Application of MsMYBS3 gene or biological material thereof in regulation and control of plant cold resistance

Abstract

The invention provides application of an MsMYBS3 gene or a biological material thereof in regulation and control of plant cold resistance, and belongs to the technical field of plant genetic engineering. The amino acid sequence coded by the MsMYBS3 gene disclosed by the invention is as shown in SEQ ID NO. 4. The invention provides an application of a medicago sativa MYB transcription factor gene MsMYBS3 in the aspect of regulating and controlling the cold resistance of medicago sativa. A gene editing technology is adopted to directionally change the development and the cold resistance of the medicago sativa. The cold resistance of an MsMYBS3 gene editing strain constructed by the invention is reduced, and the MsMYBS3 gene has the effect of regulating and controlling the cold resistance of alfalfa, so that a theoretical basis and a candidate gene are provided for cultivating new varieties of high-resistance leguminous forage, and the application of alfalfa in production is promoted.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a... MsMYBS3 Application of genes or their biological materials in regulating plant cold resistance. Background Technology

[0002] alfalfa ( Medicago Sativa L Alfalfa is a highly adaptable and palatable forage grass. With its significant symbiotic nitrogen-fixing characteristics and excellent feed protein quality, it holds core value in agricultural production and livestock breeding, earning it the title of "King of Forage." Due to the wide distribution and significant differences between alfalfa varieties, coupled with varying environmental and production factors across regions (such as salinity, drought, and frost damage), different regions require different alfalfa varieties. Selected varieties should possess good stress resistance and root systems with strong ecological and absorptive functions. Therefore, accelerating the development of more new alfalfa varieties with strong resistance, high yield, and high quality has become a research focus for many in the forage industry, thereby promoting the development of the forage industry.

[0003] MYB transcription factors constitute the largest family of transcription factors in plants, characterized by the presence of a conserved MYB-type DNA-binding domain. This domain consists of 1-4 non-repeating R motifs. Based on the number of R motifs, MYB transcription factors can be divided into four subclasses: 1R-MYB (1R motif), R2R3-MYB (2R motif), 3R-MYB (3R motif), and 4R-MYB (4R motif). Among these, R2R3-MYB constitutes the vast majority and plays a crucial role in plant growth, development, secondary metabolism, and environmental adaptation. For example, the opposite control of MtMYB61 and MtMYB3 increases the freeze tolerance of truncate alfalfa by mitigating the inhibition of C-repetition sequence binding factors.

[0004] In the field of legume research, there is limited research on stress resistance related to MYB transcription factors, and even less research on CRISPR-Cas9 technology based on stable genetic transformation in alfalfa. Exploring the function and application potential of MYB transcription factors in alfalfa, and making them a core target for stress resistance mechanism research and molecular design breeding, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a MsMYBS3 Application of genes or their biological materials in regulating plant cold resistance.

[0006] To achieve the above objectives, the present invention provides a MsMYBS3 The application of genes or their biological materials in regulating the cold resistance of alfalfa, the aforementioned MsMYBS3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.4.

[0007] Preferably, the MsMYBS3 The gene polynucleotides are shown in (a), (b), or (c): (a) Polynucleotides as shown in SEQ ID NO.3; (b) A polynucleotide that has more than 90% homology with the polynucleotide shown in SEQ ID NO.3; (c) A polynucleotide mutant with the same functional activity obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID NO.3.

[0008] Preferably, the biomaterial comprises any one of the following (1) to (3): (1) An expression cassette containing the polynucleotides described in (a), (b) or (c); (2) A recombinant vector containing the polynucleotides described in (a), (b) or (c) or a recombinant vector containing the expression cassette described in (1); (3) A recombinant microorganism containing the polynucleotides described in (a), (b) or (c), a recombinant microorganism containing the expression cassette described in (1) or a recombinant microorganism containing the recombinant vector described in (2).

[0009] Preferably, the regulation is any one of the following: S1, by reducing the MsMYBS3 Gene expression levels and / or activity reduce the cold resistance of the alfalfa. S2, By improving the above MsMYBS3 The expression level and / or activity of the gene improve the cold resistance of the alfalfa.

[0010] Preferably, reducing the MsMYBS3 Gene expression levels include suppression, silencing, or knockout using the CRISPR / Cas system. MsMYBS3 Gene; The CRISPR / Cas system targets gene editing sites 1 and 2, with the sequence of target 1 shown in SEQ ID NO.9 and the sequence of target 2 shown in SEQ ID NO.10.

[0011] The present invention also provides MsMYBS3 The application of the gene or its biological material in the breeding of cold-resistant transgenic alfalfa, wherein the amino acid sequence encoded by the MsMYBS3 gene is shown in SEQ ID NO.4.

[0012] Preferably, the biomaterial comprises any one of the following: V1, reduce the MsMYBS3Recombinant vectors for gene expression levels and / or activity; V2, Recombinant bacteria containing the recombinant vector described in V1; The MsMYBS3 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0013] The present invention also provides a method for regulating the cold resistance of alfalfa, the method comprising reducing the cold resistance of the receptor alfalfa. MsMYBS3 The steps for determining the expression level of genes to obtain transgenic alfalfa; the transgenic alfalfa has lower cold resistance than the recipient alfalfa.

[0014] Preferably, reducing the MsMYBS3 Gene expression levels include suppression, silencing, or knockout using the CRISPR / Cas system. MsMYBS3 Gene.

