roles of cytokines (CMLs) in plant growth and development. CML10 interacts with phosphomannose mutase (AtPMM) to promote ascorbic acid biosynthesis, thereby regulating the oxidative stress resistance of Arabidopsis thaliana (Cho et al., 2016). In Arabidopsis, CML24 can regulate the oxidative stress resistance of Arabidopsis thaliana through a Ca-dependent pathway. 2+ The signal regulation of plant circadian rhythms, CML24, plays a role by establishing a connection with the circadian rhythm oscillator through the TOC1 (TIMING OF CAB2 EXPRESSION 1) pathway (Martiet et al., 2018). Arabidopsis CML25, on the other hand, participates in the regulation of Ca2+ signaling. 2+ K mediates pollen germination and pollen tube elongation. +The influx of ABA into plants is a significant factor (Wang et al., 2015). CMLs also participate in regulating plant stress responses, including those to pests, diseases, light, salt, drought, and temperature. For example, CML9 may be a regulator involved only in the defense against nutrient-dependent pathogens, but its function is unrelated to JA signaling (Heyer et al., 2018). Meanwhile, studies have found that CML8 and CML9 can enhance plant immunity to the plant pathogen *Pseudomonas* (Zhu et al., 2017). The Arabidopsis CML37 knockout mutant cml37 is highly sensitive to drought stress, while its ABA content is significantly reduced, indicating that CML37 negatively regulates drought resistance (Scholz et al., 2015). Alfalfa CML42 regulates plant cold tolerance and flowering time (Sun et al., 2021), while alfalfa CML10 maintains ROS homeostasis at low temperatures, promotes sugar accumulation, and enhances plant cold tolerance through interactions with cytoplasmic GST8 and FBA6 (Yu et al., 2022). Alfalfa is one of the most important leguminous forage crops, known as the "King of Forages." Identifying genes regulating alfalfa cold tolerance can provide target genes for molecular breeding of alfalfa with cold tolerance. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a calmodulin CML50 gene for alfalfa plants that enhances their cold resistance.

[0006] Another object of the present invention is to provide the protein encoded by the calmodulin CML50 gene of alfalfa mentioned above.

[0007] Another object of the present invention is to provide the application of the above-mentioned calmodulin CML50 gene in alfalfa.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A calmodulin CML50 gene for alfalfa, the nucleotide sequence of which is as follows (1) or (2):

[0010] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0011] (2) A nucleotide sequence that has more than 90% homology with and has the same function as the nucleotide sequence described in (1).

[0012] The protein encoded by the calmodulin CML50 gene of alfalfa, as described above, has the following amino acid sequence (a) or (b):

[0013] (a) The amino acid sequence as shown in SEQ ID NO.2;

[0014] (b) A sequence with equivalent function formed by substituting, deleting or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO.2.

[0015] Biological materials containing the above-mentioned alfalfa calmodulin CML50 gene or biological materials used to silence or interfere with the above-mentioned alfalfa calmodulin CML50 gene, wherein the biological material is at least one of a recombinant vector, expression cassette, cell line and host bacteria.

[0016] The above-mentioned alfalfa calmodulin CML50 gene, the above-mentioned alfalfa calmodulin CML50, or the above-mentioned biological materials are used in improving plant cold resistance or cultivating transgenic plants with improved cold resistance.

[0017] The above-described application involves overexpressing the alfalfa calmodulin CML50 gene in target plants to improve their cold resistance.

[0018] A method to improve plant cold hardiness involves overexpressing the alfalfa calmodulin CML50 gene in a target plant to enhance its cold hardiness.

[0019] As a preferred technical solution, the process of overexpressing the alfalfa calmodulin CML50 gene in the target plant is as follows: constructing an overexpression vector of the alfalfa calmodulin CML50 gene as described above, and transferring the overexpression vector into the target plant through Agrobacterium-mediated transformation to obtain transgenic plants with improved cold resistance.

[0020] The plants mentioned above are monocotyledonous or dicotyledonous plants, such as alfalfa, but are not limited to these.

[0021] During the research, technicians constructed the overexpression vector 35S::MsCML50 and the interference vector MsCML50-RNAi using pCAMBIA3301 and pFGC5941, respectively, with glufosinate as the selection pressure. Mature leaves of alfalfa cuttings at 8-12 weeks of age were selected for genetic transformation. Transgenic alfalfa was obtained through a series of processes including infection, co-culture, callus induction, callus regeneration, and rooting of regenerated seedlings.

[0022] The recombinant vector containing the alfalfa calmodulin CML50 gene is a recombinant expression vector, which is obtained by linking the nucleotide sequence of the alfalfa calmodulin CML50 gene to a plant expression vector; the plant overexpression vector is preferably pCAMBIA3301.

[0023] An interference vector for interfering with the expression of calmodulin CML50 gene in alfalfa was prepared, with pFGC5941 being the preferred vector.

[0024] A method for preparing a recombinant expression vector containing the alfalfa calmodulin CML50 gene (MsCML50) includes the following steps:

[0025] (1) Primer design

[0026] Primer (1) is the upstream primer Y4600 for MsCML50 amplification:

[0027] 5'-ATGGCAACCAATCC-3';

[0028] Primer (2) is the downstream primer Y4601 for MsCML50 amplification:

[0029] 5'-CTAATTGCTATTGGCTTG-3';

[0030] Primer (3) is the upstream primer Y4602 that introduces the Nco I restriction site:

[0031] 5'-GGGGACTCTTGACCATGGCAATGGCAACCAATCC-3';

[0032] Primer 4 is the downstream primer Y4603 that introduces the BstE II restriction site:

[0033] 5'-GAAATTCGAGCTGGTCACCCTAATTGCTATTGGCTTG-3';

[0034] Primer (5) is the upstream primer Y4604 that introduces the Asc I restriction site:

[0035] 5'-ACCATGGGGCGCGCCCTGAAACTACCACCACCAA-3';

[0036] Primer 6 is the downstream primer Y4605 that introduces the Asc I restriction site:

[0037] 5'-TCATCGATTGGGCGCGCCGGCCGTTCTTGTCCTTGTC-3';

[0038] Primer 7 is the downstream primer Y4606 that introduces the Xba I restriction site:

[0039] 5'-CTTAATTAACTCTCTAGACTGAAACTACCACCACCAA-3';

[0040] Primer (8) is the downstream primer Y4607 that introduces the BamHI restriction site:

[0041] 5'-TTGCAGGTATTTGGATCCGGCCGTTCTTGTCCTTGTC-3';

[0042] Primer 9 is the upstream primer Y4800 for amplifying the upstream promoter of MsCML50.

