Application of alfalfa FFT2 gene in cultivation of cold-resistant plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and provides application of an alfalfa FTa2 gene in cultivation of cold-resistant plants, and the nucleotide sequence of the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3. The invention further provides a preparation method of the alfalfa FTa2 gene. The alfalfa cold resistance related gene FFT2 is found, the alfalfa cold resistance related gene FFT2 negatively regulates the cold resistance of alfalfa, the FFT2 is knocked out or knocked down through a genome editing technology, the cold resistance of plants can be remarkably improved, and particularly, the survival rate of the plants after freezing treatment is increased, and the relative conductivity of leaves is reduced. The invention provides important normal forms and gene resources for cultivating cold-resistant plant varieties.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering technology. Specifically, this application provides the application of the alfalfa FTa2 gene in the cultivation of cold-resistant plants. Background Technology

[0002] Plants are subject to various biotic and abiotic stresses during their growth and development. In recent years, with the changes in global climate, the resulting drastic temperature fluctuations have significantly inhibited plant growth and threatened crop productivity. Among these, low-temperature stress is a serious natural disaster that not only affects plant growth, development, yield, and quality, but also limits the geographical distribution of many plant species.

[0003] In-depth research into cold-resistance-related genes in plants can help elucidate the mechanisms by which plants resist low-temperature stress. Furthermore, by using plant genetic engineering to modify plants, we can further enhance their tolerance to low-temperature stress, reduce the dependence of food on the environment, and thus increase food yields under low-temperature conditions, benefiting humankind.

[0004] Alfalfa is the world's fourth most important economic crop and an important source of feed. Discovering the key genes in alfalfa that resist low temperatures can provide genetic resources for breeding cold-resistant plant varieties. Summary of the Invention

[0005] On the one hand, this application provides the application of the alfalfa FTa2 gene in the cultivation of cold-resistant plants, wherein the nucleotide sequence of the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3.

[0006] Furthermore, in the application, knocking out or down the alfalfa FTa2 gene in the plant, or reducing the level or activity of the protein encoded by the alfalfa FTa2 gene in the plant, can achieve one or more effects selected from (A1)-(A3):

[0007] (A1) Reduce the relative electrical conductivity of the blades;

[0008] (A2) Improve the survival rate of plants under low temperature conditions;

[0009] (A3) Improve the growth status of plants at low temperatures.

[0010] Furthermore, the application uses one or more tools selected from (B1)-(B5) to knock out or knock down the alfalfa FTa2 gene in the plant, or to reduce the level or activity of the protein encoded by the alfalfa FTa2 gene in the plant:

[0011] (B1) A tool based on the CRISPR-Cas9 method; preferably, the tool based on the CRISPR-Cas9 method introduces a mutation through gene editing to cause premature termination of translation of the alfalfa FTa2 gene; more preferably, the target sites in the gene editing are SEQ ID NO.5 and SEQ ID NO.6; or SEQ ID NO.13 and SEQ ID NO.14;

[0012] (B2) Tools based on homologous recombination methods;

[0013] (B3) Tools based on the ZFN method;

[0014] (B4) The RNA interference tool for the alfalfa FTa2 gene; preferably, siRNA, miRNA, shRNA, or dsRNA;

[0015] (B5) The ASO of the alfalfa FTa2 gene;

[0016] (B6) Use an inhibitor of the protein encoded by the alfalfa FTa2 gene, preferably an antibody.

[0017] The tools described above can be conventionally designed, obtained, and modified by those skilled in the art using known tools, and can be carried using carriers known in the art.

[0018] Furthermore, the application is used for one or more of (C1)-(C3).

[0019] (C1) Improve the cold resistance of plant plants;

[0020] (C2) Provide plant propagation materials with improved cold resistance; preferably seeds, tissue culture materials, and organs that can be used for propagation;

[0021] (C3) Provides plant strains with improved cold resistance.

[0022] Furthermore, the plant in question is a plant of the genus *Alfalfa*.

[0023] Furthermore, the plant is alfalfa or trichome.

[0024] On the other hand, this application provides a method for improving the cold resistance of plants, the method comprising knocking out or knocking down the alfalfa FTa2 gene in the plant, wherein the nucleotide sequence of the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3.

[0025] On the other hand, this application provides the alfalfa FTa2 gene, the nucleotide sequence of which is SEQ ID NO.1 or SEQ ID NO.3.

[0026] On the other hand, this application provides the protein encoded by the aforementioned alfalfa FTa2 gene.

[0027] Furthermore, the amino acid sequence of the protein is SEQ ID NO.2 or SEQ ID NO.4.

[0028] On the other hand, this application provides the application of any one of the tools (D1)-(D3) for detecting the expression level of the alfalfa FTa2 gene or the expression level of the protein encoded by the alfalfa FTa2 gene:

[0029] (D1) Determine the cold resistance of alfalfa plants;

[0030] (D2) Predicting the cold resistance of alfalfa propagation material;

[0031] (D3) Breed cold-resistant alfalfa varieties;

[0032] The tool for detecting the expression level of the alfalfa FTa2 gene or the expression level of the protein encoded by the alfalfa FTa2 gene is one or more of the following:

[0033] (E1) Primer;

[0034] (E2) Probe;

[0035] (E3) PCR reagents;

[0036] (E4) chip;

[0037] (E5) Antibody;

[0038] (E6) ELISA reagent;

[0039] The nucleotide sequence of the FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3; the nucleotide sequence of the protein encoded by the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3.

[0040] The tools described above can be conventionally designed, obtained, and modified by those skilled in the art using known tools, and can be used in conjunction with other self-made or commercially available reagents / kits, such as PCR kits, to achieve detection.

[0041] The low temperature range described in this application includes, but is not limited to, below 8°C, below 4°C, and below 0°C.