[0015] Preferably, the gene editing targets of the CRISPR / Cas system are target 1 and target 2, the sequence of target 1 is shown in SEQ ID NO.9, and the sequence of target 2 is shown in SEQ ID NO.10.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides MsMYBS3 The application of genes or their biological materials in regulating the cold resistance of alfalfa, the aforementioned MsMYBS3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.4. Gene editing technology can be used to suppress, silence, or knock out [the gene]. MsMYBS3 Genes were used to directionally alter the development and cold resistance of alfalfa. This invention conducted cold resistance tests on the obtained transgenic lines and found... MsMYBS3 Gene-edited strains exhibit decreased cold resistance. MsMYBS3 The gene positively regulates the cold resistance of alfalfa, providing a new theoretical basis and candidate genes for breeding new leguminous forage varieties with high resistance, which will greatly promote the application of alfalfa in production. Attached Figure Description

[0017] Figure 1 Alfalfa under different stress treatments in Example 2 MsMYBS3 Results of gene expression pattern analysis.

[0018] Figure 2 In Example 2 MsMYBS3 Results of relative gene expression levels in different tissues of wild-type alfalfa 'Zhongmu 1'.

[0019] Figure 3 In Example 3 MsMYBS3- Subcellular localization results of GFP fusion protein, where scale bar: 25 μm.

[0020] Figure 4 In Example 4 MsMYBS3 Schematic diagram of gene editing vector structure.

[0021] Figure 5 alfalfa in Example 4 MsMYBS3 Gene mutant identification results, where A is MsMYBS3 PCR validation results of mutant knockout lines, M represents DL2000 Marker; 1-7 represent different mutant lines; CK + Represents a positive control; CK - Represents a negative control; CK -(ddH 2 O) B represents the blank control; B represents alfalfa. MsMYBS3 Mutant strains MsMYBS3 Gene expression level, **representing the level compared to WT. P Significant differences exist at the <0.01 level; C represents alfalfa. MsMYBS3 Sequencing results of gene mutant lines targeting specific targets; ED-2, ED-6, and ED-7 represent, respectively. MsMYBS3 Genetically modified knockout lines.

[0022] Figure 6 alfalfa in Example 4 MsMYBS3 Sequencing peak diagram of target sites in gene mutant strains.

[0023] Figure 7 The wild-type alfalfa 'Zhongmu No. 1' and alfalfa grown under normal conditions in Example 5. MsMYBS3 Growth characteristics of mutant lines: A represents the phenotype of alfalfa lines after 4 weeks of normal growth; B represents the leaf phenotype after 4 weeks of normal growth; C represents plant height; D represents the number of leaves; E represents the number of primary branches; F represents the number of secondary branches; G represents the number of main stem nodes; and H represents fresh weight and dry weight.

[0024] Figure 8 alfalfa under cold stress in Example 5 MsMYBS3 Results of cold tolerance tests on mutant and wild-type alfalfa lines. In this paper, A shows the phenotypic figures of wild-type and mutant alfalfa before cold stress and after 2 days of recovery at room temperature; B shows the electrical conductivity; and C shows the survival rate. Detailed Implementation

[0025] This invention provides MsMYBS3 The application of genes or their biological materials in regulating the cold resistance of alfalfa, the aforementioned MsMYBS3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.4.

[0026] In this invention, the MsMYBS3 The gene polynucleotides are shown in (a), (b), or (c): (a) Polynucleotides as shown in SEQ ID NO.3; (b) A polynucleotide that has more than 90% homology with the polynucleotide shown in SEQ ID NO.3; (c) A polynucleotide mutant with the same functional activity obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID NO.3.

[0027] In this invention, the biomaterial includes any one of the following (1) to (3): (1) An expression cassette containing the polynucleotides described in (a), (b) or (c); (2) A recombinant vector containing the polynucleotides described in (a), (b) or (c) or a recombinant vector containing the expression cassette described in (1); (3) A recombinant microorganism containing the polynucleotides described in (a), (b) or (c), a recombinant microorganism containing the expression cassette described in (1) or a recombinant microorganism containing the recombinant vector described in (2).

[0028] In this invention, the regulation is any one of the following: S1, by reducing the MsMYBS3 Gene expression levels and / or activity reduce the cold resistance of the alfalfa. S2, By improving the above MsMYBS3 The expression level and / or activity of the gene improve the cold resistance of the alfalfa.

[0029] In this invention, the reduction of the MsMYBS3 Gene expression levels include suppression, silencing, or knockout using the CRISPR / Cas system. MsMYBS3 The gene editing targets of the CRISPR / Cas system are target 1 and target 2, the sequence of target 1 is shown in SEQ ID NO.9, and the sequence of target 2 is shown in SEQ ID NO.10. The CRISPR / Cas system used in this invention is based on Chinese Patent 202110884895.X – “Alfalfa CRISPR / Cas9 Genome Editing System and Its Application”. Based on the alfalfa CRISPR / Cas9 dual-mutation system in the above-referenced patent, alfalfa… MsMYBS3 Design and construct based on gene sequence to target MsLEA3 Gene editing vectors were used to transform the alfalfa variety 'Zhongmu 1' using Agrobacterium-mediated transformation, thus constructing alfalfa... MsMYBS3 Gene-edited strains. This invention has discovered that gene-edited strains of alfalfa...MsMYBS3 After gene knockout, compared to wild type, MsMYBS3 The mutant lines have reduced survival rates, higher levels of cell membrane damage, reduced cell membrane permeability, and are more sensitive to cold treatment. MsMYBS3 Positively regulating the cold resistance of alfalfa, knocking out [a specific substance] in mutants. MsMYBS3 It can significantly reduce the adaptability of alfalfa to cold stress.

[0030] The present invention also provides MsMYBS3 The application of the gene or its biological material in the breeding of cold-resistant transgenic alfalfa, wherein the amino acid sequence encoded by the MsMYBS3 gene is shown in SEQ ID NO.4.