[0043] 5'-CCATTTTGGTCCTTGAATGTGCAAC-3';

[0044] Primer 10 is for the upstream promoter of MsCML50 to amplify the downstream primer Y4801:

[0045] 5'-TGTTTGATTGAGAGAGAGAAGAGGA-3'

[0046] Primer 11 is the upstream primer Y4803 for introducing the EcoRI restriction site:

[0047] 5'-TATGACCATGATTACGAATTCCCATTTTGGTCCTTGAATGTGCAAC-3';

[0048] Primer 12 is the downstream primer Y4804 that introduces the Nco I restriction site:

[0049] 5'-AATTTACCCTCAGATCTACCATGGTGTTTGATTGAGAGAGAGAAGAGGA-3';

[0050] Primer ⒀ is the upstream primer Y4805 that introduces the Xba I restriction site:

[0051] 5'-TCCGGAGCTAGCTCTAGAATGGCAATGGCAACC-3';

[0052] Primer 14 is the upstream primer Y4806 that introduces the BamHI restriction site:

[0053] 5'-CTTGCTCACCATGGATCCATTGCTATTGGCTTG-3';

[0054] (2) Obtaining the MsCML50 gene fragment:

[0055] Using alfalfa cDNA as a template, the MsCML50 gene fragment was amplified using primers (1) and (2), and homologous arms were ligated using this as a template to clone the pCAMBIA3301-MsCML50 homologous arm fragment and the interference sense strand MsCML50-RNAi-1 and antisense strand MsCML50-RNAi-2 homologous arm fragments.

[0056] (3) Construction of recombinant expression vectors pCAMBIA3301-MsCML50 and pFGC5941-MsCML50 containing the MsCML50 gene:

[0057] Using the MsCML50 gene fragment as a template, primers (3) and (4) were used to introduce the MsCML50 gene fragment into the Nco I and BstE II restriction sites, and then ligated it with the plant expression vector pCAMBIA3301 to construct the recombinant expression vector pCAMBIA3301-MsCML50 containing the MsCML50 gene.

[0058] Using the MsCML50 gene fragment as a template, primers (5) and (6) were used to introduce the MsCML50 gene fragment into the Asc I restriction site, and then ligated with the plant expression vector pFGC5941 to obtain the interference positive strand vector. Using this vector as a template, double digestion with Xba I and BamHI was performed to obtain the large fragment of the vector. Primers (7) and (8) were used to introduce the MsCML50 gene fragment into the Xba I and BamHI restriction sites, and then ligated with the double-digested large fragment of the vector to construct the pFGC5941-MsCML50 interference vector.

[0059] Transgenic plants containing the recombinant expression vector pCAMBIA3301-MsCML50 of the MsCML50 gene and transgenic plants containing the interference vector pFGC5941-MsCML50 were obtained by transforming alfalfa leaf tissue with the above recombinant expression vector or interference vector and culturing the transformed plant tissue.

[0060] The preferred plant is alfalfa;

[0061] The method for preparing transgenic plants expressing alfalfa MsCML50 includes the following steps:

[0062] (1) The recombinant expression vector pCAMBIA3301-MsCML50 and the interference vector pFGC5941-MsCML50 were introduced into Agrobacterium rhizogenes EHA105 by electroporation, and Agrobacterium-positive colonies containing the recombinant expression vector pCAMBIA3301-MsCML50 and the interference vector pFGC5941-MsCML50 were obtained, respectively.

[0063] (2) Agrobacterium tumefaciens EHA105 containing recombinant expression vector pCAMBIA3301-MsCML50 and interference vector pFGC5941-MsCML50 was used to infect alfalfa leaves. After co-culture and glufosinate resistance screening, transgenic alfalfa was obtained.

[0064] This invention cloned the cDNA sequence MsCML50 of alfalfa calmodulin CML50 from alfalfa, and ligated the obtained MsCML50 gene into a plant expression vector to construct recombinant overexpression vectors and interference vectors. These vectors were then used to transform alfalfa leaves, resulting in transgenic plants. The MsCML50 gene is induced by low-temperature stress; MsCML50 is located in the nucleus and cytoplasm; and MsCML50 exhibits GUS activity in Arabidopsis leaves, roots, anthers, and stigmas, which is largely consistent with the MsCML50 expression results detected in alfalfa. This invention provides a method for cultivating cold-resistant plants using the MsCML50 gene. Overexpression of this gene in alfalfa improves the cold resistance of transgenic alfalfa; conversely, interference of this gene in alfalfa reduces the cold resistance of transgenic alfalfa.

[0065] The present invention also provides an alfalfa calmodulin CML50 promoter, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0066] The application of the above-mentioned alfalfa calmodulin CML50 promoter in the following (1) or (2) is also within the scope of protection of this invention:

[0067] (1) Drives GUS expression;

[0068] (2) Regulate the expression of downstream genes in response to abiotic stress; the abiotic stress is cold stress, drought stress or hormone (e.g. ABA) stress.

[0069] The present invention has the following advantages and effects compared with the prior art:

[0070] (1) The present invention cloned the cDNA sequence of MsCML50 from alfalfa, the expression of which is induced by low temperature.

[0071] (2) The present invention performs subcellular localization analysis on the obtained MsCML50 protein, which is located in the cell nucleus and cytoplasm.

[0072] (3) The present invention connects the obtained MsCML50 gene with a plant expression vector to construct a recombinant expression vector suitable for alfalfa and transforms it into alfalfa. Transgenic alfalfa that overexpresses MsCML50 has significantly improved cold resistance, while RNAi plants have significantly reduced cold resistance.

[0073] (4) This invention provides a method for cultivating stress-tolerant plants using the MsCML50 gene. Attached Figure Description

[0074] Figure 1This is a schematic diagram of the expression cassette of the recombinant expression vector pCAMBIA3301-MsCML50.

[0075] Figure 2 This is an agarose gel electrophoresis image of the PCR amplification of the open reading frame sequence of the MsCML50 gene;

[0076] Where M is a standard DNA molecule; lane 2 is an amplified fragment of the MsCML50 gene.

[0077] Figure 3 This is an agarose gel electrophoresis image of the PCR identification of the recombinant expression vector pCAMBIA3301-MsCML50;

[0078] In this context, M is the standard DNA molecule; lanes 1, 2, and 4 are positive recombinants; and lane 3 is a negative recombinant.

[0079] Figure 4 This is an agarose gel electrophoresis image of the PCR identification of the recombinant expression vector pFGC5941-MsCML50;

[0080] In this context, M is the standard DNA molecule; lanes 1, 2, 3, 4, 5, and 6 are positive recombinants.

[0081] Figure 5 This is the subcellular localization of MsCML50.

