Application of molecular module for regulating and controlling cold resistance of alfalfa in improving cold resistance of alfalfa

Abstract

The invention belongs to the technical field of agricultural biology, and provides application of a molecular module for regulating and controlling the cold resistance of alfalfa in improving the cold resistance of alfalfa, and the molecular module for regulating and controlling the cold resistance of alfalfa is MsmiR319-MsTCP3-MsXTH9. Through overexpression of MsmiR31, inhibition of MsTCP3 or / and inhibition of expression of MsXTH9, the cold resistance of medicago sativa can be significantly improved. The MsmiR319-MsTCP3-MsXTH9 module has an important application value in the genetic improvement of the medicago sativa L..

Description

Technical Field

[0001] This application relates to the field of agricultural biotechnology, and specifically, this application provides the application of a molecular module for regulating alfalfa cold resistance in improving the cold resistance of alfalfa. Background Technology

[0002] Alfalfa (Medicago sativa L.) is one of the most important legume forage crops, playing a vital role in the stable and healthy development of my country's grassland animal husbandry. In Inner Mongolia and other major livestock bases, the overwintering of alfalfa is crucial for the food supply of cattle and sheep and the healthy development of animal husbandry in the following year. Alfalfa is a perennial plant, and its ability to regrow in spring after enduring low autumn temperatures and severe winter cold has a significant impact on its yield and quality. Therefore, identifying functional genes for cold resistance in alfalfa and elucidating their molecular mechanisms is of great significance for creating new alfalfa materials with significantly improved cold resistance. miR319 was the first miRNA discovered through positive gene mutation screening (Xue Jukun, et al., 2019). As one of the most conserved miRNAs, it plays an important role in plant organ morphogenesis, hormone signal transduction, and stress resistance. The TEOSINTE BRANCHED 1 / CYCLOIDEA / PCF (TCP) transcription factor is plant-specific, containing a bHLH domain. It is a basic helix-loop-helix structure composed of 59 amino acid residues and can interact with DNA or proteins (Koyama T, et al., 2017). miR319 primarily functions by forming the miR19-TCP molecular regulatory pathway with downstream TCP transcription factors. Summary of the Invention

[0003] On the one hand, this application provides the application of a molecular module for regulating alfalfa cold resistance in improving the cold resistance of alfalfa, wherein the molecular module for regulating alfalfa cold resistance is MsmiR319-MsTCP3-MsXTH9.

[0004] Furthermore, the application uses overexpression of MsmiR319, inhibition of MsTCP3 expression, and / or inhibition of MsXTH9 expression to improve the cold resistance of alfalfa.

[0005] Furthermore, the nucleotide sequence of MsmiR319 is SEQ ID NO.7.

[0006] Furthermore, MsmiR319 was overexpressed by introducing an overexpression vector.

[0007] Furthermore, the nucleotide sequence of MsTCP3 is SEQ ID NO.9.

[0008] Furthermore, the function of MsTCP3 can be achieved by inhibiting its transcriptional activation activity in the application; for example, MsTCP3SRDX with the nucleotide sequence SEQ ID NO.11 can inhibit MsTCP3 expression.

[0009] Furthermore, the nucleotide sequence of MsXTH9 is SEQ ID NO.13.

[0010] Furthermore, the improvement of alfalfa's cold resistance includes increasing the chlorophyll content and soluble sugar content in the plant, thereby increasing the plant's survival rate.

[0011] On the other hand, this application provides the application of the above-mentioned application in the breeding of cold-resistant varieties of alfalfa.

[0012] On the other hand, this application provides a cold-resistant alfalfa variety in which MsmiR319 is overexpressed, MsTCP3 expression is inhibited, and / or MsXTH9 expression is inhibited.

[0013] Overexpression in this application can be performed using various known and available overexpression vectors. Inhibition of expression can be performed by knockout or knockdown of expression levels. Specifically, it can be performed using interference methods such as siRNA and shRNA, SRDX transcription factor inhibition methods, mutation methods, CRISPR-based gene editing methods, or other known biological methods in the research.