[0042] This application identifies the alfalfa cold resistance-related gene FTa2, which negatively regulates alfalfa cold resistance. Knocking out or downregulating FTa2 using genome editing technology significantly improves plant cold resistance, specifically by increasing plant survival rate after freezing treatment and decreasing leaf relative electrical conductivity. Conversely, overexpressing FTa2 using a constitutive promoter weakens plant cold resistance, specifically by decreasing plant survival rate after freezing treatment and increasing leaf relative electrical conductivity. This invention successfully obtained alfalfa with significantly enhanced cold resistance by knocking out FTa2 using genome editing technology, providing an important paradigm and gene resource for breeding cold-resistant plant varieties. Attached Figure Description

[0043] Figure 1 This is a structural diagram of the gene editing binary vector p6401-MtFTa2.

[0044] Figure 2 The mutation modes of the mtfta2-20 and mtfta2-22 mutant lines are shown, with the target sequence marked in the box. In the mutant sequences, the mutation mode is described at the end of the sequence, where "-" indicates a deleted base and lowercase letters indicate an inserted base.

[0045] Figure 3 This image shows the phenotype of the mtfta2 mutant line under low-temperature stress, as well as statistical graphs of survival rate and relative conductivity, with three independent biological replicates. Part A presents morphological photographs of the wild-type Alfalfa R108 and the mtfta2 mutant lines before and after low-temperature stress. Part B is a schematic diagram of the survival status of the plants in Part A after low-temperature stress. × represents surviving plants, and × represents dead plants. Part C is a statistical graph showing the survival rate of wild-type R108 and mtfta2 mutant lines of alfalfa after low-temperature stress. Part D is a statistical graph showing the relative conductivity of wild-type R108 and mtfta2 mutant lines of alfalfa after low-temperature stress.

[0046] Figure 4 This is a structural diagram of the gene editing binary vector p6401-MsFTa2.

[0047] Figure 5 The image shows the PCR results for transgenic identification of the msfta2 mutant line of alfalfa. Wild-type alfalfa ZM1 is the negative control, msfta2-9 is the transgenic negative line, and M represents the standard molecular weight of DNA.

[0048] Figure 6 The mutation modes of the msfta2-5 and msfta2-34 mutant lines are shown, with the target sequences marked in boxes. In the mutant sequences, the mutation mode is described at the end of the sequence, where "-" indicates a deleted base.

[0049] Figure 7 This image shows the phenotype of the msfta2 mutant lines under low-temperature stress, as well as statistical graphs of survival rate and relative conductivity, with three independent biological replicates. Part A contains morphological photographs of the control group (control group-1) and the msfta2 mutant lines before and after low-temperature stress. Part B is a schematic diagram of the survival status of the plants in Part A after low-temperature stress. × represents surviving plants, and × represents dead plants. Part C is a statistical graph showing the survival rate of the control group (control group-1) and the msfta2 mutant line after low-temperature stress. Part D is a statistical graph showing the relative conductivity of the control group (control group-1) and the msfta2 mutant line after low-temperature stress.

[0050] Figure 8 This figure shows the transgenic identification PCR results of regenerated plants (MtFTa2 OE-1 to MtFTa2 OE-10) obtained by Agrobacterium tumefaciens transformation into the pCAMBIA1307-MtFTa2 vector. In the figure, wild-type alfalfa R108 is the negative control, pCAMBIA1307-MtFTa2 plasmid (P) is the positive control, and M is the standard molecular weight of DNA.

[0051] Figure 9 To detect MtFTa2 protein in MtFTa2 overexpression lines (MtFTa2 OE-1 to MtFTa2 OE-10) using Western blotting. In the figure, wild-type Alfalfa R108 serves as the negative control, Anti-FLAG is the antibody for detecting MtFTa2 protein, and Anti-β-actin is the antibody for detecting the internal reference protein β-actin.

[0052] Figure 10 This image shows the phenotypes of MtFTa2-overexpressing lines under low-temperature stress, as well as statistical graphs of survival rate and relative electrical conductivity, with three independent biological replicates. Part A presents morphological photographs of wild-type Alfalfa R108 and MtFTa2-overexpressing lines before and after low-temperature stress. Part B is a schematic diagram of the survival status of plants from Part A after low-temperature stress. × represents surviving plants, and × represents dead plants. Part C is a statistical graph showing the survival rate of wild-type R108 and MtFTa2 overexpressing lines of alfalfa after low-temperature stress. Part D is a statistical graph showing the relative conductivity of wild-type R108 and MtFTa2 overexpressing lines of alfalfa after low-temperature stress.

[0053] Figure 11 The figures show the transgenic identification PCR results of regenerated plants (MsFTa2 OE-1 to MsFTa2 OE-13) obtained by Agrobacterium tumefaciens transformation into the pCAMBIA1307-MsFTa2 vector. Wild-type alfalfa ZM1 is the negative control, and M represents the standard molecular weight of DNA.

[0054] Figure 12 To detect MsFTa2 protein in MsFTa2 overexpressing lines (MsFTa2 OE-1, MsFTa2 OE-6, MsFTa2 OE-11, and MsFTa2 OE-12) using Western blotting. In the figure, wild-type alfalfa ZM1 serves as the negative control, MsFTa2 OE-12 is the transgenic negative line, Anti-FLAG is the antibody for detecting MsFTa2 protein, and Anti-β-actin is the antibody for detecting the internal reference protein β-actin.

[0055] Figure 13 This image shows the phenotypes, survival rates, and relative electrical conductivity of MsFTa2-overexpressing lines under low-temperature stress, with three independent biological replicates. Part A contains morphological photographs of the control group (control group-2) and the MsFTa2-overexpressing lines before and after low-temperature stress. Part B is a schematic diagram of the survival status of the plants in Part A after low-temperature stress. × represents surviving plants, and × represents dead plants. Part C is a statistical graph showing the survival rate of the control group (control group-2) and the MsFTa2 overexpressing lines after low temperature stress. Part D is a statistical graph showing the relative conductivity of the control group (control group-2) and the MsFTa2 overexpressing lines after low temperature stress. Detailed Implementation

[0056] Main methods and reagents

[0057] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. In the examples, Prism10 software was used for Student's t-test significance analysis. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and ns represents no significant difference.

[0058] Unless otherwise specified, alternation of light and dark means 16 hours of light / 8 hours of darkness.

[0059] Culture medium: It is prepared by mixing 5 parts vermiculite and 2 parts perlite.