[0031] In the application of this invention in the breeding of cold-resistant transgenic alfalfa, the biological material includes any one of the following: V1, reduce the MsMYBS3 Recombinant vectors for gene expression levels and / or activity; V2, Recombinant bacteria containing the recombinant vector described in V1; The MsMYBS3 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0032] In this invention, the reduction of the MsMYBS3 Methods for expressing gene expression levels and / or activity using expression vectors include gene editing, preferably performed using a CRISPR / Cas9 system. As an optional implementation, the expression vector includes... p 6401 carrier and / or p 5CBC vector.

[0033] The present invention also provides a method for regulating the cold resistance of alfalfa, the method comprising reducing the cold resistance of the receptor alfalfa. MsMYBS3 The step of obtaining transgenic alfalfa by adjusting gene expression levels; the transgenic alfalfa exhibits lower cold hardiness than the recipient alfalfa. Lowering the expression level of the gene... MsMYBS3 Gene expression levels include suppression, silencing, or knockout using the CRISPR / Cas system. MsMYBS3 Gene; the gene editing targets of the CRISPR / Cas system are target 1 and target 2, the sequence of target 1 is shown in SEQ ID NO.9, and the sequence of target 2 is shown in SEQ ID NO.10.

[0034] This invention uses gene editing technology to suppress, silence, or knock out... MsMYBS3 Genes were used to directionally alter the development and cold resistance of alfalfa. This invention conducted cold resistance tests on the obtained transgenic lines and found... MsMYBS3 Gene-edited strains exhibit decreased cold resistance.MsMYBS3 The gene positively regulates the cold resistance of alfalfa, providing a new theoretical basis and candidate genes for breeding new leguminous forage varieties with high resistance, which will greatly promote the application of alfalfa in production.

[0035] In the following embodiments of the present invention, the experimental material is alfalfa 'Zhongmu No. 1' ( Medicago sativa L .), Benedict's tobacco ( Nicotiana tabacum L All of these are known varieties. The alfalfa used in this invention was kindly provided by Professor Yu Linqing of the School of Life Sciences, Inner Mongolia University; *Tobacco Benzovia* (…) Nicotiana tabacum L (This was kindly donated by Professor Hada of the School of Life Sciences, Inner Mongolia University.)

[0036] In the following embodiments, p 6401 carrier and p 5CBC vector: See “ZHU, F., YE, Q., CHEN, H., DONG, J. & WANG, T. 2021. Multigene editing reveals that MtCEP1 / 2 / 12 redundantly regulate lateral root and nodule number in Medicago truncatula. J. Exp. Bot.”, kindly provided by Professor Jiangli Dong of the College of Biological Sciences, China Agricultural University.

[0037] p The BE-35S-GFP vector, see Chinese patent CN202310897060.7, describes the application of the MfERF026 gene in regulating the growth, development, and stress tolerance of alfalfa, and was kindly provided by Professor Hou Xiaowan of the South Subtropical Crops Research Institute, Chinese Academy of Agricultural Sciences.

[0038] In the following examples, the formulations of each culture medium are shown below: SH3α liquid culture medium formula: sucrose 30 g / L, 2,4-D 4 mg / L, 6-BA 0.5 mg / L, Timetin 200 mg / L, Hygromycin B 10 mg / L, MgSO4·7H2O 185 mg / L, KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, CaCl2·2H2O 166 mg / L, KH2PO4 400 mg / L, MnSO4·H2O 10 mg / L, H3BO3 5.0 mg / L, ZnSO4·7H2O 1.0 mg / L, KI 1.0 mg / L, Na2MoO4·2H2O 0.1 mg / L, CuSO4·5H2O 0.2 mg / L, CoCl2·6H2O The medium contained 0.1 mg / L EDTA-FeNa 140 mg / L, thiamine hydrochloride 5.0 mg / L, pyridoxine hydrochloride 5.0 mg / L, nicotinic acid 5.0 mg / L and inositol 100 mg / L, with a pH of 5.9.

[0039] SH3α co-culture medium formulation: sucrose 30 g / L, acetylsylgenin 100 μmol / L, 2,4-D 4 mg / L, 6-BA 0.5 mg / L, plant gel 3 g / L, MgSO4·7H2O 185 mg / L, KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, CaCl2·2H2O 166 mg / L, KH2PO4 400 mg / L, MnSO4·H2O 10 mg / L, H3BO3 5.0 mg / L, ZnSO4·7H2O 1.0 mg / L, KI 1.0 mg / L, Na2MoO4·2H2O 0.1 mg / L, CuSO4·5H2O 0.2 mg / L, CoCl2·6H2O 0.1 mg / L, EDTA-FeNa 140 mg / L, thiamine hydrochloride 5.0 mg / L, pyridoxine hydrochloride 5.0 mg / L, nicotinic acid 5.0 mg / L and inositol 100 mg / L; the pH of the culture medium was 5.9.

[0040] SH3α selective medium formulation: sucrose 30 g / L, 2,4-D 4 mg / L, 6-BA 0.5 mg / L, Timetin 200 mg / L, Hygromycin B 10 mg / L, plant gel 3 g / L, MgSO4·7H2O 185 mg / L, KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, CaCl2·2H2O 166 mg / L, KH2PO4 400 mg / L, MnSO4·H2O 10 mg / L, H3BO3 5.0 mg / L, ZnSO4·7H2O 1.0 mg / L, KI 1.0 mg / L, Na2MoO4·2H2O 0.1 mg / L, CuSO4·5H2O 0.2 mg / L, CoCl2·6H2O 0.1 mg / L, EDTA-FeNa The medium contained 140 mg / L thiamine hydrochloride, 5.0 mg / L pyridoxine hydrochloride, 5.0 mg / L nicotinic acid, and 100 mg / L inositol, with a pH of 5.9.