[0014] Overexpression of MsmiR319 or inhibition of MsTCP3 expression can significantly increase the cold resistance of alfalfa. The MsmiR319-MsTCP3-MsXTH9 module has important potential application value in the genetic improvement of alfalfa. Attached Figure Description

[0015] Figure 1 The results of PCR identification and quantitative detection of miR319-OX plants are as follows (Part A, PCR identification results of miR319-OX plants: M, DNA ladder AL2000, +, pZH01-miR319-OX plasmid positive control, -, wild-type (WT) alfalfa DNA negative control, 1-21, resistant seedling DNA samples; Part B, expression level of mature miR319 in wild-type and transgenic seedlings; Note: Values ​​represent mean ± SD (n=3)).

[0016] Figure 2PCR identification and quantitative detection results of MIM319 transgenic plants (Part A, PCR identification results of MIM319 transgenic plants: M, DNA Marker, +, positive control; Part B, quantitative analysis of miR319B in MIM319B (M) gene-positive plants; Note: Values ​​represent mean ± SD (n=3)).

[0017] Figure 3 The results of PCR identification and expression level detection of MsTCP3-SRDX plants are as follows (Part A, PCR identification of MsTCP3-SRDX plants: M, DNA ladder AL2000, +, pZH01-MsTCP3-SRDX plasmid positive control, -, wild-type alfalfa DNA negative control, 1-21, resistant seedling DNA sample; Part B, relative expression level detection of MsTCP3-SRDX; Note: Values ​​represent mean ± SD (n=3)).

[0018] Figure 4 Results of obtaining and identifying MsXTH-OX transgenic seedlings (Part A: WT and transgenic seedling photos; Part B: PCR identification of MsXTH-OX transgenic seedlings: M, DNA ladder AL5000, +, PCGW-MsXTH-OX plasmid positive control, -, wild-type (WT) alfalfa DNA negative control; 1, 2, 5, 10, DNA samples of resistant seedlings; Note: Values ​​represent mean ± SD (n=3)).

[0019] Figure 5 Results of cold resistance experiments on WT, MIM319, and OE319 lines (A: WT and miR319-OX plants treated at 0d and 21d, scale bar 5cm; B: Close-up of damaged leaves, scale bar 1cm; C: WT and MIM319 plants treated at 0d and 21d, scale bar 5cm; D: Close-up of damaged leaves, scale bar 1cm; E, F: Chlorophyll content and soluble sugar content of WT and miR319-OX plants treated at 0d and 21d; G, H: Chlorophyll content and soluble sugar content of WT and MIM319 plants treated at 0d and 21d; I, J: Phenotype and survival rate of WT and miR319-OX plants treated at -7℃; K, L, Phenotypic and survival rate of WT and MIM plants treated at -7℃; Note: Values ​​represent mean ± SD (n=3); different lowercase letters indicate significant differences (P<0.05); all indicators are converted to fresh weight.

[0020] Figure 6The results of the cold resistance and freezing tolerance experiments of WT and SR-TCP3 lines are as follows (Part A: WT and SR-TCP3 plants treated at 0d and 21d, scale bar is 5cm; Part B: close-up of damaged leaves, scale bar is 1cm; Parts C and D: chlorophyll content and soluble sugar content of WT and SR-TCP3 plants treated at 0d and 21d; Parts E and F: phenotype and survival rate of WT and SR-TCP3 plants treated at -7℃; Note: Values ​​represent mean ± SD (n=3); different lowercase letters represent significant differences (P<0.05); all indicators are converted to fresh weight).

[0021] Figure 7 Results of MsTCP3 activating MsXTH9 expression (Part A, MsXTH9 expression level in SR-TCP3 plants; Part B, yeast single-hybrid verification that MsTCP3 can bind to the MsXTH9 promoter; Part C, dual-luciferase verification that MsTCP3 can bind to the MsXTH9 promoter; Part D, EMSA verification that MsTCP3 can bind to the MsXTH9 promoter; Note: Values ​​represent mean ± SD (n=3); different lowercase letters indicate significant differences (P<0.05)).

[0022] Figure 8 The results of cold resistance and freezing tolerance experiments for WT and OEXTH lines are as follows (Part A: WT and OEXTH plants treated at 0d and 35d, scale bar 5cm; Part B: Close-up of damaged leaves, scale bar 1cm; Parts C and D: Chlorophyll content and soluble sugar content of WT and OEXTH plants treated at 0d and 35d; Parts E and F: Phenotype and survival rate of WT and OEXTH plants treated at -7℃; Note: Values ​​represent mean ± SD (n=3); different lowercase letters indicate significant differences (P<0.05); all indicators are converted to fresh weight). Detailed Implementation

[0023] Example 1: Cloning and expression vector construction of MsmiR319-MsTCP3-MsXTH9 gene