[0060] The wild-type alfalfa seeds used in the examples are alfalfa R108 seeds. The wild-type alfalfa plants in the examples refer to alfalfa R108 plants. The wild-type alfalfa plants in the examples refer to alfalfa plant No. 1 (represented by ZM1).

[0061] The p6401 vector and p5CBC are described in the following literature: Zhu F, Ye Q, Chen H, Dong J, Wang T (2021) Multigene editing reveals that MtCEP1 / 2 / 12 redundantly control lateralroot and nodule number in Medicago truncatula. J Exp Bot 72: 3661-3676.

[0062] The pCAMBIA1307 vector is described in the following literature: Zhu F, Deng J, Chen H, Liu P, Zheng L, Ye Q, Li R, Brault M, Wen J, Frugier F et al (2020) A CEP Peptide Receptor-Like Kinase Regulates Auxin Biosynthesis and Ethylene Signaling to Coordinate Root Growth and Symbiotic Nodulation in Medicago truncatula. Plant Cell 32:2855-2877.

[0063] The culture medium formulations used in the examples are as follows:

[0064] The formulation of SH3a liquid culture medium (1L) is as follows: 100mL of 10×N6 large volume stock solution, 1mL of 1000×SH micro volume stock solution, 1mL of 1000×SH organic stock solution, 20mL of 50×EDFS iron salt stock solution, 0.4mL of 2,4-D stock solution, 0.5mL of 6-BAP stock solution, 100mg of inositol, 30g of sucrose, pH adjusted to 5.85, and the remainder is water. 3.2g of plant gel is added to the solid culture medium.

[0065] The formulation of SH9 liquid culture medium (1L) is as follows: 100mL of 10×N6 large volume stock solution, 1mL of 1000×SH micro volume stock solution, 1mL of 1000×SH organic stock solution, 20mL of 50×EDFS iron salt stock solution, 100mg of inositol, 20g of sucrose, pH adjusted to 5.85, and the remainder is water. 8g of agar is added to the solid culture medium.

[0066] The formulation of 1 / 2 MS liquid medium (1L) is as follows: 2.22g MURASHIGE & SKOOG (MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories™), 12g sucrose, pH adjusted to 5.85, and the remainder is water. 8g agar is added to the solid medium.

[0067] The formulation of SM4 liquid medium (1L) is as follows: MURASHIGE&SKOOG(MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories™) 4.43g, 2,4-D stock solution (10mg / mL) 0.4mL, 6-BAP stock solution (1mg / mL) 0.2mL, sucrose 30g, pH adjusted to 5.85. 3.2g of plant gel is added to the solid medium.

[0068] The MSBK liquid medium (1L) formulation is as follows: MURASHIGE&SKOOG(MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories™) 4.43g, kinetin (1mg / mL) 1mL, 6-BAP stock solution (1mg / mL) 0.5mL, sucrose 30g, pH adjusted to 5.85. Add 3.2g of plant gel to the solid medium.

[0069] 10×N6 large-volume mother liquor (1L): MgSO4·7H2O 1.85g, KNO3 28.3g, (NH4)2SO4 4.63g, CaCl2·2H2O 1.66g, KH2PO4 4g, H2O added to 1L.

[0070] 1000×SH micro-volume stock solution (100mL): MnSO4·H2O 1g, H3BO3 500mg, ZnSO4·7H2O 100mg, KI 100mg, Na2MoO4·2H2O 10mg, CuSO4·5H2O 20mg, CoCl2·6H2O 10mg, add H2O to 1L.

[0071] 1000×SH Organic Mother Liquor (100mL): Nicotinic acid 500mg, pyridoxine hydrochloride 500mg, thiamine hydrochloride 500mg, H2O added to 1L.

[0072] 50×EDFS iron salt mother liquor (500mL): NaFe·EDTA 3.487g, H2O added to 1L.

[0073] The MtFTa2 gene was obtained by PCR amplification using cDNA from wild-type Alfalfa R108 as a template.

[0074] MtFTa2 cds sequence (SEQ ID NO.1):

[0075] ATGGCAAGTGGTAGCA GACCGAATCCTCTTGCTGT TGGGCGTGTAATAGGGGATGTATTAGACCCCTTTGAAAGTACTATTCCTCTCTTAATCACCTATG GTAATAGGACTGTTACCAA TGGTGGTGAGCTTAAACCTTCCCAAGTTGCTAATCAACCCCAAGTGATTATTGGCGTAAATGACCCAACAGCCCTCTACACCCTGGTTTTGGTAGATCCAGATGCTCCTAGCCCCAGTTACCCCAGTTTTAGGGAGTACCTTCATTGGATGGTGACTGATATTCCAGCAACTAATGCGGCTAGTTTTGGTAATGAGGTTGTAAGTTA TGAAAAGCCACGACCCAATTTAGGGATTCATCGTTTCGTGTTTGTATTATTGCATCAAAAGTGTAGACAAAGAGTCTATGCTCCGGGATGGCGACAAAATTTCAATACAAGAGAATTCATTGAATTTTACAATCTTGGATCGCCGGTTGCTGCTGTCTTCTTCAATTGTCAAAGGGAAACTGGTTCTGGGGGAAGAACCTTTAGATGA

[0076] MtFTa2 protein sequence (SEQ ID NO.2):

[0077] MASGSRPNPLAVGRVIGDVLDPFESTIPLLITYGNRTVTNGGELKPSQVANQPQVIIGVNDPTALYTLVLVDPDAPSPSYPSFREYLHWMVTDIPATNAASFGNEVVSYEKPRPNLGIHRFVFVLLHQKCRQRVYAPGWRQNFNTREFIEFYNLGSPVAAVFFNCQRETGSGGRTFR

[0078] The MsFTa2 gene was obtained by PCR amplification using cDNA from alfalfa cultivar 1 as a template.