[0082] Figure 6 This is a bar chart analyzing the results of low-temperature induced MsCML50 gene expression.

[0083] The value represents the effect of low temperature on MsCML50 gene transcription; the letters a, b, and c above the column indicate significant differences between different treatments (P≤0.05), that is, when the letters above the data columns are the same, it means there is no significant difference, and when the letters above the data columns are different, it means there is a significant difference.

[0084] Figure 7 This is an agarose gel electrophoresis image of the PCR amplification of the MsCML50 gene promoter sequence;

[0085] Where M is a standard DNA molecule; lanes 1, 2, 3, 4, 5, and 6 are amplified fragments of the MsCML50 gene promoter sequence.

[0086] Figure 8 This is an agarose gel electrophoresis image of the PCR identification of the recombinant interference vector pFGC5941-P:MsCML50;

[0087] In this context, M is the standard DNA molecule; lane 7 is the positive recombinant; and lanes 1, 2, 3, 4, 5, 6, 8, and 9 are the negative recombinants.

[0088] Figure 9 This is a tissue-specific expression analysis of the MsCML50 gene.

[0089] Figure A shows the expression of the MsCML50 gene in different tissues of alfalfa; the letters a, b, and c above the columns indicate significant differences between different treatments (P≤0.05), meaning that the same letter above the data column indicates no significant difference, and different letters above the data column indicate significant differences. Figure B shows the GUS activity in transgenic Arabidopsis thaliana driven by the MsCML50 promoter.

[0090] Figure 10 This is a graph showing the results of real-time quantitative PCR identification of transgenic alfalfa overexpressing the MsCML50 gene.

[0091] Wherein, WT represents wild type; OE represents positive strain;

[0092] Figure 11 This is a real-time quantitative PCR identification result of the interference transgenic alfalfa with the MsCML50 gene inserted.

[0093] Wherein, WT represents wild type; RNAi represents positive line;

[0094] Figure 12 This is an analysis of the cold resistance test results of transgenic alfalfa with the MsCML50 gene introduced;

[0095] (A) represents the half-lethal temperature of the transgenic lines after low-temperature treatment; (B) represents the survival rate of the transgenic lines after freezing treatment; the letters a, b, and c above the column indicate significant differences between different materials (P < 0.05). Detailed Implementation

[0096] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0097] The alfalfa from Baoding is owned by this laboratory.

[0098] Agrobacterium tumefaciens EHA105: purchased from Beijing Tianenze Gene Technology Co., Ltd. pCAMBIA3301 overexpression vector thermos: preserved in our laboratory. pFGC5941 interference vector: preserved in our laboratory. Escherichia coli DH5α: purchased from Shanghai Chaoyan Biotechnology Co., Ltd. All the above biological materials are conventional biological materials known to those skilled in the art.

[0099] Example 1: Cloning of the MsCML50 gene

[0100] I. Preparation of alfalfa cDNA template

[0101] The alfalfa variety used was Baoding alfalfa. Alfalfa was sown in 15 cm diameter plastic pots containing a mixed nutrient soil (peat:perlite = 3:1:1 (v / v)) and cultured in a greenhouse (25℃) under natural light. After 40 days of growth, mature leaves were harvested, and total RNA was extracted using the Trizol method. Reverse transcription was performed using the PrimeScript RT reagent Kit with gDNAErase (TaKaRa) to obtain cDNA templates, which were then stored at -20℃ for later use.

[0102] II. Primers for amplifying the MsCML50 gene

[0103] Based on the cDNA sequence of the CML50 gene from the genome database of alfalfa Xinjiang large-leaf, primers were designed to amplify the cDNA open reading frame of alfalfa MsCML50 (synthesized by Tsingk).

[0104] The upstream primer for amplifying MsCML50 was Y4600, with the sequence: 5'-ATGGCAACCAATCC-3';

[0105] The downstream primer for amplifying MsCML50 was Y4601, with the sequence: 5'-CTAATTGCTATTGGCTTG-3'.

[0106] III. Amplification of the MsCML50 gene and construction of a vector

[0107] PCR amplification was performed using the template prepared in step one and the primers designed in step two:

[0108] PCR reaction system (50 μL): KOD FX polymerase (TOYOBO, 1 U / μL) -1 )1μL, 2×PCR buffer for KOD FX 25μL, dNTPs (2mmol·L -1 10 μL of upstream primer (0.3 μmol·L⁻¹) -1 1.5 μL of the primer and 0.3 μmol·L⁻¹ of the downstream primer. -1 1.5 μL of first-strand cDNA was reverse transcribed, and the volume was brought to 50 μL with deionized water.

[0109] PCR reaction program: PCR reaction program: 94℃ for 2 min; 98℃ for 0.1 min, 68℃ for 1 min, 35 cycles; 72℃ for 10 min; the amplification products were detected by agarose gel electrophoresis with a mass fraction of 0.8%;

[0110] A sequence of approximately 504 bp was obtained. Figure 2 The obtained PCR products were recovered using a DNA gel recovery kit (AXYGEN).

[0111] Example 2: Construction of overexpression vector pCAMBIA3301-MsCML50 and interference vector pFGC5941-MsCML50

[0112] I. Construction of the overexpression vector pCAMBIA3301-MsCML50

[0113] Based on the MsCML50 open reading frame sequence obtained in Example 1, amplification primers with Nco I and BstE II restriction sites were designed:

[0114] Introducing upstream primer Y4602 with an Nco I restriction site:

[0115] 5'-GGGGACTCTTGACCATGGCAATGGCAACCAATCC-3';

[0116] The downstream primer Y4603, which introduces the BstE II restriction site, is as follows:

[0117] 5'-GAAATTCGAGCTGGTCACCCTAATTGCTATTGGCTTG-3';

[0118] The PCR-recovered fragments were amplified using homologous arms and ligated into a vector. The pCAMBIA3301 expression vector was double-digested with restriction endonucleases Nco I (TaKaRa) and BstE II (TaKaRa). The target fragment amplified from the homologous arms of the recovered MsCML50 gene and the digested vector fragment were ligated using Clon Express II recombination reaction solution at a molar ratio of 1:2. The reaction system was as follows: 4 μL of 5×CE II Buffer, 2 μL of Exnase II, 0.06 pmol of the recovered target fragment, and water was added to a final volume of 20 μL. The ligation was carried out at 37°C for 30 min, and the product was obtained after cooling on ice.