[0024] Cloning primers covering the entire cDNA sequence were designed based on three generations of database data (MsMIR319B_XbaF: TCTAGACCATCTTATATTTAACCAACAATGT, SEQ ID NO.1; MsMIR319B_KpnR: GGTACCATGTGCGAACCTGTTGGAATC, SEQ ID NO.2). RNA was extracted from alfalfa (Zhongmu 1), and cDNA was reverse transcribed into a template. PCR was performed using primers MsMIR319B_XbaF: TCTAGACCATCTTATATTTAACCAACAATGT, SEQ ID NO.1; MsMIR319B_KpnR: GGTACCATGTGCGAACCTGTTGGAATC, SEQ ID NO.2 to clone the MsMIR319B coding region sequence. The PCR product was digested with restriction endonucleases XbaI and KpnI. The digested product was then ligated to the vector backbone obtained by double digestion of pZH01 vector with XbaI and KpnI. The recombinant vector with the correct sequence was labeled as pZH01-Ms miR319.

[0025] Using Target mimicry™ technology, the miR399 binding site sequence in the Arabidopsis thaliana IPS1 gene was replaced with an incompletely complementary miR319 sequence, forming a miR319 interfering TM sequence (MIM319B). Figure 2-4 The MIM319B gene sequence was inserted into the plant expression vector pZh01 between XbalⅠ and SalⅠ using a double enzyme digestion and ligation reaction, and expression was initiated by the 35S promoter. The hygromycin gene was used as a selection marker gene.

[0026] Cloning primers covering the entire cDNA sequence were designed based on three generations of database data (MsTCP3SRDX_XbaF: TCTAGAAATGAACGGTGGAGAAATAGTGG, SEQ ID NO.3; MsTCP3SRDX_KpnR: GCTTCCTCTGATTCTCACCGTGGATCCCTGGATCTGGATCTGGAACTGCGCCTGGGCTTTGCGTAAGTCGAC, SEQ ID NO.4). RNA was extracted from alfalfa (Zhongmu 1), and cDNA was reverse transcribed into a template. PCR was performed using primers MsTCP3SRDX_XbaF: TCTAGAAATGAACGGTGGAGAAATAGTGG, SEQ ID NO.3; MsTCP3SRDXKpnR: GCTTCCTCTGATTCTCACCGTGGATCCCTGGATCTGGATCTGGAACTGCGCCTGGGCTTTGCGTAAGTCGAC, SEQ ID NO.4, to clone the MsTCP3 coding region sequence. The PCR product was digested with restriction endonucleases XbaI and KpnI. The digested product was then ligated to the vector backbone obtained by double digestion of pZH01 vector with XbaI and KpnI. The recombinant vector with the correct sequence was labeled as pZH01-MsTCP3SRDX.

[0027] Cloning primers covering the entire cDNA sequence were designed based on three generations of database data (MsXTH9_XbaF: TCTAGAATGACTAGGATGTCTTGTCTC, SEQ ID NO.5; MsXTH9_BamHIR: GGATCCCTAGTGGTGGTGACGAACAC, SEQ ID NO.6). RNA was extracted from alfalfa (Zhongmu 1), and cDNA was reverse transcribed into a template. PCR was performed using primers MsXTH9_XbaF: TCTAGAATGACTAGGATGTCTTGTCTC, SEQ ID NO.5; MsXTH9_BamHIR: GGATCCCTAGTGGTGGTGACGAACAC, SEQ ID NO.6 to clone the MsTCP3 coding region sequence. The PCR product was digested with restriction endonucleases XbaI and BamHI. The digested product was then ligated to the vector backbone obtained by double digestion of pZH01 vector with XbaI and BamHI. The recombinant vector with the correct sequence was labeled as PC-GW-MsXTH9.