[0079] MsFTa2 cds sequence (SEQ ID NO.3):

[0080] ATGGCAACTGGTAGCA GGCCAAATCCTCTTGCTGT TGGGCGTGTAATAGGGGATGTATTAGACCCCTTTGAAAGTTCTATTCCTCTCTTAGTCACCTACG GTAATAGGACTGTTACCAA TGGTCGTGAGCTTAAACCTTCCCAAGTTGCCAATCAACCCCAAGTGATTATTGGCGTCAATGACCCAACAACCTTCTACACCCTGGTTTTGGTAGATCCAGATGCTCCTAGTCCAAGTTACCCCAGTTTTAGGGAGTACCTTCATTGGATGGTGACTGATATTCCAGCAACTAATGCGGCTAGTTTTGGTAATGAGGTTGTAAGTTATGAAAAGCCACGACCCAATTTAGGGATTCATCGTTACGTGTTTGTATTATTGCGTCAACGGTGCAGTCAAAGAGTTTATGCTCCGGGATGGCGACAAAATTTCAATACAAGAGAATTCATTGAATTTTACGATCTTGGATCACCGGTTGCTGCTGTCTTCTTCAATTGTCAAAGGGAAACTGGCTCTGGGGGAAGAACCTTTAGATGA

[0081] MsFTa2 protein sequence (SEQ ID NO.4):

[0082] MATGSRPNPLAVGRVIGDVLDPFESSIPLLVTYGNRTVTNGRELKPSQVANQPQVIIGVNDPTTFYTLVLVDPDAPSPSYPSFREYLHWMVTDIPATNAASFGNEVVSYEKPRPNLGIHRYVFVLLRQRCSQRVYAPGWRQNFNTREFIEFYDLGSPVAAVFFNCQRETGSGGRTFR.

[0083] Example 1: Cultivating Medicago truncatula mtfta2 mutant lines with enhanced cold tolerance

[0084] Construction of gene editing binary vector p6401-MtFTa2:

[0085] A binary gene-editing vector targeting the MtFTa2 gene in the *Alfalfa truncatula* genome was constructed. The two selected targets are as follows: Target 1: 5′-GACCGAATCCTCTTGCTGT-3′ (SEQ ID NO. 5); Target 2: 5′-GTAATAGGACTGTTACCAA-3′ (SEQ ID NO. 6). Nucleotides 17-35 are target 1, and nucleotides 101-119 are target 2.

[0086] sgRNA module construction: Using primers MtFTa2-BsF, MtFTa2-F0, MtFTa2-R0, and MtFTa2-BsR, PCR amplification was performed using p5CBC as a template to amplify the sgRNA module MtFTa2-5CBC carrying the target sequence. The primer sequences are as follows: MtFTa2-BsF: 5′-ATATATGGTCTCGCTTG GACCGAATCCTCTTGCTGT GTT-3′ (SEQ ID NO.7); MtFTa2-F0: 5′-G GACCGAATCCTCTTGCTGT GTTTTAGAGCTAGAAATAGC-3′ (SEQ ID NO.8); MtFTa2-R0: 5′-AAC TTGGTAACAGTCCTATTAC CAATTTAATGGTTCGCTTGTA-3′ (SEQ ID NO.9); MtFTa2-BsR: 5′-ATTATTGGTCTCGAAAC TTGGTAACAGTCCTATTAC C-3′ (SEQ ID NO.10).

[0087] Construction of recombinant plasmid p6401-MtFTa2: Golden Gate enzyme digestion and ligation reaction was performed. The amplified MtFTa2-5CBC fragment carrying the target sequence and the p6401 vector were digested with BsaI and ligated using T4 DNA ligase. The resulting recombinant vector with the correct sequence was p6401-MtFTa2. Figure 1 ).

[0088] Obtaining the mtfta2 mutant line of alfalfa tribulus:

[0089] The plant transformation vector p6401-MtFTa2 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. The recombinant Agrobacterium was inoculated into 200 mL of YEP liquid medium containing Rifampicin (75 mg / L) and Kanamycin (50 mg / L) and incubated at 28 °C and 230 rpm until OD. 600nmThe concentration should be 0.6-0.8. Transfer the bacterial culture to a sterile centrifuge bottle, centrifuge at 5000 rpm for 10 min at room temperature, remove the supernatant, and resuspend in 200 mL of SH3a liquid medium (containing 0.1 mM acetylsuccinyl ketone) to prepare the infection solution.

[0090] Wild-type seeds of *Alfalfa tribulus* were treated with 1 mL of 98% H₂SO₄ for 8 min, followed by rinsing five times with pre-cooled deionized water. Then, 1 mL of 0.5% NaClO aqueous solution (containing 0.1% Triton X100) was added for sterilization for 12 min, followed by rinsing five times with sterile water in a clean bench. The seeds were then spread evenly on 0.8% water agar plates and incubated upside down in the dark at 4°C for 3 days. Subsequently, the seeds were incubated upside down at room temperature in the dark for 12 h, and the germinated seeds were seeded on 1 / 2 MS medium in a clean bench. After 3-4 weeks of alternating light and dark growth at 22°C, the seeds were ready for genetic transformation. Fully expanded leaves of R108 sterile seedlings were collected in a clean bench, and each leaf was cut into 2-4 pieces for use as explants.

[0091] Immerse the explants in the prepared infection solution, evacuate to -0.09 MPa and maintain for 30 min, then slowly release the gas; then place them on a horizontal shaker at 60 rpm and incubate in the dark for 1.5 h; then remove the explants, spread them evenly on SH3a solid medium containing 0.1 mM acetylsyl syringone covered with a single layer of filter paper, and incubate at 22 ℃ in the dark for 3 days.

[0092] Explants were transferred to SH3a solid medium containing 10 mg / L hygromycin B and 200 mg / L termethin and cultured at 22°C in the dark. Subcultures were performed every 2 weeks for a total of 6 weeks.

[0093] The callus was transferred to SH9 solid medium containing 10 mg / L hygromycin B and 120 mg / L termethin, and cultured at 22°C under alternating light and dark conditions. Subcultured every 3 weeks until regenerated seedlings were obtained.

[0094] The regenerated seedlings were transferred to 1 / 2 MS solid medium and cultured at 22°C under alternating light and dark conditions. Subculture was performed every 3-4 weeks until the plants rooted, which were then designated as T0 generation plants. Each T0 generation plant was numbered sequentially.