[0041] MSO medium formulation: MS basal medium 2.22 g / L, sucrose 5 g / L and plant gel 3 g / L.

[0042] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0044] Example 1 alfalfa MsMYBS3 Acquisition of transcription factors Total RNA was extracted from leaf tissue of wild-type alfalfa 'Zhongmu 1' plants using liquid nitrogen grinding and Takara Bio's Trizol RNA extraction reagent. cDNA was synthesized by reverse transcription according to the instructions of Takara Bio's reverse transcription kit (catalog number: 6210A).

[0045] by MsMYBS3 -F and ​ -R is the primer for PCR.

[0046] ​ -F:ATGGGCAGAAAGTGCTCAC(SEQ ID NO.1); ​ -R: TTAAGTGACACTAATTGGCCCAAT (SEQ ID NO. 2).

[0047] PCR conditions were as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10 sec; annealing at 58 ℃ for 30 sec; extension at 68 ℃ for 1 min, denaturation to extension for 30 cycles; final extension at 68 ℃ for 10 min; storage at 4 ℃.

[0048] The PCR reaction system is shown in Table 1 below: Table 1 PCR reaction system

[0049] After the PCR reaction, the target fragment (approximately 888 bp) was recovered using the SanPrep Column DNA Gel Recovery Kit from Sangon Biotech Co., Ltd., following the kit's instructions. Alfalfa was obtained using the above method. ​ Gene CDS sequence. The recovered fragments were then compared with... ​ ® -Blunt-Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd.) connection obtained ​ ®-Blunt- ​ Transformed Escherichia coli ( ​ DH5α competent cells were selected, and single clones were sent to Shanghai Sangon Biotech for sequencing.

[0050] ​ The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0051] ​ The nucleotide sequence of the gene is as follows: ATGGGCAGAAAGTGCTCACATTGTGGAAACATAGGCCATAATTCAAGGACTTGTAATTCATTAAGAGGAAGTAGTAGTTTTGTTGGAGTTAGACTTTTTGGTGTTCAACTTGATTTATCTTCTTCTTGTGTTTCCATGAAAAAGAGTTTTAGCATGGATTCTTTTCCTACTTCATCTTCTTCTCCTACTTCTTCCTTTTCTTCTTCAAGATTATCCATTGATGATAGAGCCTCTATTGGTTATCTTTCAGATGGTCTCATAGTTCGTACACAAGAAAGGAAAAAAGGAGTTCCATGGACAGAAGAAGAGCATAGAAAATTCCTTGTTGGACTTGAGAAGCTTGGAAAGGGAGATTGGAGGGGTATCTCTAGAAACTATGTGACTACAAGAACACCAACACAAGTTGCAAGCCATGCTCAAAAGTATTTTATTCGACTTGCAACCTTGAATAAGAAGAAGAGACGTTCAAGTCTCTTTGACATGGTTGGTAGTGGCAAGACAAACAAAGCAGTTGATCCAAATAACAGCTCAAAAAGCAAGTCAGGTGATTCAGTTTGTAGACATGATGATCATGAAGTTGAGAAGGATGGAACTTTGTCACTACTAATAAACTCACTTCAACAACAAACCAAATCAGATGATTATGATATGCAAAAAATTGAAGATGTTGAAGTTGAAGATGATTCTGAAGAAGCAGAACATAAAGATGTGCCAGATTGGCTACATCCTCTAACAAAGTCTTTAAATATGACATTGGTGATACCAAATTCTTCTAATGTAGCAGCACCTGATCTTGAGCTAACTCTGGCAGGGTCCAATTCAAATAACATGGAACAAGATAAAACATCCCCAAGTTCATTTCTTATTGGGCCAATTAGTGTCACTTAA(SEQ ID NO.3).

[0052] ​ The amino acid sequence encoded by the gene is as follows: MGRKCSHCGNIGHNSRTCNSLRGSSSFVGVRLFGVQLDLSSSCVSMKKSFSMDSFPTSSSSPTSSFSSSRLSIDDRASIGYLSDGLIVRTQERKKGVPWTEEEHRKFLVGLEKLGKGDWRGISRNYVTTRTPTQVASHAQKYFIRLATL NKKKRRSSLFDMVGSGKTNKAVDPNNSSKSKSGDSVCRHDDHEVEKDGTLSLLINSLQQQTKSDDYDMQKIEDVEVEDDSEEAEHKDVPDWLHPLTKSLNMTLVIPNSSNVAAPDLELTLAGSNSNNMEQDKTSPSSFLIGPISVT(SEQ ID NO.4).

[0053] Example 2 alfalfa ​ Analysis of expression patterns of 3 genes (1) Select wild-type alfalfa 'Zhongmu No. 1' that is 4 weeks old and grows normally, and perform stress treatment according to the following method.

[0054] Low temperature stress: After 4 weeks of cultivation, alfalfa seedlings were subjected to low temperature stress in a 0 ℃ low temperature incubator, and samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. Salt stress: NaCl was added to 1 / 2 Hoagland nutrient solution to a final concentration of 250 mM. Samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after NaCl treatment. Mannitol stress: D-mannitol was added to 1 / 2 Hoagland nutrient solution to a final concentration of 400 mM. Samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after D-mannitol treatment. Salicylic acid treatment: Salicylic acid was added to 1 / 2 Hoagland nutrient solution to a final concentration of 1 mM. Samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after salicylic acid treatment.

[0055] At different stress time points, whole alfalfa plants were wrapped in aluminum foil, labeled (stress time, stress type), flash-frozen in liquid nitrogen, and stored at -80 ℃.