[0028] CDS sequence:

[0029] MsmiR319b, SEQ ID NO.7

[0030] CCATCTTATATTTAACCAACAATGT TTGTTGAATATGTATATATCACACAGATCTAGTTGATAGAATCATGGAAGGATGCATGGCGAATGGTAATGAGAGAGAGAGAGCTTTCTTTAGTCCACTCATGGGTTGCCGTAAGATTTAATTAGCTGCTGACTCATTCATCCAAATGCTGAGTGAATATACTTTTAAGTATACTCAGCAAATGAGTGAATGATGCGGGAGACAAATTGAATCTTAAGCTTCCTGTATTTGGACTGAAGGGAGCTCCCTTTCTTTCTTTCTTTCTTTCT TTCTTCCTATTATCTCTTTCATTTTGCTTCTGATCTCTTTCTTTTAATCTCTTTTTCCTTTAAATTTTCATATTTGTGTTTAGTTATTGCATACACATGAGATTTATCTAACTAAAAGTACCTGCACGTTCTTACCCCTTTAAGGAAATATAATTAATTAATTAAACCTTTCTTCTTGTTATGACTGGCTTGTGTAAGATCTTGGACTATACTGTATTTACTTTAATAGTAATTAACAATGGTTGTGGGGTTCCTTTGGTACGGTAAAATG GATTCCAACAGGTTCGCACAT

[0031] >MIM319B SEQ ID NO. 8

[0032] AAAACACCACAAAAACAAAAGAAAAATGGCCATCCCCTAGCTAGGTGAAGAAGAATGAAAACCTCTAATTTATCTAGAGGTTATTCATCTTTTAGGGGATGGCCTAAATACAAAATGAAAACTCTCTAATTAAGTGGTTTTGTGTTCATGTAAGGAAAGCGTTTTAAGATATGGAGCAATGAAGACTGCAGAAGGCTGATTCAGACTGCGAGTTTTGTTTATCTCCCTCTAGAAA GGGAGCTCCTCTATTCAGTCCAAAGCTTCGGTTCCCCTCGGAATCAGCAGATTATGTATCTTTAATTTTGTAATACTCTCTCTCTTCTCTATGCTTTGTTTTTCTTCATTATGTTTGGGTTGTACCCACTCCCGCGCGTTGTGTGTTCTTTGTGTGAGGAATAAAAAAATATTCGGATTTGAGAACTAAAACTAGAGTAGTTTTATTGATATTCTTGTTTTTCATTTAGTATCTAATAAGTTTGGAGAATAGTCAGACCAGTGCATGTAAATTTGCTTCCGATTCTCTTTATAGTGAATTCCTCTT

[0033] > MsTCP3,SEQ ID NO.9

[0034] ATGAACGGTGGAGAAATAGTGGAAGTTGACGGAGGTCACGTTATAAGGTCAAGAGGTCGAAAAGATCGT CATAGTAAAGTTTGCACGGCGAAAGGGCCGAGAGACAGACGTGTCCGTCTCTCGGCCCATACCGCTATTGAATTTTA CGATGTTCAAGATCGACTGGGCTTTGATAGGCCCAGTAAAGCACTTGATTGGCTCATTAATAAAGCTAAGCCTGCCA TCGACCAACTCGCTCATCTTCCTCCATGGAAACCTACTCTTTTCAAACAGCAACAGCAAAAGCAAAACGACGACGTT TTAGAGAAACAGAATCAAAACGAAAACGAAAACAACGAGTTTCGTTTTCTTCAGAATTTTAGCAATAATAGTAACAG CAACTGTTTCATTCCGTTTCAAAATGAAATACCTGAAACGGTGTCGTTTAGTAATGATTTGAAGCTTTCGCTTCAAC AGAATCATAATCAGAATCAACACGTGCAACAGCACGTGCTTTTCGCCGGGAATTTTGATGGAATGGTGACGTGGAAT TCCGGTGGTGGTGCTGCTACTATTGATACTGGTAGTGGTGGCGGTGGCGGTGGTGGTGATGGTTTTGTGTTTCGTGG TTCTTCGCCGTCGCCGGTGGTTTTTCCGGCGATGATGTATGGTCAGAATCAGTATCTTTCACAGAGGGGACCCCTTC AGTCCAGTTACAATCCTTCAGTTCGTGCTTGGATAGATGCGCCGGCGACATTTGTTGCGGCTGATTATCGACGTCAA GCGGGTGGTTCGGGTGCCGGATTTGCTTCTGGTGGCTTTTCTGGTTTCCGTGTGCCGGCACGAATTGGAGGTGATGA TGAGGAGATGCATGGCGGTTTATCTGACAGGCCGTCGTCTGCTTCCTCTGATTCTCACCGTTGA