[0119] Preparation of competent *E. coli* cells: *E. coli* DH5α bacterial suspension stored at -80℃ was taken with an inoculation loop, streaked onto LB solid medium, and incubated overnight at 37℃. A single colony of *E. coli* DH5α was picked and inoculated into 1 mL of LB liquid medium, and cultured on a shaker at 37℃ with shaking at 200 rpm for approximately 6 hours. The culture was then inoculated into 200 mL of LB liquid medium and cultured on a shaker at 37℃ with shaking at 200 rpm until OD (Organic Dose) was reached. 550=0.6; collect bacterial cells by centrifugation at 2500 rpm for 10 min, wash with pre-cooled 50% glycerol (glycerol:LB liquid medium), centrifuge to collect bacteria; repeat washing with pre-cooled 10% glycerol (glycerol:LB liquid medium), centrifuge to collect bacteria, finally aliquot the bacteria and store at -80℃ for later use.

[0120] Heat shock transformation of E. coli with ligation product: The ligation product was added to 100 μL of DH5α competent cells and placed on ice for 30 min; after heat shock at 42℃ for 45 s, it was placed on ice for 2 min; the heat-shocked cells were transferred to 1 mL of LB broth and cultured on a shaker at 37℃ with shaking at 200 rpm for 1 h. 100 μL of the bacterial culture was spread onto LB solid medium (containing 200 μg / mL Kan) and incubated upside down overnight at 37℃.

[0121] Screening, purification, and sequencing of recombinant plasmid pCAMBIA3301-MsCML50: White colonies were picked for colony PCR detection. PCR-positive colonies were picked and inoculated into 2 mL of LB liquid medium containing 200 μg / mL kanamycin, and cultured overnight at 37°C with shaking at 200 rpm. 2 mL of the bacterial culture was then used to extract the plasmid using a plasmid DNA purification kit (Qiagen), and stored at 4°C for later use. The purified recombinant plasmid was named pCAMBIA3301-MsCML50.

[0122] PCR detection showed that the correct-sized fragment (approximately 500 bp) was amplified using the pCAMBIA3301-MsCML50 plasmid as a template and primers (3) and (4). This indicated that the recombinant vector contained the MsCML50 gene. Figure 3 Further sequencing of the insert fragment showed that its sequence was completely identical to the coding region of MsCML50, and the restriction enzyme sites at both ends of the insert fragment were also correct, thus proving the successful construction of the recombinant expression vector pCAMBIA3301-MsCML50. A schematic diagram of the expression cassette of this recombinant expression vector is shown below. Figure 1 .

[0123] II. Construction of the interference vector pFGC5941-MsCML50

[0124] Based on the MsCML50 open reading frame sequence obtained in Example 1, a band of approximately 200 bp was selected. Amplification primers with an Asc I (TaKaRa) restriction site were designed for PCR amplification to obtain the homologous arm of the positive strand. At the same time, the vector pFGC5941 was digested with Asc I (TaKaRa). After recovering the PCR product and the digestion product, the target fragment was ligated to the vector fragment using homologous recombination.

[0125] Primer (5) is the upstream primer Y4604 that introduces the Asc I restriction site:

[0126] 5'-ACCATGGGGCGCGCCCTGAAACTACCACCACCAA-3';

[0127] Primer 6 is the downstream primer Y4605 that introduces the Asc I restriction site:

[0128] 5'-TCATCGATTGGGCGCGCCGGCCGTTCTTGTCCTTGTC-3';

[0129] The recovery method, homologous recombination method, and plasmid transformation method for E. coli are the same as those described above.

[0130] PCR detection using pFGC5941-MsCML50-1 plasmid as a template and primers (5) and (6) amplified a fragment of the correct size, with a band size of approximately 200 bp. This indicates that the recombinant vector contains the 200 bp MsCML50 gene. Further sequencing of the inserted fragment showed that its sequence was completely identical to the MsCML50 coding region, and the restriction enzyme sites at both ends of the inserted fragment were also completely correct. This confirms the successful construction of a correct vector plasmid containing the positive strand fragment, named pFGC5941-MsCML50-1.

[0131] Based on the ligation rules and methods of interference vectors, amplification was further designed with double restriction sites of Xba I (TaKaRa) and BamHI (TaKaRa). The MsCML50 gene fragment obtained in Example 1 was used as a template for PCR amplification to obtain antisense homologous arms. At the same time, the pFGC5941-MsCML50-1 vector obtained in the above steps was double-digested with Xba I (TaKaRa) and BamHI (TaKaRa). After recovering the PCR product and the digestion product, the target fragment was ligated to the vector fragment using homologous recombination.

[0132] Primer 7 is the downstream primer Y4606 that introduces the Xba I restriction site:

[0133] 5'-CTTAATTAACTCTCTAGACTGAAACTACCACCACCAA-3';

[0134] Primer (8) is the downstream primer Y4607 that introduces the BamHI restriction site:

[0135] 5'-TTGCAGGTATTTGGATCCGGCCGTTCTTGTCCTTGTC-3';

[0136] The recovery method, homologous recombination method, and plasmid transformation method for E. coli are the same as those described above.

[0137] PCR detection, using the plasmid as a template, amplified the correctly sized fragment with a band size of approximately 200 bp using primers (7) and (8), indicating that the recombinant vector contained the 200 bp MsCML50 gene. Further sequencing of the insert fragment showed that its sequence was completely identical to the MsCML50 coding region, and the restriction enzyme sites at both ends of the insert fragment were also completely correct. This proved the successful construction of a correct interference vector plasmid containing both sense and antisense strands, forming a hairpin structure, named pFGC5941-MsCML50. Figure 4 ).

[0138] Example 3: Subcellular localization of MsCML50

[0139] Based on the amplified CDS sequence of MsCML50, the stop codon-removed MsCML50 CDS sequence was constructed into the pCAMBIA1305.1 vector. The MsCML50 sequence was fused with GFP, and Agrobacterium-mediated transformation of Nicotiana benthamiana was performed for transient expression in tobacco. The specific steps are as follows:

[0140] Primer ⒀ is the upstream primer Y4805 that introduces the Xba I restriction site:

[0141] 5'-TCCGGAGCTAGCTCTAGAATGGCAATGGCAACC-3';

[0142] Primer 14 is the upstream primer Y4806 that introduces the BamHI restriction site:

[0143] 5'-CTTGCTCACCATGGATCCATTGCTATTGGCTTG-3';

[0144] (1) Inoculate newly activated Agrobacterium monoclonal containing MsCML50-GFP expression vector into YEP medium containing Rif and Kan, and culture overnight on a shaker (28℃) at 200 rpm.

[0145] (2) When the bacterial solution OD 600nm When the concentration is 0.6-0.8, centrifuge at 5000 rpm for 10 min and collect the bacterial pellet.