[0095] Transfer T0 generation plants to the culture medium, cover with plastic wrap to maintain humidity for 5 days, then remove the wrap and cultivate in a greenhouse. Greenhouse conditions: 22℃, 16h light / 8h darkness, humidity 70%-80%.

[0096] The T0 generation plants are self-pollinated and their seeds are harvested. The plants that grow from these seeds are the T1 generation plants. The T1 generation plants are self-pollinated and their seeds are harvested. The plants that grow from these seeds are the T2 generation plants. The T2 generation plants use the same numbering system as the T0 generation parent plants.

[0097] Identification of the *Alfalfa tribulus* mtfta2 mutant strain:

[0098] GMO identification

[0099] Leaves from T0 generation plants or wild-type alfalfa plants were collected, and genomic DNA was extracted from them. Using the genomic DNA as a template, PCR was performed using primers MtFTa2-F0 and MtFTa2-R0. If an 811bp target band was obtained, the identification result was positive, indicating that the plant was a regenerated plant transformed into the p6401-MtFTa2 vector.

[0100] Mutant Genotyping

[0101] Using genomic DNA from regenerated plants transformed with the p6401-MtFTa2 vector as a template, and MtFTa2-F and MtFTa2-R as primers, PCR amplification was performed. The band size in wild-type alfalfa was 384 bp. The amplified band was excised for subsequent Sanger sequencing. The primers used for mutant genotyping were as follows: MtFTa2-F: 5′-CTCTGGGCTGGTGTTGTTG-3′ (SEQ ID NO. 11); MtFTa2-R: 5′-CTAGGAGCATCTGGATCTA-3′ (SEQ ID NO. 12).

[0102] Two mutant lines were screened based on the sequencing results and named mtfta2-20 and mtfta2-22, respectively. Their sequencing results are shown below. Figure 2 The specific mutation methods are as follows:

[0103] The mutant line mtfta2-20 is a biallelic homozygous mutant. Compared with the wild-type MtFTa2 gene of alfalfa, this mutant gene has an insertion of a T base between positions 33 and 34, and the TAGGACTGTTAC at positions 105-116 is replaced with a base A, resulting in premature termination of protein translation at position 23.

[0104] The mutant line mtfta2-22 is a biallelic homozygous mutant. Compared with the wild-type MtFTa2 gene of alfalfa, this mutant lacks the TGTTTGGGCGTGTAATAGGGGATGTATTAGACCCCTTTGAAAGTACTATTCCTCTCTTAATCACCTATGGTAATAGGACTGTTA at positions 33-115, resulting in premature termination of protein translation at position 14.

[0105] Phenotypic identification of the alfalfa mtfta2 mutant lines:

[0106] Seeds tested: T2 generation seeds of mtfta2 mutants (mtfta2-20 and mtfta2-22) and wild-type seeds of alfalfa tribulus.

[0107] The test seeds were placed in 2 mL centrifuge tubes and treated with 1 mL of 98% H2SO4 for 8 min, followed by rinsing five times with pre-cooled deionized water. Then, 1 mL of 0.5% NaClO aqueous solution (containing 0.1% Triton X100) was added for sterilization for 12 min, followed by rinsing five times with sterile water. The seeds were then spread evenly on 0.8% water agar plates and incubated upside down in the dark at 4°C for 3 days. The germinated seeds were then sown in a culture medium, with 5 alfalfa seeds planted in each pot (8.5 cm high, 10 cm wide). The plants were incubated in a greenhouse for four weeks under the following conditions: 22°C, 16 h light / 8 h dark, and 70%-80% humidity. Three replicates were set up, with each replicate containing more than 15 seeds.

[0108] The test plants were photographed morphologically and then placed in a low-temperature incubator in the dark. The temperature was gradually reduced from 0°C to -4°C, starting at 1°C per hour. After maintaining the temperature at -4°C for one hour, the plants were kept at 4°C overnight. The plants were then returned to the greenhouse for 3 days of cultivation. Morphological photographs were taken and the survival rate and relative electrical conductivity of the plant leaves were recorded.

[0109] like Figure 3 As shown, the survival rate of the mtfta2-20 mutant line was 78.53%, and the relative conductivity was 35.52%; the survival rate of the mtfta2-22 mutant line was 71.96%, and the relative conductivity was 39.40%; while the survival rate of the wild type was 50.89%, and the relative conductivity was 51.47%. These results indicate that the survival rate of the mtfta2 mutant line was significantly higher than that of the wild type, while its relative conductivity was significantly lower.

[0110] Example 2: Cultivating msfta2 mutant lines with enhanced cold resistance

[0111] Construction of the gene editing binary vector p6401-MsFTa2

[0112] A binary gene-editing vector targeting the MsFTa2 gene in the alfalfa genome was constructed. The two selected targets are as follows: Target 1: 5′-GGCCAAATCCTCTTGCTGT-3′ (SEQ ID NO.13); Target 2: 5′-GTAATAGGACTGTTACCAA-3′ (SEQ ID NO.14). Nucleotides 17-35 of the gene are targeted at target 1, and nucleotides 101-119 are targeted at target 2.

[0113] sgRNA module construction: Using primers MsFTa2-BsF, MsFTa2-F0, MsFTa2-R0, and MsFTa2-BsR, PCR amplification was performed using p5CBC as a template to amplify the sgRNA module MsFTa2-5CBC carrying the target sequence. The primer sequences are as follows: MsFTa2-BsF: 5′-ATATATGGTCTCGCTTG GGCCAAATCCTCTTGCTGT GTT-3′ (SEQ IDNO.15); MsFTa2-F0: 5′-G GGCCAAATCCTCTTGCTGT GTTTTAGAGCTAGAAATAGC-3′ (SEQ ID NO. 16) MsFTa2-R0: 5′-AAC TTGGTAACAGTCCTATTAC CAATTTAATGGTTCGCTTGTA-3′ (SEQ ID NO. 17) MsFTa2-BsR: 5′-ATTATTGGTCTCGAAAC TTGGTAACAGTCCTATTA CC-3′ (SEQ ID NO.18).