[0035] Amino acid sequence

[0036] > MsTCP3,SEQ ID NO.10

[0037] MNGGEIVEVDGGHVIRSRGRKDRHSKVCTAKGPRDRRVRLSAHTAIEFYDVQDRLGFDRPSKALDWLIN KAKPAIDQLAHLPPWKPTLFKQQQQKQNDDVLEKQNQNENENNEFRFLQNFSNNSNSNCFIPFQNEIPETVSFSNDL KLSLQQNHNQNQHVQQHVLFAGNFDGMVTWNSGGGAATIDTGSGGGGGGGDGFVFRGSSPSPVVFPAMMYGQNQYLS QRGPLQSSYNPSVRAWIDAPATFVAADYRRQAGGSGAGFASGGFSGFRVPARIGGDDEEMHGGLSDRPSSASSDSHR

[0038] > MsTCP3SRDX,SEQ ID NO.11

[0039] ATGAACGGTGGAGAAATAGTGGAAGTTGACGGAGGTCACGTTATAAGGTCAAGAGGTCGAAAAGATCGT CATAGTAAAGTTTGCACGGCGAAAGGGCCGAGAGACAGACGTGTCCGTCTCTCGGCCCATACCGCTATTGAATTTTA CGATGTTCAAGATCGACTGGGCTTTGATAGGCCCAGTAAAGCACTTGATTGGCTCATTAATAAAGCTAAGCCTGCCA TCGACCAACTCGCTCATCTTCCTCCATGGAAACCTACTCTTTTCAAACAGCAACAGCAAAAGCAAAACGACGACGTT TTAGAGAAACAGAATCAAAACGAAAACGAAAACAACGAGTTTCGTTTTCTTCAGAATTTTAGCAATAATAGTAACAG CAACTGTTTCATTCCGTTTCAAAATGAAATACCTGAAACGGTGTCGTTTAGTAATGATTTGAAGCTTTCGCTTCAAC AGAATCATAATCAGAATCAACACGTGCAACAGCACGTGCTTTTCGCCGGGAATTTTGATGGAATGGTGACGTGGAAT TCCGGTGGTGGTGCTGCTACTATTGATACTGGTAGTGGTGGCGGTGGCGGTGGTGGTGATGGTTTTGTGTTTCGTGG TTCTTCGCCGTCGCCGGTGGTTTTTCCGGCGATGATGTATGGTCAGAATCAGTATCTTTCACAGAGGGGACCCCTTC AGTCCAGTTACAATCCTTCAGTTTCGTGCTTGGATAGATGCGCCGGCGACATTTGTTGCGGCTGATTATCGACGTCAA GCGGGTGGTTCGGGTGCCGGATTTGCTTCTGGTGGCTTTTCTGGTTTCCGTGTGCCGGCACGAATTGGAGGTGATGA TGAGGAGATGCATGGCGGTTTATCTGACAGGCCGTCGTCTGCTTCCTCTGATTCTCACCGTGGATCCCTGGATCTGG ATCTGGAACTGCGCCTGGGCTTTGCGTGA

[0040] >MsTCP3SRDX,SEQ ID NO.12

[0041] MNGGEIVEVDGGHVIRSRGRKDRHSKVCTAKGPRDRRVRLSAHTAIEFYDVQDRLGFDRPSKALDWLINKAKPAIDQLAHLPPWKPTLFKQQQQKQNDDVLEKQNQNENENNEFRFLQNFSNNSNSNCFIPFQNEIPETVSFSNDLKLSLQQNHNQNQHVQQHVLFAGNFDGMVTWNSGGGAATIDTGSGGGGGGGDGFVFRGSSPSPVVFPAMMYGQNQYLSQRGPLQSSYNPSVRAWIDAPATFVAADYRRQAGGSGAGFASGGFSGFRVPARIGGDDEEMHGGLSDRPSSASSDSHRGSLDLDLELRLGFA