[0146] (3) Resuspend the bacterial culture in working solution (10mM MgCl2, 10mM MES, 200μM acetylsalicylic acid) and adjust to OD600. nm=0.1, mix equal volumes of different combinations of bacterial solutions for later use;

[0147] (4) Gently inject the substance into the underside of leaves of 1-month-old Nicotiana benthamiana using a syringe. Place the plants in a growth chamber and grow at 25°C for 48-72 hours. Observe and photograph the transformed leaves using a laser confocal scanning microscope (Zeiss LSM800, Germany).

[0148] MsCML50-GFP fluorescence was observed in both the cytoplasm and nucleus, and overlapped with the fluorescence of mCherry, a nuclear marker protein, while GFP fluorescence transformed with the control vector was observed throughout the cell. These results indicate that MsCML50 is localized in both the cytoplasm and nucleus. Figure 5 ).

[0149] Example 4: Low-Temperature Induced Expression Analysis of MsCML50

[0150] I. Obtaining alfalfa cDNA template under low temperature conditions

[0151] Alfalfa seedlings grown under natural light in a greenhouse for 40 days were selected and placed in an artificial climate chamber for low-temperature (5℃) treatment. Leaves were cut off at 0h, 1h, 2h, 6h and 12h, respectively, and placed in 2ml centrifuge tubes and then rapidly frozen in liquid nitrogen. They were then stored in a -80℃ freezer for later use.

[0152] Take out the samples stored at -80℃ for later use, add liquid nitrogen to grind the samples, extract total RNA from the leaves using TRE-Trizol reagent (TaKaRa), and perform reverse transcription using Prime Script RT reagent Kit with gDNAErase (TAKaRa) to prepare cDNA templates.

[0153] II. Design of Specific Detection Primers

[0154] The MsCML50 gene cDNA sequence was analyzed using the website https: / / sg.idtdna.com / scitools / Applications / RealTimePCR / , and primers for real-time quantitative PCR were designed.

[0155] The upstream primer for the MsCML50 gene is Y2213: 5'-TACGACAAGGACAAGAAC-3';

[0156] The downstream primer for the MsCML50 gene is Y2214: 5'-CGTCACCATCAGAATCAA-3';

[0157] The upstream primer for the Actin gene is Y3213: 5'-CCCACTGGATGTCTGTAGGTT-3';

[0158] The downstream primer for the Actin gene, Y3214: 5'-AGAATTAAGTAGCAGCGCAAA-3';

[0159] III. Real-time quantitative PCR detection of MsCML50 gene expression

[0160] The template cDNA prepared in step one was diluted 30-fold and used as the template for real-time quantitative PCR. The reaction system was 10 μL: 5 μL ChamQ Universal SYBR qPCR Master Mix (2×), 0.2 μL each of upstream and downstream primers (10 μM), 4 μL cDNA template, and 0.6 μL sterile water. The instrument used was a TaKaRa Thermal Cycler Dice™ RealTime System. The PCR reaction conditions were set as follows: 95℃ for 30 s; 94℃ for 10 s, 60℃ for 60 s, for 40 cycles. No cDNA template was used as a negative control. Each sample was set up in 3 replicates, and the housekeeping gene Actin was used as an internal reference gene. After the reaction, melting curve analysis was performed. The PCR amplification efficiency was above 95%, and the relative expression level of the gene was calculated.

[0161] The results of real-time quantitative PCR showed that MsCML50 was induced after 12 hours of low-temperature treatment, and its expression level increased significantly, indicating that low temperature induces the expression of the MsCML50 gene. Figure 6 ).

[0162] Example 5: Tissue-specific expression analysis of MsCML50

[0163] I. Obtaining the MsCML50 gene promoter fragment

[0164] First, the sequence of the MsCML50 upstream promoter, approximately 2000 bp in length, was obtained using TBtools software. Based on this sequence, upstream primer (9) and downstream primer (10) were designed to amplify the promoter. Using Xinjiang bigleaf DNA as a template, high-fidelity enzyme (KOD) was used for PCR amplification.

[0165] Primer 9 is the upstream primer Y4800 for amplifying the upstream promoter of MsCML50.

[0166] 5'-CCATTTTGGTCCTTGAATGTGCAAC-3';

[0167] Primer 10 is for the upstream promoter of MsCML50 to amplify the downstream primer Y4801:

[0168] 5'-TGTTTGATTGAGAGAGAGAAGAGGA-3'

[0169] PCR reaction system: KOD FX polymerase (TOYOBO, 1 U / μL) -1 )1μL, 2×PCR buffer for KODFX 25μL, dNTPs (2mmol·L -1 10 μL of upstream primer (0.3 μmol·L⁻¹) -1 1.5 μL of the primer and 0.3 μmol·L⁻¹ of the downstream primer. -1 1.5 μL of Xinjiang bigleaf DNA, and bring the total volume to 50 μL with deionized water;

[0170] PCR reaction program: 94℃ for 2 min; 98℃ for 0.1 min, 68℃ for 1 min, 35 cycles; 72℃ for 10 min; the amplified products were detected by agarose gel electrophoresis at a mass fraction of 0.8%. Figure 7 );

[0171] A sequence approximately 1857 bp upstream of MsCML50 was obtained. Figure 7 The obtained PCR products were recovered using a DNA gel recovery kit (AXYGEN).

[0172] The nucleotide sequence of the calmodulin CML50 promoter sequence of alfalfa is shown in SEQ ID NO.3; the analysis of the promoter elements of the MsCML50 gene is shown in Table 1.

[0173] Table 1The Promoter cis-acting elements of MsCML50

[0174]

[0175] II. P MsCML50 Construction of GUS expression vector

[0176] Based on the promoter sequence upstream of MsCML50 obtained in step one, a homologous arm amplification was performed using EcoRI and NcoI restriction sites. Simultaneously, the pFGC5941 expression vector was double-digested with restriction endonucleases EcoRI (TaKaRa) and NcoI (TaKaRa). The recovered p:MsCML50 promoter homologous arm amplification target fragment and the digested vector fragment were then ligated using Clon Express II recombination reaction solution at a molar ratio of 1:2. The reaction system was as follows: 5×CEII Buffer 4 μL, Exnase II 2 μL, recovered target fragment 0.06 pmol, deionized water added to 20 μL, ligation at 37℃ for 30 min, followed by cooling on ice to obtain the ligation product. The recombination reaction solution was transformed into DH5α, single clones were picked and sent for sequencing, and the correctly sequenced bacterial cultures and plasmids were preserved. Figure 8 The recombinant expression vector pFGC5941-P:MsCML50 was further introduced into Agrobacterium tumefaciens EHA105 using electroporation transformation.