[0056] RNA was extracted separately, and cDNA was obtained through reverse transcription. This cDNA was then used as a template... ​ -QF and ​-QR is the primer, and the procedure was performed according to the instructions of the TAKARA Biotech Real-Time Fluorescence Kit (catalog number: RR047Q).

[0057] ​ -QF:TCTTTCAGATGGTCTCATAGTTCGT(SEQ ID NO.5); ​ -QR: CCTCCAATCTCCCTTTCCAA (SEQ ID NO. 6).

[0058] The results are as follows ​ As shown: Alfalfa ​ The gene can be expressed in response to low temperature, NaCl stress, and salicylic acid treatment.

[0059] (2) Plant materials: Wild-type alfalfa 'Zhongmu No. 1' at 4 weeks of normal growth were selected. Root, stem, and leaf tissues were quick-frozen in liquid nitrogen and stored at -80 ℃. RNA was extracted from each tissue, and cDNA was obtained by reverse transcription. This cDNA was used as a template for... ​ -QF (SEQ ID NO.5) and ​ -QR (SEQ ID NO.6) was used as the primer, and the procedure was performed according to the instructions of the TAKARA Biotech Real-Time Fluorescence Responsive Kit (catalog number: RR047Q).

[0060] The results are as follows ​ As shown: Results are displayed ​ The relative expression level of the gene is highest in the leaves.

[0061] Example 3 alfalfa ​ Subcellular localization analysis 3.1 Construction of recombinant plasmids p BE-GFP- ​ Prepared in Example 1 ​ ®-Blunt- ​ Use as a template p BE- ​ -F and p BE- ​ -R gets with ​ I and ​ I. Enzyme cleavage site ​ Full-length cDNA.

[0062] p BE- ​ -F: GACTCTAGAGCAGTCGACGATGGGCAGAAAGTGCTCAC (SEQ ID NO.7); p BE- ​ -R: GGCGACCGGTGGATCCCGAGTGACACTAATTGGCCCAAT (SEQ ID NO. 8).

[0063] The PCR system is shown in Table 2: Table 2 PCR system

[0064] PCR conditions were as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 10 sec; annealing at 58 ℃ for 30 sec; extension at 68 ℃ for 1 min, followed by 30 cycles of denaturation to extension; final extension at 68 ℃ for 10 min; storage at 4 ℃. p BE-35S-GFP (pBE for short) empty vector plasmid for use ​ H Ⅰ and ​ I. Perform double enzyme digestion, as shown in Table 3. After mixing the system as shown in Table 3, digest at 37 °C for 1 hour. Use TaKaRa's In-Fusion enzyme. ® The HD Cloning Kit is used to extract the target fragment (with...) ​ I and ​ I. Enzyme cleavage site ​ Homologous recombination ligation of full-length cDNA and pBE vector yields recombinant plasmids. p BE-GFP- ​ The ligation product was transformed into E. coli DH5α, and after colony PCR detection, it was sent to Shanghai Sangon Biotech for sequencing. The positive clones verified by sequencing were amplified and cultured, and plasmids were extracted for later use.

[0065] Table 3 Double enzyme digestion system

[0066] 3.2 Recombinant plasmids p BE-GFP- ​ Transformation of Agrobacterium GV3101 Take 5 μL of the recombinant plasmid prepared above. p BE-GFP- ​ and no load pBE-35S-GFP was added to Agrobacterium GV3101 competent cells. After gentle mixing, the cells were incubated on ice for 5 min, then in liquid nitrogen for 5 min. They were then transferred to a 37 ℃ water bath for 5 min, followed by an ice-water bath for 5 min. In a clean bench, 700 μL of LB liquid medium equilibrated to room temperature was added, and the cells were incubated at 28 ℃ and 200 rpm for 2 h. After centrifugation at 6000 rpm for 1 min, 500 μL of the supernatant was discarded. The remaining liquid was suspended, and 80 μL was spread onto LB solid medium containing 50 mg / L Kanamycin, 50 mg / L Gentamicin, and 25 mg / L Rifampin. The cells were incubated upside down at 28 ℃ for 36–48 h until single colonies appeared.

[0067] 3.3 Injection into the lower epidermis of native tobacco leaves Single colonies of positive Agrobacterium GV3101 were picked and cultured in YEB liquid medium containing Kan, Gent, and Rif at 28°C with shaking for 24 hours. Then, 500 μL of the bacterial culture was added to 40 mL of YEB triple-antibiotic (Kan, Gent, and Rif) liquid medium containing 500 μL of 1M MES and 5 μL of 200 mMAS, and cultured at 28°C with shaking until the OD600 value was between 1.2 and 1.5. After collection, the colonies were resuspended in an invasion dye solution (containing 1 mL of 1M MES, 1 mL of 1M MgCl2, and 100 μL of 200 mMAS per 100 mL ddH2O) to achieve an OD600 value of 1.0. After standing in the dark for 4 hours, the colonies were injected into the lower epidermis of 4-week-old tobacco leaves and cultured in a plant tissue culture room for 36 hours.

[0068] 3.4 Observation of leaf fluorescence using laser confocal microscopy Will contain p BE- ​ Agrobacterium-mediated activation of the GFP plasmid, followed by the preparation of a resuspension and injection into tobacco lower epidermal cells to transform the empty vector. p Tobacco containing BE-35S-GFP was used as a blank control. The location of green fluorescent GFP was observed under a laser confocal microscope.

[0069] Tobacco leaves from the infected area were cut and placed in a petri dish. DAPI (4',6-diamidinyl-2-phenylindole) was dropped onto the leaves and stained for 20 min. The leaves were then washed twice with PBS buffer, 20 min each time. The stained leaves were placed on a glass slide and the lower epidermis was observed using a laser confocal microscope.