[0042] > MsXTH9,SEQ ID NO.13

[0043] ATGACTAGGATGTCTTGTCTCTTGGGACTTTCTTTGTGTTTTTTGTTTGTTGGGTTAGTTGCTTCTTCTAAATTTGAGGAACTCTTCCAACCTGGTTGGGCTATGGACCATTTTGTCCATGAAGGAGATTTGCTTAAACTCAAACTTGACAATTATTCTGGTGCTGGATTTCAATCTAAGAGCAAGTATATGTTTGGGAAAGTGACTGTGCATCTCAAACTTGTGGAGGGTGATTCTGCTGGAACTGTAACTGCTTTCTATATGTCATCTGAAGGCCCAACTCATAATGAATTTGATTTTGAGTTTTTGGGCAACACCACTGGTGAACCTTACTCAGTCCAAACCAATGTGTATGTGAATGGTGTTGGTAATAGGGAGCAAAGACTTAACCTATGGTTTGATCCATCTAAGGATTTTCACACCTACTCAATCTTTTGGAACCAACGTCAAGTAGTGTTTCTAGTGGATGAGACACCAATAAGGGTACACACAAATTTGGAACACAAAGGAATTCCCTTCCCAAAAGACCAAGCAATGGGTGTATACAGTTCTATATGGAATGCAGATGATTGGGCAACACAAGGTGGTCGTGTGAAGACAGATTGGAGTCATGCACCTTTCATTGCTACCTACAAAGATTTTGAGATCAATGCTTGTGAAGTTGCTGTGCCAGTGACATCAAATGATAATGCTAAGAAATGTGCTAGTAGTGAAGATAAGAAATACTGGTGGGATGAACCAATGTTGAATGAGCTCACTATTCATCAGAGTCATCAACTTATTTGGGTTAGGGCTAATCATATGGTCTATGACTATTGCGCCGATACCGCTAGGTTCCCGGCCATACCACTTGAATGTGTTCGTCACCACCACTAG

[0044] Amino acid sequence:

[0045] >MsXTH9, SEQ ID NO.14

[0046] MTRMSCLLGLSLCFLFVGLVASSKFEELFQPGWAMDHFVHEGDLLKLKLDNYSGAGFQSKSKYMFGKVTVHLKLVEGDSAGTVTAFYMSSEGPTHNEFDFEFLGNTTGEPYSVQTNVYVNGVGNREQRLNLWFDPSKDFHTYSIF WNQRQVVFLVDETPIRVHTNLEHKGIPFPKDQAMGVYSSIWNADDWATQGGRVKTDWSHAPFIATYKDFEINACEVAVPVTSNDNAKKCASSEDKKYWWDEPMLNELTIHQSHQLIWVRANHMVYDYCADTARFPAIPLECVRHHH

[0047] Carrier information:

[0048] For information on the pZh01 vector, see: Xiao H., Wang Y., Liu D., Wang W., Li X., Zhao X., Xu J., Zhai W., Zhu L. (2003) Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference. Plant Molecular Biology, 52, 957-966.

[0049] For PC-GW vector information, see: Dalal J, Yalamanchili R, La Hovary C, et al. Anovel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants. Plasmid. 2015;81:55-62.

[0050] Example 2: Obtaining transgenic plants with overexpression of MsmiR319 and suppression of MsTCP3 expression

[0051] Agrobacterium-mediated genetic transformation: The constructed vectors pZH01-Ms miR319, pZH01-MsTCP3SRDX, and PC-GW-MsXTH9 were transduced into Agrobacterium tumefaciens EHA105 using a heat shock transformation method. Successful transformation of the plant expression vectors into Agrobacterium tumefaciens EHA105 was confirmed by screening on YEP medium containing 100 mg / L kanamycin and 50 mg / L rifampin, and by colony PCR. Genetic transformation was then performed using the Agrobacterium-mediated genetic transformation method previously published in our laboratory, with the recipient plant being alfalfa cultivar Zhongmu 1. See: [1] Li Juan, Zhang Wanjun, Wang Tao. Study on the establishment of high-frequency plant regeneration system of alfalfa[J]. Acta Grasslandica Sinica, 2014, 22(04): 834-839; [2] Wang Kexin; Liu Yanrong; Teng Fengkui; Cen Huifang; Yan Jianping; Lin Shiwen; Li Dayong; Zhang Wanjun*; Heterogeneous expression of Osa-MIR156bc increases abioticstress resistance and forage quality of alfalfa, The Crop Journal, 2021: https: / / doi.org / 10.1016 / j.cj.2020.11.009.

[0052] Hygromycin-resistant plants were obtained by screening with pZH01-MsmiR319, transforming the MsmiR319 gene. Twenty-one hygromycin-resistant plants were obtained by pZH01-MsTCP3SRDX, transforming the MsTCP3SRDX gene. Four hygromycin-resistant plants overexpressing the MsXTH9 gene were obtained using PC-GW-MsXTH9.