[0177] Primer 11 is the upstream primer Y4803 for introducing the EcoRI restriction site:

[0178] 5'-TATGACCATGATTACGAATTCCCATTTTGGTCCTTGAATGTGCAAC-3';

[0179] Primer 12 is the downstream primer Y4804 that introduces the Nco I restriction site:

[0180] 5'-AATTTACCCTCAGATCTACCATGGTGTTTGATTGAGAGAGAGAAGAGGA-3'.

[0181] III. Genetic transformation to obtain transgenic Arabidopsis thaliana with GUS driven by the MsCML50 promoter.

[0182] (1) Streak the preserved Agrobacterium EHA105 onto YEP medium containing kanamycin and rifampin resistance, and incubate at 28°C for two days; pick single colonies and add them to YEP liquid medium, and incubate at 28°C and 200 rpm. One day before transformation, add the bacterial culture to 50 mL of YEP liquid medium at a ratio of 1:50, and incubate in the dark at 28°C and 200 rpm until OD600. nm =0.6-0.8.

[0183] (2) Centrifuge at 4℃ and 5000rpm for 15min and remove the supernatant.

[0184] (3) Resuspend the precipitate in a 5% (g / 100ml) sucrose solution (containing 0.03% silwetl-77, v / v) to achieve an OD600 of 0.03%. nm =0.6-0.8, the sucrose solution needs to be prepared fresh for use and does not require sterilization.

[0185] (4) Immerse the healthy Arabidopsis thaliana flower buds in the resuspension solution while gently rotating the Arabidopsis thaliana plant. This step should be done in a way that avoids the conversion solution from entering the substrate to prevent damage to the Arabidopsis thaliana root system.

[0186] (5) Cover the plant with plastic wrap to keep it moist, and place it in the dark for 12 hours before restoring it to normal growth conditions.

[0187] (6) Homozygous MsCML50 promoter transgenic Arabidopsis was obtained by screening with glufosinate and PCR identification.

[0188] IV. Obtaining transgenic Arabidopsis thaliana with homozygous MsCML50 promoter and performing GUS staining.

[0189] Preparation of GUS staining:

[0190] (1) X-Gluc stock solution (20mM): X-Gluc powder was dissolved in NN-dimethylamide (DMF), 100μL per tube, and stored at -20℃ for later use;

[0191] (2) X-Gluc base solution (50mM PBS, pH 7.0): Add 0.78g NaH2PO4, 16.5mg K3[Fe(CN)6] (potassium ferricyanide), 1.79g Na2HPO4, 100uL Triton-100, 0.372g Na2EDTA, 21.1mg K4[Fe(CN)6] (potassium ferrocyanide) and 80mL to a beaker and mix well. Dilute to 100mL with distilled water using a graduated cylinder and store at 4℃ for later use.

[0192] (3) Preparation of staining solution: 50uL X-Gluc stock solution + 450uL X-Gluc base solution (prepare fresh for use). Place transgenic Arabidopsis seedlings, stems, leaves, flowers, and pods into the GUS staining solution and vacuum for 10 minutes. Place at 37℃ in the dark for 2-24 hours until the color changes, then discard the staining solution. Decolorize with 70% ethanol 3-5 times and take pictures using a stereomicroscope.

[0193] Using the Actin gene from alfalfa as an internal control, quantitative PCR was performed to detect the expression of the MsCML50 gene in different tissues of alfalfa. The quantitative results showed that MsCML50 expression was lowest in alfalfa stems and higher in flowers. Simultaneously, some expression was observed in roots and stems, indicating a constitutive expression distribution of MsCML50 in alfalfa. Figure 9 A). Meanwhile, homozygous transgenic Arabidopsis lines with GUS driven by the MsCML50 promoter were obtained. GUS staining results showed high GUS activity in the roots of seedlings, and also in the leaf veins, stems, and anthers of mature flowers of mature plants, while expression was weak in other tissues. These results indicate that MsCML50 is mainly expressed in plant flowers, leaves, and roots. Figure 9 B).

[0194] Example 6: Production and Molecular Detection of Transgenic Alfalfa

[0195] I. The Development of Genetically Modified Alfalfa

[0196] 1. The recombinant expression vector pCAMBIA3301-MsCML50 and the interference vector pFGC5941-MsCML50 were introduced into Agrobacterium tumefaciens EHA105.

[0197] The preparation of EHA105 competent cells was based on the method of J. Sambrook (Huang Peitang, Wang Jiaxi, Zhu Houchu. J. Sambrook, D.W. Russell, author [J]. Molecular Cloning: A Laboratory Manual, 2002: 27-30) with modifications: EHA105 bacterial culture was streaked on YEP+Kan+Rif plates and incubated at 28℃ for 48 h. Single colonies were picked and inoculated into 50 mL of liquid LB and incubated overnight at 28℃. 0.5 mL of the bacterial culture was then inoculated into 500 mL of liquid YEP and incubated at 28℃ for 8 h until OD reached the target value. 600nmThe solution was set to 0.6, cooled in an ice bath for 10 min, poured into a sterile 200 mL centrifuge tube, and centrifuged at 4000 rpm for 10 min at 4°C after equilibration. The cells were collected, the LB solution was discarded, and the tube was placed upside down on a sterile paper towel to drain. 50 mL of pre-chilled 10% glycerol (glycerol:YEP liquid medium) was added, and the cells were shaken on ice to suspend them. The cells were centrifuged at 4000 rpm for 15 min at 4°C to collect them. The cells were washed once more, and 2 mL of pre-chilled 10% glycerol (glycerol:YEP liquid medium) was added to suspend them. The cells were aliquoted into 25 μL / tube, flash-frozen in liquid nitrogen, and stored at -80°C. Thaw competent cells on ice. Pre-cool 0.2 cm inner diameter electroporation cuvettes on ice. In a clean bench, add 1.5 μL of pCAMBIA3301-MsCML50 plasmid and pHSE401-MsCML50 (20 ng / μL) to 20 μL of thawed competent cells. Gently tap the tube wall to mix. After incubating on ice for 1 min, transfer to an electroporation cuvette and place it between the electrodes of an electroporator (MicroPulser, Bio-RAD). Select the Agr program and perform electroporation. After electroporation, quickly pour 1 mL of YEP liquid culture medium into the electroporation cuvette in the clean bench, then transfer it to a shaker tube using a pipette. Incubate at 28°C with gentle shaking for 2 h. Take 0.3 mL of the bacterial culture and spread it on a YEP plate (containing 35 mg / L rifampin and 50 mg / L kanamycin). Incubate upside down in a 28°C incubator for 48 h.