[0070] Observation results as follows ​ As shown, in plants injected with empty vectors, fluorescence was localized to the cell nucleus, cell membrane, and cytoplasm. p BE- ​Tobacco containing the -GFP fusion vector exhibits green fluorescence located in the cell nucleus. (The above...) ​ The results showed ​ Transcription factor proteins are located in the cell nucleus, which is consistent with the classic functional characteristics of transcription factors.

[0071] Example 4 Gene editing ​ Obtaining genetically modified alfalfa 4.1 Gene editing vector p5CBC- p 6401- ​ Construction Gene editing methods for constructing alfalfa using the CRISPR / Cas9 genome editing system of alfalfa and its application (Chinese Patent 202110884895.X). ​ Gene-edited strains.

[0072] Based on the above alfalfa CRISPR / Cas9 dual mutant system, with alfalfa ​ The genome sequence was obtained, and the optimal dual target sites were selected using the online website https: / / crispor.gi.ucsc.edu / crispor.py. Target 1 was GGACTTGTAATTCATTAAGAGG (SEQ ID NO. 9), and target 2 was TTGATGATAGAGCCTCTATTGG (SEQ ID NO. 10). Primers were designed using the target sequences to amplify the dual target sites and ligate them into p5CBC and p6401 vectors. The resulting vectors were then ligated using enzyme digestion to form gene editing vectors containing two sgRNAs, such as... ​ As shown.

[0073] The specific steps are as follows: (1) Construction of sgRNA module: primers designed for two target sites ​ -DT1-BsF、 ​ -DT1-F0、 ​ -DT2-R0、 ​ -DT2-BsR and p The 5CBC vector was used to amplify sgRNA modules carrying target sequences via PCR. p 5CBC- ​ -DT1DT2.

[0074] Primers were constructed using the p5CBC empty plasmid and the editing vector. ​ -DT1-BsF、 ​ -DT1-F0、 ​ -DT2-R0、 ​ -DT2-BsR obtains targets ​-p5CBC-DT1T2 fragment, amplification system as shown in Table 4.

[0075] ​ -DT1-BsF: ATATATGGTCTCGCTTGGGACTTGTAATTCATTAAGGTT (SEQ IDNO.11); ​ -DT1-F0:GGGACTTGTAATTCATTAAGGTTTTAGAGCTAGAAATAGC(SEQ IDNO.12); ​ -DT2-R0:AACATAGAGGCTCTATCATCAACAATTTAATGGTTCGCTTGTA(SEQ IDNO.13); ​ -DT2-BsR: ATTATTGGTCTCGAAACATAGAGGCTCTATCATCAAC (SEQ ID NO. 14).

[0076] Table 4 ​ - p 5CBC-DT1T2 reaction system

[0077] PCR conditions were as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 15 sec; annealing at 60 ℃ for 30 sec; extension at 68 ℃ for 1 min, followed by 30 cycles of denaturation to extension; final extension at 68 ℃ for 5 min; recovery. ​ -p5CBC-DT1T2 fragment.

[0078] After the bridging PCR reaction was completed, the products were detected by 1% agarose gel electrophoresis. The gel block containing the target fragment was excised and the fragment was recovered using a gel recovery kit.

[0079] The gel-recovered fragments were sent for sequencing to obtain... ​ - p The sequence of the 5CBC-DT1T2 fragment is shown below.

[0080] ​-p5CBC-DT1T2 (807 bp): GGACTTGTAATTCATTAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTGCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCTTCGGCGTTCAATTTCTGGGGTTTTCTCTTCGTTTTCTGTAACTGAAACCTAAAATTTGACCTAAAAAAAATCTCAAATAATATGATTCAGTGGTTTTGTACTTTTCAGTTAGTTGAGTTTTGCAGTTCCGATGAGATAAACCAATAGAGTGTCGTTTTAGTAAAAAAAATTATTTTAAAATGAATATCATCACTTTTCAATATAGAATTATTATTTTACTTCCAATTATACCCTCTAATTAATTTCCAAAGCATTATACCAATAGTAAATAAAGTTAGTTTAGTAAAATTGTCATATCTTTTAACATTATTATTAGATTTCTTAATTTGTGTTTAAAAGCTTTAAACGATGATCATTTTTAAACAGAGAGTATAAAGTAGTAAAATAGTACTATTAGAAATGAATTGACGTGACATGCTATGAAAAGTCTGGAAGAGTATCGATAAAAGGCTACACTAGAGGTAGCTACTTATATGCGCAGGAACTGAAATCAAAAATGAAATAAAGGAGAAGGAAGATGCATGTTGTGTTATATAAGTGAAGGAGAAGGACTTGCATGTTGTGTTATATTTGCTTGTTTTAGTCCCACATCGACTGAAACAGAAAGTATCTCGGCGTTTATATACTACAAGCGAACCATTAAATTGTTGATGATAGAGCCTCTAT (SEQ ID NO.15).

[0081] (2) Construction of gene editing binary vector: Use p 6401 empty plasmid and ​The 5CBC-DT1T2 fragment was digested and ligated using Golden Gate enzymes. The reaction system is shown in Table 5. After mixing, the reaction was incubated at 37 ℃ for 5 h, 50 ℃ for 5 min, and 80 ℃ for 10 min. The resulting fragment was then transformed into *E. coli* DH5α. ​ -DT1-BsF、 ​ -DT2-BsR was used as primers. Positive colonies were screened by colony PCR. After the reaction, 10 μL of PCR product was taken and electrophoresed on a 1% agarose gel for detection.