[0114] Construction of recombinant plasmid p6401-MsFTa2: Golden Gate enzyme digestion and ligation reaction was performed. The MsFTa2-5CBC fragment with the target sequence obtained in step 2 and the p6401 vector were digested with BsaI and ligated with T4 DNA ligase. The resulting recombinant vector with the correct sequence was p6401-MsFTa2. Figure 4 ).

[0115] Obtaining the msfta2 mutant line of alfalfa:

[0116] The recombinant plasmid p6401-MsFTa2 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium.

[0117] Recombinant Agrobacterium was inoculated into 200 mL of YEP liquid medium containing Rifampicin (75 mg / L) and Kanamycin (50 mg / L), and incubated at 28°C and 230 rpm until OD was reached. 600nm The concentration should be 0.2-0.4. Transfer the bacterial culture to a sterile centrifuge bottle, centrifuge at 5000 rpm for 10 min at room temperature, remove the supernatant, and resuspend in 200 mL of SM4 liquid medium (containing 0.1 mM acetylsuccinyl ketone) to prepare the infection solution.

[0118] Take leaves from wild-type alfalfa plants, soak them in 0.1% Tween 20 solution for 5 minutes, then rinse them 3 times with deionized water, then soak them in 30% bleach solution for 10 minutes, and then rinse them 3 times with deionized water.

[0119] Transfer the leaves to a sterile empty glass bottle, add the prepared infection solution, first evacuate for 5 minutes, then sonicate for 2 minutes, then evacuate for 5 minutes again, then take the leaves, remove the surface liquid with filter paper, blow dry, and cut into small pieces with a blade, which are the explants.

[0120] The explants were spread evenly on SM4 solid medium and cultured in the dark at room temperature for 3 days.

[0121] Explants were transferred to SM4 solid medium containing 200 mg / L termethin and 10 mg / L hygromycin B and cultured at 24°C under light until callus tissue grew. Subcultured every 2 weeks.

[0122] The callus tissue was transferred to MSBK solid medium containing 200 mg / L termethin and 10 mg / L hygromycin B and cultured for 2-3 weeks.

[0123] The callus tissue was transferred to SH9 solid medium containing 200 mg / L termethin and 10 mg / L hygromycin B and cultured at 22°C under alternating light and dark conditions. Subculture was performed every 3 weeks until regenerated seedlings were obtained.

[0124] Transfer the regenerated seedlings to 1 / 2 MS solid medium and culture at 22°C under alternating light and dark conditions. Subculture every 3-4 weeks until the plants root, which are then designated as T0 generation plants. Number each T0 generation plant sequentially.

[0125] Identification of alfalfa msfta2 mutant lines

[0126] Transgenic identification: Genomic DNA was extracted from leaves of T0 generation plants or wild-type alfalfa plants. Using the genomic DNA as a template, PCR was performed using primers MsFTa2-F0 and MsFTa2-R0. A positive result (811 bp target band) indicated a regenerated plant transformed with the p6401-MsFTa2 vector. Results showed that msfta2-O-5, msfta2-O-21, and msfta2-34 were transgenic positive lines, while msfta2-9 was a transgenic negative line, which could be used as a negative control for subsequent experiments. Crossing msfta2-O-5 and msfta2-O-21 yielded F1 generation msfta2-5, which was a transgenic positive line. Figure 5 ).

[0127] Genotyping of mutants: Using genomic DNA from regenerated plants transformed with the p6401-MsFTa2 vector as a template, and MsFTa2-F and MsFTa2-R as primers, PCR amplification was performed. The band size in wild-type alfalfa was 1468 bp. The amplified band was excised for subsequent Sanger sequencing. If a double peak was observed, the corresponding PCR amplification product was ligated into the pLB vector, and multiple single clones were selected for sequencing again to identify the msfta2 mutant with the edited MsFTa2 gene. The primers used for mutant genotyping were as follows: MsFTa2-F: 5′-CTCTGGGCTGGTGTTGTTG-3′ (SEQ ID NO.19); MsFTa2-R: 5′-ACCTGCAACCCTCATTTCCTT-3′ (SEQ ID NO.20).

[0128] Two triellic mutation lines, msfta2-5 and msfta2-34, were identified based on sequencing results. (Sequencing results are shown below.) Figure 6 The specific mutation methods are as follows:

[0129] The mutant line msfta2-5 is a triallelic heterozygous mutant. Compared with the MsFTa2 gene in wild-type alfalfa, this mutant has the following deletions: the first allele deletion is GGACTGTTAC at positions 107-116, which causes premature termination of protein translation at position 39; the second allele deletion is AC at positions 115-116, which causes premature termination of protein translation at position 53; the third allele deletion is TGCT at positions 30-33 and GACTGTTACA at positions 108-117, which causes premature termination of protein translation at position 15; the fourth allele is not mutated.

[0130] The mutant line msfta2-34 is a triallelic heterozygous mutant. Compared with the MsFTa2 gene in wild-type alfalfa, this mutant has the following deletions: the first allele deletion of TGTTACCA at positions 111-118 causes premature termination of protein translation at position 39; the second allele deletion of TGTTACC at positions 111-117 causes premature termination of protein translation at position 53 of SEQ ID No. 6; the third allele deletion of CCTTTGCTGTTGGGCGTGTAATAGGGGATGTATTAGACCCCT at positions 25-67 and AC at positions 115-116 causes premature termination of protein translation at position 15; the fourth allele is not mutated.

[0131] Phenotypic identification of alfalfa msfta2 mutant lines:

[0132] Test plants: msfta2 mutant (msfta2-5 and msfta2-34) plants and transgenic negative plant msfta2-9 (control group-1).

[0133] Stems were cut from the test plants, and equal-length stem segments were inserted into flowerpots (10cm high, 10cm wide) filled with culture medium, with 4-6 stem segments per pot. After 5 weeks of cultivation in a greenhouse, the test plants were cut at the base (5cm from the surface of the culture medium) and allowed to continue growing for another 4 weeks. Greenhouse conditions: 22℃, 16h light / 8h darkness, humidity 70%-80%. Three replicates were set up, with more than 15 stem segments in each replicate.