[0198] 2. Identification of Agrobacterium-positive colonies containing recombinant expression vector pCAMBIA3301-MsCML50 and interference vector pFGC5941-MsCML50

[0199] Single colonies were picked from the plates for colony PCR detection and labeled. PCR-positive colonies were then picked and added to 3 mL of YEB liquid medium (containing 35 mg / L rifampin and 50 mg / L kanamycin) and incubated at 28°C with shaking for 40 h. 2 mL of the bacterial culture was used to extract plasmids using the alkaline lysis method. The expression vector pCAMBIA3301-MsCML50 was digested with restriction endonucleases Nco I (TaKaRa) and BstE II (TaKaRa) to confirm the presence of the plant expression vector pCAMBIA3301-MsCML50 in the positive clones. The expression vector pFGC5941-MsCML50 was digested with restriction endonucleases Xba I (TaKaRa) and BamH... I (TaKaRa) was used for enzyme digestion detection to confirm that the positive clone contained the plant expression vector pFGC5941-MsCML50; 0.8 mL of Agrobacterium tumefaciens solution was taken, 0.2 mL of 50% glycerol (glycerol: YEB liquid medium) was added, mixed well and stored at -80℃ for later use.

[0200] 3. Obtaining transgenic alfalfa plants

[0201] (1) Genetic transformation and identification of alfalfa

[0202] The plasmids of the successfully constructed plant overexpression vectors pCAMBIA3301-MsCML50 and pFGC5941-MsCML50 and interference vectors were transformed into Agrobacterium. Positive clones were screened for further genetic transformation of plants. The screening marker was glufosinate.

[0203] Take leaves of alfalfa that have grown for about 40 days, wash off surface impurities with tap water, disinfect the surface with 6.25% NaClO for 30 minutes, and wash with sterile distilled water 3 to 5 times to remove NaClO.

[0204] (2) Activation and suspension of Agrobacterium

[0205] Agrobacterium EHA105 stock solution containing recombinant expression vector pCAMBIA3301-MsCML50 and interference vector pFGC5941-MsCML50 was streaked onto YEP plates (containing 35 mg / L rifampin and 50 mg / L kanamycin) and incubated at 28°C. Well-isolated single colonies were picked and inoculated into 2 mL of liquid YEB (containing 35 mg / L rifampin and 50 mg / L kanamycin) and incubated in the dark with shaking at 28°C for 24 h. 20 μL of the bacterial culture was spread onto YEP plates containing the same antibiotics and incubated upside down in the dark at 28°C for 36 h. Newly grown Agrobacterium were scraped off, resuspended and diluted with YEP liquid medium to allow OD to rise. 600nm It ranges from 0.6 to 0.8;

[0206] (3) Infection, co-culture and the production of transgenic seedlings

[0207] Inside a clean bench, cut off the petioles and leaf margins, and cut the leaves into small pieces for transformation experiments. After cutting off the petioles and leaf margins, transfer the pieces to the infection solution, mix thoroughly, and vacuum for 10 minutes; sonicate at 40 kHz for 3-5 minutes (water temperature should be below 20℃), then vacuum for another 10 minutes, at 28℃, 50-60 rpm, for 1.5 hours, keeping it in the dark; remove the leaves, remove as much Agrobacterium as possible from the surface of the slices on sterile filter paper, and then transfer them to SM4 solid medium containing 100 uM acetylsylgenone, ensuring the leaf surface is facing up, and co-culture in the dark for 2 days; after co-culturing, transfer the leaves to SM4 solid medium containing glufosinate, timetin, and cefotaxime (ensuring the leaf surface is facing up), and culture under light for 4-5 weeks, rotating the plate every 2 weeks. Once the callus tissue matures, it is transferred to MSBK selective medium containing glufosinate, timetin, and cefotaxime for 2 weeks, and then transferred to 1 / 2 MS medium for approximately 6-8 weeks, at which point the embryo begins to develop into small plantlets. The small plantlets are then transferred to tissue culture flasks containing 1 / 2 MS medium for 2-3 weeks for rooting culture.

[0208] (4) Growth of transgenic alfalfa

[0209] Once rooting is complete, transfer the plants to a mixed nutrient soil and cultivate them under greenhouse conditions. After the regenerated seedlings have grown 6-7 leaves, DNA can be extracted from the leaves for positive seedling identification. Seedlings originating from the same callus tissue are considered to have undergone the same transformation event. Positive seedlings are harvested and propagated by cuttings for phenotypic verification.

[0210] PCR detection of transgenic alfalfa

[0211] 1. PCR detection of transgenic alfalfa

[0212] Leaves were cut from transgenic alfalfa seedlings that had grown under natural light in a greenhouse for 30 days. The leaves were placed in 2ml centrifuge tubes and then quickly frozen in liquid nitrogen. They were then stored at -80℃ for later use.

[0213] Take out the samples stored at -80℃ for later use, add liquid nitrogen to grind the samples, extract total RNA from the leaves using TRE-Trizol reagent (TaKaRa), and perform reverse transcription using Prime Script RT reagent Kit with gDNAErase (TAKaRa) to prepare cDNA templates.

[0214] 2. Design specific detection primers

[0215] The MsCML50 gene cDNA sequence was analyzed using the website https: / / sg.idtdna.com / scitools / Applications / RealTimePCR / , and primers for real-time quantitative PCR were designed.

[0216] The upstream primer for the MsCML50 gene is Y2213: 5'-TACGACAAGGACAAGAAC-3';

[0217] The downstream primer for the MsCML50 gene is Y2214: 5'-CGTCACCATCAGAATCAA-3';

[0218] The upstream primer for the Actin gene is Y3213: 5'-CCCACTGGATGTCTGTAGGTT-3';

[0219] The downstream primer for the Actin gene, Y3214: 5'-AGAATTAAGTAGCAGCGCAAA-3';

[0220] The template cDNA prepared in step one was diluted 30-fold and used as the template for real-time quantitative PCR. The reaction system was 10 μL: 5 μL ChamQ Universal SYBR qPCR Master Mix (2×), 0.2 μL each of upstream and downstream primers (10 μM), 4 μL cDNA template, and 0.6 μL sterile water. The instrument used was a TaKaRa Thermal Cycler Dice™ RealTime System. The PCR reaction conditions were set as follows: 95℃ for 30 s; 94℃ for 10 s, 60℃ for 60 s, for 40 cycles. No cDNA template was used as a negative control. Each sample was set up in 3 replicates, and the housekeeping gene Actin was used as an internal reference gene. After the reaction, melting curve analysis was performed. The PCR amplification efficiency was above 95%, and the relative expression level of the gene was calculated.

[0221] in, Figure 10 This is a real-time quantitative PCR identification result of transgenic alfalfa overexpressing the MsCML50 gene; WT indicates wild type; OE indicates positive line. Figure 11 This is a real-time quantitative PCR identification result of the interference transgenic alfalfa with the MsCML50 gene introduced. WT represents wild type; RNAi represents positive lines.