[0082] Table 5 Golden Gate enzyme digestion and ligation reaction

[0083] 4.2 Gene Editing Vectors p 5CBC- p 6401- ​ Transformation of Agrobacterium tumefaciens EHA105 Gene editing vectors with correct sequencing p 5CBC- p 6401- ​ Transformation of Agrobacterium EHA105. The transformation method is as follows: Add 10 μL of recombinant plasmid and 50 μL of EHA105 Agrobacterium competent cells to a 1.5 mL Eppendorf tube and place on ice for 5 min. After flash freezing in liquid nitrogen for 5 min, transfer to a 37 ℃ metal bath for heat shock for 5 min, add 1 mL of LB medium, and incubate at room temperature for 2 h. Incubate at 28 ℃ and 150 rpm for 3 h. Centrifuge at 8000 rpm for 10 min. Discard 250 μL of supernatant, mix the cells again by pipetting, and spread 100 μL onto LB solid medium (50 mg / L kan, 50 mg / L Rif) and incubate upside down at 28 ℃ for 48 h.

[0084] 4.3 Agrobacterium EHA105-mediated leaf disc transformation of alfalfa (1) Planting of alfalfa: Select wild-type alfalfa seeds of 'Zhongmu No. 1' with plump and uniform size, treat with concentrated sulfuric acid for 5 min, wash with sterile water 6 times, disinfect with sodium hypochlorite rinsing solution for 6 min, wash with sterile water 8 times, use moist sterile filter paper, germinate in the dark, transplant into flower pots containing vermiculite and nutrient soil in a 3:2 (v:v) ratio, and cultivate in a greenhouse under the following conditions: 16 h light / 8 h darkness, temperature 22 ℃, light intensity of 200 umol / m2 / s, and humidity 60%.

[0085] (2) Agrobacterium activation: Activation of bacteria containing the target gene ​A single colony of Agrobacterium EHA105 was inoculated into 5 mL of LB liquid medium. Rifampicin (50 mg / L) and kanamycin (50 mg / L) were added at a ratio of 1:1000. The culture was shaken at 28 °C and 200 rpm for 12 h. 1 mL of the culture was then inoculated into 30 mL of LB liquid medium for reactivation until the OD600 value reached 0.6-0.8. An appropriate amount of SH3α liquid medium was added to resuspend the cells until the OD600 value reached 0.2-0.3.

[0086] (3) Explant preparation, infection, and co-culture: Use sterile scissors to cut four-week-old leaves in good growth condition. Disinfect the leaves with sterile water and hypochlorous acid rinsing solution (containing Tween 20), mix with infection solution, and in a vacuum dryer, infiltrate under vacuum of 0.09 MPa for 10 min. Then, perform ultrasonic treatment in a Branson 2510 ultrasonic cleaner for 5 min. Repeat the vacuuming once. Finally, place the leaves on SH3α co-culture medium.

[0087] (4) Screening, plant regeneration, and rooting: The explants were transferred to SH3α selective medium, and the corresponding medium was changed regularly. Subsequently, they were transferred to different mediums for subculture according to time. They were transferred to MSO medium for rooting culture and then transplanted into flower pots with a 3:2 (v:v) ratio of vermiculite and nutrient soil.

[0088] 4.4 Detection of Gene-Edited Transgenic Alfalfa The transgenic lines were validated using PCR, RT-qPCR, and Sanger sequencing.

[0089] First, DNA was extracted from leaves of transgenic plants that had survived (approximately 4 weeks) for PCR identification. ​ As shown in A, the primers used ​ -DT1-BsF and ​ -DT2-BsR, PCR conditions were as follows: pre-denaturation at 98℃ for 5 min; denaturation at 98℃ for 10 sec; annealing at 57℃ for 30 sec; extension at 68℃ for 1 min, denaturation to extension for 30 cycles; final extension at 68℃ for 10 min; storage at 4℃.

[0090] RNA was extracted from leaves of positive strains and reverse transcribed into cDNA. Then, quantitative real-time RT-qPCR was used to calculate the cDNA values ​​for each strain. MsMYBS3 The relative expression level. MsMYBS3 -QF and MsMYBS3 -QR is the primer, and the procedure was performed according to the instructions of the TAKARA Biotech Real-Time Fluorescence Kit (RR047Q).

[0091] like Figure 5As shown in B, compared to wild-type (WT), gene editing... MsMYBS3 The expression levels of transgenic lines 2, 3, 4, 5, 6, and 7 were significantly reduced.

[0092] DNA was collected from leaves of the three lines with the lowest expression levels (ED-2, ED-6, and ED-7) that tested positive for gene editing. Genotyping of these gene-edited lines was performed, and primers containing two target sequences were designed. MsMYBS3 -p6401-F (sequence shown in SEQ ID NO.16) and MsMYBS3 -p6401-R (sequence shown in SEQ ID NO.17) was cloned under the same PCR conditions as in Example 1, and then mixed with pEASY. ® -Blunt was ligated and transformed into E. coli, and positive bacteria were verified by colony PCR and sent to bioengineering for testing.

[0093] MsMYBS3 -p6401-F:ATTAAATAGTGTGGTGTGCGTCC (SEQ ID NO.16); MsMYBS3 -p6401-R: CTCTTCTTCTGTCCATGGAACTC (SEQ ID NO. 17).

[0094] Target sequencing results as follows Figure 5 As shown in C, the sequencing peak diagram is as follows: Figure 6 As shown. The results show that it contains MsMYBS3 The gene-editing vector containing the gene target sequence has been transferred into the alfalfa genome and three gene edits have been obtained. MsMYBS3 Transgenic lines. Lines ED-2, ED-6, and ED-7 each have at least three different knockout patterns. Lines ED-2 and ED-6 have undergone ternary allele editing and retained one wild-type (WT) allele, while line ED-7 has all four alleles mutated. Therefore, mutant lines were selected. MsMYBS3 -ED-2、 MsMYBS3 -ED-6、 MsMYBS3 -ED-7 will be used for subsequent related experiments.