[0134] Morphological photographs were taken of the test plants, and then they were placed in a low-temperature incubator under dark conditions. Gradual cooling was used, starting from 0°C and decreasing by 2°C per hour until -6°C. After maintaining at -6°C for three hours, the plants were kept at 4°C overnight. The plants were then returned to the greenhouse for 7 days of cultivation, morphological photographs were taken, and the survival rate and relative electrical conductivity of the plant leaves were recorded.

[0135] like Figure 7 As shown, the survival rate of the msfta2-5 mutant line was 58.32%, and the relative conductivity was 37.06%; the survival rate of the msfta2-34 mutant line was 57.01%, and the relative conductivity was 38.96%; while the survival rate of the control group-1 was 44.33%, and the relative conductivity was 51.11%. These results indicate that the survival rate of the msfta2 mutant line was significantly higher than that of the control group, while its relative conductivity was significantly lower.

[0136] Example 3: Cultivation of MtFTa2 overexpression lines in alfalfa with reduced cold resistance

[0137] Construction of vector pCAMBIA1307-MtFTa2:

[0138] Amplification was performed using alfalfa cDNA as a template, with primers 1307-MtFTa2-F and 1307-MtFTa2-R. The amplified products and the pCAMBIA1307 vector were digested with SalI and XbaI enzymes, followed by ligation to obtain the pCAMBIA1307-MtFTa2 plasmid.

[0139] Obtaining MtFTa2 overexpression lines in alfalfa:

[0140] The recombinant plasmid pCAMBIA1307-MtFTa2 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Other procedures were the same as in Example 1.

[0141] Identification of MtFTa2 overexpression lines in alfalfa

[0142] Test plants: T0 generation plants of various transgenic lines or wild-type alfalfa plants.

[0143] DNA level identification: Genomic DNA was extracted from leaves of the tested plants. Using the genomic DNA as a template, PCR amplification was performed using primers 1307-MtFTa2-F and FLAG-R, followed by agarose gel electrophoresis. If a 620bp amplification product was observed, the PCR identification result was positive, and the tested plant was a transgenic positive plant. Figure 8 The primers used for identification are as follows: 1307-MtFTa2-F: 5′-GCGTCGACATGGCAAGTGGTAG-3′ (SEQ ID NO.21); FLAG-R: 5′-CTACTTATCGTCATCGTCCTTGTA-3′ (SEQ ID NO.22).

[0144] RNA level identification: Total RNA was extracted from leaves of the tested plants and reverse transcribed to obtain cDNA. Using cDNA as a template, the transcription level of the MtFTa2 gene was detected by semi-quantitative RT-PCR. Results were detected by 2.0% agarose gel electrophoresis, and the RT-PCR amplification product of MtActin4A was used as an internal standard for total RNA template quantification. Primers used for detecting the MtFTa2 gene were as follows: MtFTa2-SqF: 5′-CGTAAATGACCCAACAGCC-3′ (SEQ ID NO.23); FLAG-R: 5′-CTACTTATCGTCATCGTCCTTGTA-3′ (SEQ ID NO.24). Primers used for detecting MtActin4A were as follows: MtActin4A-F: 5′-CCAAAGGCCAACAGAGAAAA-3′ (SEQ ID NO.25); MtActin4A-R: 5′-ACGACCAGCAAGATCCAAAC-3′ (SEQ ID NO.26).

[0145] Protein level identification: Total protein was extracted from leaves of the tested plants and subjected to SDS-PAGE gel electrophoresis. The protein was then transferred to a cellulose acetate membrane for Western blot hybridization. The primary antibody was FLAG-Tag Mouse mAb. The secondary antibody was peroxidase-labeled goat anti-mouse IgG. The MtFTa2 OE-2, MtFTa2 OE-3, and MtFTa2 OE-10 lines all showed a target band of approximately 23 kDa, indicating they were all immunoblot-positive plants. Figure 9 ).

[0146] Phenotypic identification of MtFTa2 overexpression lines

[0147] Test seeds: T2 generation seeds of MtFTa2 OE plants (MtFTa2 OE-2, MtFTa2 OE-3, and MtFTa2 OE-10) and wild-type seeds of alfalfa. Other procedures were the same as in Example 1.

[0148] like Figure 10 As shown, the survival rate of MtFTa2 OE-2 plants was 36.04%, and the relative conductivity was 77.38%; the survival rate of MtFTa2OE-3 overexpression lines was 37.68%, and the relative conductivity was 80.53%; the survival rate of MtFTa2 OE-10 overexpression lines was 31.72%, and the relative conductivity was 76.08%; and the survival rate of wild-type was 46.03%, with a relative conductivity of 60.31%. These results indicate that the survival rate of MtFTa2 OE plants was significantly lower than that of the wild type, while their relative conductivity was significantly higher.

[0149] Example 4: Cultivation of MsFTa2 overexpression lines with reduced cold resistance

[0150] Construction of vector pCAMBIA1307-MsFTa2

[0151] cDNA from wild-type alfalfa plants was amplified using primers 1307-MsFTa2-F and 1307-MsFTa2-R as a template. The amplified products and the pCAMBIA1307 vector were digested with SalI and XbaI enzymes, followed by ligation to obtain the pCAMBIA1307-MsFTa2 plasmid.

[0152] Obtaining MsFTa2 overexpression lines in alfalfa

[0153] The recombinant plasmid pCAMBIA1307-MsFTa2 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Other procedures were the same as in Example 2.

[0154] Identification of MsFTa2 overexpression lines in alfalfa

[0155] Test plants: T0 generation plants of the obtained transgenic lines or wild-type alfalfa plants.

[0156] DNA level identification: Genomic DNA was extracted from leaves of the test plants. Using the genomic DNA as a template, PCR amplification was performed using primers 1307-MsFTa2-F and FLAG-R, followed by agarose gel electrophoresis. Figure 11Except for MsFTa2OE-5 and MsFTa2 OE-12, all other strains showed a 620bp amplification product, and the PCR identification results were positive. The primers used for identification are as follows: 1307-MsFTa2-F: 5′-GCGTCGACATGGCAACTGGTAGC-3′ (SEQ ID NO.27); FLAG-R: 5′-CTACTTATCGTCATCGTCCTTGTA-3′ (SEQ ID NO.28).