[0222] Example 7 Identification of Low-Temperature Semi-Lethality of Transgenic Alfalfa

[0223] I. Low-Temperature Half-Lethality Determination

[0224] Transgenic alfalfa (OE5, OE6, RNAi3, RNAi7) and its wild-type S-type plants (WT) that have been grown in a plant incubator for 40 days were selected, and the relative conductivity (Ion leakage) of the second-to-last leaf was collected.

[0225] The determination method is as follows:

[0226] Take alfalfa leaves, rinse them clean, blot the surface moisture with filter paper, and trim off any excess petioles. Place three single leaves into the same glass test tube (pre-cooled on ice), and add a small piece of crushed ice to each test tube. Each sample should have three replicates. Place the test tubes in a low-temperature freezing cycler, starting at 0℃ (-2℃ after cold acclimatization), and then decrease the temperature by 2℃ / h for 1 hour at each temperature. Set five temperatures for each treatment. Remove the test tube at each 2℃ drop and thaw it overnight at 4℃. Add 6 mL of deionized water to each test tube (the conductivity of the water is C0), place it on a shaker at 25℃ for 4 hours, and then measure the conductivity C1. Place the test tubes in a boiling water bath for 40 minutes, cool to room temperature, and measure the conductivity C2. Calculate the relative conductivity using the formula.

[0227] from Figure 12 As shown in Figure A, after 7 days of low-temperature treatment, the results indicated that, compared to the wild-type (WT), both overexpressing transgenic lines exhibited lower TEL50 values ​​under both non-acclimation (NA) and acclimation (CA) conditions, while the RNAi lines showed higher TEL50 values ​​under both NA and CA conditions. Simultaneously, the TEL50 of the non-acclimated (NA) plants was significantly higher than that of all acclimated (CA) plants. Figure 12 A) This indicates that cold acclimatization improved the cold tolerance of alfalfa, and overexpression of MsCML50 also led to enhanced cold tolerance in alfalfa. This suggests that under low-temperature stress, transgenic alfalfa exhibited improved cold tolerance compared to wild-type plants.

[0228] II. Survival Rate Determination

[0229] The survival rate of transgenic alfalfa and its wild-type R108 plants grown in a plant culture box for 40 days was determined.

[0230] The determination method is as follows:

[0231] The alfalfa freeze-treatment experiment mainly followed the method of Pennycooke et al. (2008), with modifications based on the growth status of the plant materials. Alfalfa cuttings were placed in 7 cm diameter plastic pots containing a mixed nutrient soil (peat:vermiculite:perlite = 3:1:1 (v / v)) and grown for approximately 40 days in a long-day (16 h light, 8 h dark) incubator at 24°C. They were then placed in a -5°C low-temperature incubator for 1 hour, followed by overnight storage at 4°C, and then allowed to recover in a long-day incubator at 22°C. Survival rates were measured after 7 days. Alfalfa requiring cold acclimatization was moved to a long-day, 4°C incubator for one week after approximately 33 days of growth, followed by 6 hours in a -7°C low-temperature incubator. Subsequent procedures were the same as for non-cold-acclimatized materials. The survival rate formula is as follows: Survival rate = (Number of surviving plants / Total number of plants) * 100%

[0232] The result is as follows Figure 12 As shown in Figure B, the survival rate of RNAi lines under both NA and CA conditions was significantly lower than that of wild-type lines, while the survival rate of overexpression lines was higher than that of wild-type lines. Cold acclimatization (CA) treatment improved the survival rate of plants of all genotypes. These results indicate that MsCML50 positively regulates the cold tolerance of alfalfa.

[0233] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0234] sequence list

[0235] SEQ ID NO.1

[0236] ATGGCAACCAATCCAAACACTGAAACTACCACCACCAAATCCTCTGTTTACCTTGGTGACATGGACGAATTGAAGACCGTGTTCACCCGCTTTGACACCAACGGCGACGGCAAGATCTCCGTCACCGAGTTGGACAACATCCTCCGCTCCCTCGGATCCACAGTCCCTAAGGACGAGCTTCAGCGTGTCATGGAAGACCTTGACACCGACCACGACGGTTTCATCAACCTCGCTGAGTTCGCCGCCTTCTGTCGCTCTGGCTCCGCCGACGGTGATGCTTCAGAGCTTCGCGAAGCCTTTGATCTGTACGACAAGGACAAGAACGGCCTCATATCTGCAACAGAACTCTGTCAGGTGCTGAACACCCTCGGAATGAAGTGCTCCGTTGAAGAATGCCACAACATGATTAAATCCGTTGATTCTGATGGTGACGGTAACGTTAACTTTGAAGAGTTTAAGAAGATGATGAACAATAATAATAATAATCAAGCCAATAGCAATTAG

[0237] SEQ ID NO.2

[0238] MATNPNTETTTTKSSVYLGDMDELKTVFTRFDTNGDGKISVTELDNILRSLGSTVPKDELQRVMEDLDTDHDGFINLAEFAAFCRSGSADGDASELREAFDLYDKDKNGLISATELCQVLNTLGMKCSVEECHNMIKSVDSDGDGNVNFEEFKKMMNNNNNNQANSN

[0239] SEQ ID NO.3

[0240]

Claims

1. Application of the calmodulin CML50 gene of alfalfa with nucleotide sequence as shown in SEQ ID NO.1 and the protein it encodes in improving the cold resistance of alfalfa or in breeding transgenic alfalfa with improved cold resistance.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The application of biomaterials containing the calmodulin CML50 gene of alfalfa, with the nucleotide sequence shown in SEQ ID NO.1, in improving the cold resistance of alfalfa or in breeding transgenic alfalfa with improved cold resistance, characterized in that, The biological material is at least one of a recombinant vector, expression cassette, cell line, and host bacteria.

4. The application according to claim 1 or 3, characterized in that, Overexpression of the alfalfa calmodulin CML50 gene in target alfalfa improves the cold resistance of alfalfa.

5. A method for improving the cold resistance of alfalfa, characterized in that, Overexpression of the calmodulin CML50 gene, with the nucleotide sequence shown in SEQ ID NO.1, in target alfalfa improves the cold resistance of alfalfa.

6. The method according to claim 5, characterized in that, The process of overexpressing the alfalfa calmodulin CML50 gene in the target alfalfa is as follows: an overexpression vector of the alfalfa calmodulin CML50 gene is constructed, and the overexpression vector is transferred into the target alfalfa by Agrobacterium-mediated transformation to obtain transgenic plants with improved cold resistance.

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