[0095] Example 5 Developmental phenotype and cold tolerance experiments of gene-edited transgenic lines 5.1 Cuttings of wild-type and gene-edited transgenic alfalfa Select gene editing with good growth MsMYBS3Transgenic and wild-type alfalfa lines were propagated by cuttings in the same batch and allowed to grow in a greenhouse (22℃, 16 h light / 8 h dark). The cuttings were 10 cm long and contained one stem node. Each line had at least 30 replicates. After rooting for about 2 weeks, the cuttings were transplanted into square pots (10 cm × 10 cm) containing vermiculite and potting soil in a ratio of 3:2.

[0096] 5.2 Developmental phenotype of gene-edited lines Select seedlings of each line that have grown uniformly and in good condition 4 weeks after cutting. Figure 7 In the figure, A represents the phenotype of alfalfa line that has grown normally for 4 weeks; Figure 7 B in the figure represents the leaf phenotype after 4 weeks of normal growth), photographs were taken and the plant heights of the mutant lines and wild-type alfalfa plants were measured. Figure 7 C) Number of first-order lateral branches ( Figure 7 E in the middle), number of secondary branches ( Figure 7 F in the middle), number of compound leaves ( Figure 7 (D) Number of main stem nodes ( Figure 7 G in the middle), fresh weight ( Figure 7 H) and dry weight ( Figure 7 (H in the text).

[0097] according to Figure 7 As a result, compared with the control (WT) plants, the gene-edited lines showed a significant decrease in plant height, a reduction in the number of secondary branches, and an increase in the number of primary branches.

[0098] 5.3 Cold resistance experiment of gene-edited lines The selected cuttings showed uniform growth and good condition after 4 weeks of growth, indicating gene editing. MsMYBS3 Cold stress experiments were conducted on transgenic lines and wild-type alfalfa. At least 20 plants from each line were used in the cold stress experiment. The cold stress treatment conditions were as follows: plants from each line were randomly placed in a freezing room. After the temperature stabilized, it was linearly decreased to -5°C at a rate of 1°C per hour, maintained at 1 hour, and then incubated at 4°C overnight. After 2 days of recovery at room temperature, the survival rate and electrical conductivity were measured. Phenotypes before freezing and after 2 days of recovery were photographed and recorded.

[0099] like Figure 8 As shown, the results indicate that gene editing significantly improves performance compared to the control (WT). MsMYBS3 Transgenic lines have lower survival rates, poorer cell membrane permeability, are more susceptible to damage, and are significantly less cold-resistant.

[0100] The above results indicate that MsMYBS3 Positively regulating the cold resistance of alfalfa, knocking out [a specific substance] in mutants. MsMYBS3 It can significantly reduce the adaptability of alfalfa to cold stress. MsMYBS3Transcription factors can regulate the expression of stress-resistance genes and improve the cold resistance of alfalfa.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kind MsMYBS3 The application of genes or their biological materials in regulating the cold resistance of alfalfa, characterized by, The MsMYBS3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

4.

2. The application according to claim 1, characterized in that, The MsMYBS3 The gene polynucleotides are shown in (a), (b), or (c): (a) Polynucleotides as shown in SEQ ID NO.3; (b) A polynucleotide that has more than 90% homology with the polynucleotide shown in SEQ ID NO.3; (c) A polynucleotide mutant with the same functional activity obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID NO.

3.

3. The application according to claim 2, characterized in that, The biomaterial includes any one of the following (1) to (3): (1) An expression cassette containing the polynucleotides described in (a), (b) or (c); (2) A recombinant vector containing the polynucleotides described in (a), (b) or (c) or a recombinant vector containing the expression cassette described in (1); (3) A recombinant microorganism containing the polynucleotides described in (a), (b) or (c), a recombinant microorganism containing the expression cassette described in (1) or a recombinant microorganism containing the recombinant vector described in (2).

4. The application according to any one of claims 1 to 3, characterized in that, The regulation is any one of the following: S1, by reducing the MsMYBS3 Gene expression levels and / or activity reduce the cold resistance of the alfalfa. S2, By improving the above MsMYBS3 The expression level and / or activity of the gene improve the cold resistance of the alfalfa.

5. The application according to claim 4, characterized in that, Reduce the MsMYBS3 Gene expression levels include suppression, silencing, or knockout using the CRISPR / Cas system. MsMYBS3 Gene; The CRISPR / Cas system targets gene editing sites 1 and 2, with the sequence of target 1 shown in SEQ ID NO.9 and the sequence of target 2 shown in SEQ ID NO.

10.

6. MsMYBS3 The application of genes or their biological materials in the breeding of cold-resistant transgenic alfalfa, characterized in that, The MsMYBS3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

4.

7. The application according to claim 6, characterized in that, The biomaterial includes any one of the following: V1, reduce the MsMYBS3 Recombinant vectors for gene expression levels and / or activity; V2, Recombinant bacteria containing the recombinant vector described in V1; The MsMYBS3 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

8. A method for regulating the cold resistance of alfalfa, characterized in that, The method includes reducing the receptor in alfalfa as described in claim 1. MsMYBS3 The steps for determining the expression level of genes to obtain transgenic alfalfa; the transgenic alfalfa has lower cold resistance than the recipient alfalfa.

9. The method according to claim 8, characterized in that, Reduce the MsMYBS3 Gene expression levels include suppression, silencing, or knockout using the CRISPR / Cas system. MsMYBS3 Gene.

10. The method according to claim 9, characterized in that, The CRISPR / Cas system targets gene editing sites 1 and 2, with the sequence of target 1 shown in SEQ ID NO.9 and the sequence of target 2 shown in SEQ ID NO.10.

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