[0157] RNA level identification: Total RNA was extracted from leaves of the tested plants and reverse transcribed to obtain cDNA. Using cDNA as a template, MsActin4A and MsEIF4A genes were used as internal reference genes, and the transcription level of the MsFTa2 gene was detected by RT-qPCR. The primers used to detect the MtFTa2 gene are as follows: MsFTa2-qF: 5′-TGACTGATATTCCAGCAACTAATG-3′ (SEQ ID NO.29); MsFTa2-qR: 5′-CGGTGATCCAAGATCGTAAAA-3′ (SEQ ID NO.30). The primers used to detect the internal reference gene are as follows: MsActin4A-F: 5′-CCAAAGGCCAACAGAGAAAA-3′ (SEQ ID NO.31); MsActin4A-R: 5′-ACGACCAGCAAGATCCAAAC-3′ (SEQ ID NO.32); MsEIF4A-F: TTTAGCCTCCGGAAGGTTCAC (SEQ ID NO.33); MsEIF4A-R: TGCTGAATCACATCGAGACC (SEQ ID NO.34).

[0158] Protein level identification: Total protein was extracted from leaves of the tested plants and subjected to SDS-PAGE gel electrophoresis. The protein was then transferred to a cellulose acetate membrane for Western blotting. The primary antibody was FLAG-Tag Mouse mAb. The secondary antibody was peroxidase-labeled goat anti-mouse IgG. The MsFTa2 OE-1, MsFTa2 OE-6, and MsFTa2 OE-11 lines all showed a target band of approximately 23 kDa, indicating they were all immunoblotting positive plants. The MsFTa2 OE-12 line did not show a target band and was a transgenic negative plant, which could be used as a negative control for subsequent experiments. Figure 12 ).

[0159] Phenotypic identification of alfalfa MsFTa2 overexpression lines

[0160] Test plants: MsFTa2 overexpressing plants (MsFTa2 OE-1, MsFTa2 OE-6 and MsFTa2 OE-11) and transgenic negative plant MsFTa2 OE-12 (control group-2). Other procedures were the same as in Example 2.

[0161] like Figure 13 As shown, the survival rate of MsFTa2 OE-1 plants was 41.45%, and the relative conductivity was 71.70%; the survival rate of MsFTa2OE-6 overexpression lines was 39.22%, and the relative conductivity was 67.88%; the survival rate of MsFTa2 OE-11 overexpression lines was 40.46%, and the relative conductivity was 69.94%; the survival rate of the control group-2 was 51.50%, and the relative conductivity was 49.92%. These results indicate that the survival rate of MsFTa2 OE plants was significantly lower than that of the control group, while their relative conductivity was significantly higher.

Claims

1. The application of the alfalfa FTa2 gene in the breeding of cold-resistant plants, characterized by, The nucleotide sequence of the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.

3.

2. The application according to claim 1, wherein knocking out or down the alfalfa FTa2 gene in the plant, or reducing the level or activity of the protein encoded by the alfalfa FTa2 gene in the plant, achieves one or more effects selected from (A1)-(A3): (A1) Reduce the relative electrical conductivity of the blades; (A2) Improve the survival rate of plants under low temperature conditions; (A3) Improve the growth status of plants at low temperatures.

3. The application according to claim 1 or 2, wherein the application uses a tool selected from one or more of (B1)-(B5) to knock out or knock down the alfalfa FTa2 gene in the plant, or to reduce the level or activity of the protein encoded by the alfalfa FTa2 gene in the plant: (B1) A tool based on the CRISPR-Cas9 method; preferably, the tool based on the CRISPR-Cas9 method introduces a mutation through gene editing to cause premature termination of translation of the alfalfa FTa2 gene; more preferably, the target sites in the gene editing are SEQ ID NO.5 and SEQ ID NO.6; or SEQ ID NO.13 and SEQ ID NO.14; (B2) Tools based on homologous recombination methods; (B3) Tools based on the ZFN method; (B4) The RNA interference tool for the alfalfa FTa2 gene; preferably, siRNA, miRNA, shRNA, or dsRNA; (B5) The ASO of the alfalfa FTa2 gene; (B6) Use an inhibitor of the protein encoded by the alfalfa FTa2 gene, preferably an antibody.

4. The application according to any one of claims 1-3, wherein the application is used for one or more of (C1)-(C3): (C1) Improve the cold resistance of plant plants; (C2) Provide plant propagation materials with improved cold resistance; preferably seeds, tissue culture materials, and organs that can be used for propagation; (C3) Provides plant strains with improved cold resistance.

5. The application according to any one of claims 1-3, wherein the plant is an alfalfa species.

6. The application according to claim 5, wherein the plant is alfalfa or clover.

7. Alfalfa FTa2 gene, characterized by, The nucleotide sequence of the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.

3.

8. The protein encoded by the alfalfa FTa2 gene according to claim 7.

9. The protein according to claim 8, wherein the amino acid sequence of the protein is SEQ ID NO.2 or SEQ ID NO.

4.

10. Application of any one of the tools (D1)-(D3) for detecting the expression level of the alfalfa FTa2 gene or the expression level of the protein encoded by the alfalfa FTa2 gene: (D1) Determine the cold resistance of alfalfa plants; (D2) Predicting the cold resistance of alfalfa propagation material; (D3) Breed cold-resistant alfalfa varieties; The tool for detecting the expression level of the alfalfa FTa2 gene or the expression level of the protein encoded by the alfalfa FTa2 gene is one or more of the following: (E1) Primer; (E2) Probe; (E3) PCR reagents; (E4) chip; (E5) Antibody; (E6) ELISA reagent; The nucleotide sequence of the FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3; the nucleotide sequence of the protein encoded by the alfalfa FTa2 gene is SEQ ID NO.1 or SEQ ID NO.3.

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