[0053] PCR detection of MsmiR319 transgenic plants: To detect whether the vector fragment containing the target gene has been integrated into the plant genome, genomic DNA was extracted from young leaves of wild-type and resistant transgenic plants using the CTAB method. Using DNA from the resistant transgenic plants as a template, wild-type alfalfa plants (WT, i.e., recipient plants) as a negative control, and the plant expression vector as a positive control (+), a specific fragment of the MsmiR319 gene was amplified using primers:

[0054] 35S-F2: 5'-CGCACAATCCCACTATCCTTC-3' (SEQ ID NO. 15);

[0055] MsMIR319B-Kpm IR: 5'GGTACCATGTGCGAACCTGTTGGAATC-3' (SEQ ID NO. 16).

[0056] The results of the identification are as follows Figure 1 As shown, the transgenic positive plants amplified a band of the same size as the positive control (+), while the negative control (WT) did not amplify the target band, indicating that the vector fragment had been integrated into the plant genome. Twenty-one transgenic MsmiR319 positive plants were identified by PCR detection.

[0057] qRT-PCR detection of MsmiR319 transgenic plants: To detect the expression level of the target gene in transgenic plants, qRT-PCR was used to detect and analyze the expression level of the target gene in transgenic plants. Using cDNA from transgenic PCR-positive plants as a template and the housekeeping gene Actin as an internal reference gene, the relative expression level of the target gene in transgenic plants was detected. Figure 1 The quantitative primers are:

[0058] MsActin-F: CAAAAGATGGCAGATGCTGAGGAT (SEQ ID NO. 17);

[0059] MsActin-R: CATGACACCAGTATGACGAGGTCG (SEQ ID NO. 18);

[0060] MsmiR319-F: TCCACTCATGGGTTGCCGT (SEQ ID NO. 19);

[0061] MsmiR319-R: CCCGCATCATTCACTCATTTGC (SEQ ID NO. 20).

[0062] PCR detection of MIM319B transgenic plants: To detect whether the vector fragment containing the target gene has been integrated into the plant genome, genomic DNA was extracted from young leaves of wild-type and resistant transgenic plants using the CTAB method. Using DNA from the resistant transgenic plants as a template, wild-type alfalfa plants (WT, i.e., recipient plants) as a negative control, and the plant expression vector as a positive control (+), a specific fragment of the MIM319B gene was amplified using primers:

[0063] miR319_F CGGCGGTTGGACTGAAGGGT (SEQ ID NO. 21);

[0064] miR319_RGTGCAGGGTCCGAGGT (SEQ ID NO. 22).

[0065] The results of the identification are as follows Figure 2 As shown, the transgenic positive plants amplified a band of the same size as the positive control (+), while the negative control (WT) did not amplify the target band, indicating that the vector fragment had been integrated into the plant genome. Fifteen transgenic MIM319B positive plants were identified by PCR detection.

[0066] qRT-PCR detection of MIM319 transgenic plants: To detect the expression level of the target gene in transgenic plants, qRT-PCR was used to detect and analyze the expression level of the target gene in transgenic plants. Using cDNA from transgenic PCR-positive plants as a template and the housekeeping gene Actin as an internal reference gene, the relative expression level of the target gene in transgenic plants was detected. Figure 2 ).

[0067] PCR detection of MsTCP3SRDX transgenic plants: To detect whether the vector fragment containing the target gene has been integrated into the plant genome, genomic DNA was extracted from young leaves of wild-type and resistant transgenic plants using the CTAB method. Using DNA from the resistant transgenic plants as a template, wild-type alfalfa plants (WT, i.e., recipient plants) as a negative control, and the plant expression vector as a positive control (+), a specific fragment of the MsmiR319 gene was amplified using primers.

[0068] 35S-F2: 5'-CGCACAATCCCACTATCCTTC-3' (SEQ ID NO. 23);

[0069] MsTCP3-Bam-R:5'-GTCGACTTACGCAAAGCCCAGGC-3' (SEQ ID NO. 24).

[0070] The results of the identification are as follows Figure 3 As shown, the transgenic positive plants amplified a band of the same size as the positive control (+), while the negative control (WT) did not amplify the target band, indicating that the vector fragment had been integrated into the plant genome. Twenty transgenic MsTCP3SRDX gene-positive plants were identified by PCR detection.

[0071] qRT-PCR detection of MsTCP3SRDX transgenic plants: To detect the expression level of the target gene in transgenic plants, qRT-PCR was used to detect and analyze the expression level of the target gene in transgenic plants. Using cDNA from transgenic PCR-positive plants as a template and the housekeeping gene Actin as an internal reference gene, the relative expression level of the target gene in transgenic plants was detected. Figure 3 Quantitative primers:

[0072] MsTCP3-F: AAGGGCCGAGAGACAGACGTG (SEQ ID NO. 25);

[0073] MsTCP3-R: AAGGGCCGAGAGACAGACGTG (SEQ ID NO. 26).

[0074] Internal reference primer:

[0075] actin-F: CAAAAGATGGCAGATGCTGAGGAT (SEQ ID NO. 17);

[0076] actin-R: CATGACACCAGTATGACGAGGTCG (SEQ ID NO. 18).

[0077] PCR detection of MsXTH9 transgenic plants: To detect whether the vector fragment containing the target gene has been integrated into the plant genome, genomic DNA was extracted from young leaves of wild-type and resistant transgenic plants using the CTAB method. Using DNA from the resistant transgenic plants as a template, wild-type alfalfa plants (WT, i.e., recipient plants) as a negative control, and the plant expression vector as a positive control (+), a specific fragment of the MsmiR319 gene was amplified using [missing information - likely a primer name].

[0078] 35S-F2: 5'-CGCACAATCCCACTATCCTTC-3' (SEQ ID NO. 27);

[0079] MsXTH9-R:5'-GGATCCCTAGTGGTGGTGACGAACAC-3' (SEQ ID NO. 28).

[0080] The results of the identification are as follows Figure 3 As shown, the transgenic positive plants amplified a band of the same size as the positive control (+), while the negative control (WT) did not amplify the target band, indicating that the vector fragment had been integrated into the plant genome. Five transgenic MsXTH9 positive plants were identified by PCR detection. Figure 4 ).

[0081] Example 3: Analysis of Cold Resistance and Freezing Tolerance of Transgenic Alfalfa Plants Regulated by the MsmiR319-MsTCP3-MsXTH9 Module

[0082] WT and miR319-OX (OX) alfalfa were subjected to a 4℃ low-temperature treatment. At 21 days, close-up views of the terminal leaves showed that WT exhibited large areas of white frost spots. OX3, with low-level overexpression of miR319, showed similar characteristics to WT, while OX22 and OX16, with medium- and high-level overexpression, only accumulated anthocyanins and showed chlorosis at the leaf margins, with softened leaves and no obvious frost spots. Figure 5 Parts A and B).

[0083] WT and MIM319(M) alfalfa materials were treated with a 4℃ low-temperature treatment. M9, M2, and M1 were plants obtained in the laboratory earlier with suppressed MIR319B expression, and the degree of inhibition increased sequentially. After 21 days of treatment, all tested alfalfa plants showed chlorosis of the terminal leaves. With the increase of miR319 inhibition, the terminal leaves even turned white, wilted due to water loss, and curled inward on the underside of the leaves. In contrast, WT only showed chlorosis at the leaf margins. Figure 5 (Parts C and D); The above results indicate that under low temperature stress, alfalfa overexpressing MIM319 accumulates more reactive oxygen species and suffers more severe leaf damage than WT, suggesting that miR319 positively affects the cold resistance of alfalfa.

[0084] Resistance physiological indicators showed that at day 0, the soluble sugar content of all lines except OX16, which was lower than that of WT, was not different from that of WT. The soluble sugar content of the transgenic lines subjected to low temperature stress for 21 days was higher than that of WT, with the most significant difference between OX22 and WT. Figure 5 (E part). Compared with 0d, the chlorophyll content of both WT and OX decreased to varying degrees after 21d of low temperature, which is consistent with the leaf chlorosis and whitening after low temperature stress. At the same time, the chlorophyll content per unit fresh weight of OX lines was significantly higher than that of WT, with OX22 having the highest, followed by OX16, and OX3 having the lowest, which is consistent with the expression level of miR319. Figure 5 (part F).

[0085] To evaluate the damage to the physiological activities of MIM319 alfalfa under low-temperature stress, resistance physiological indicators were tested. After low-temperature stress, the soluble sugar content of all plants decreased, and the soluble sugar content of the MIM319 lines remained lower than the WT (wt), with M9 and M2 showing a significant difference. Figure 5 (Part G). Compared with 0 days of treatment at 4℃, chlorophyll content decreased in all experimental materials after 21 days. Compared with WT, M9 and M2 had significantly lower chlorophyll content, although M1 did not show a significant difference, it was still lower than WT. Figure 5 (H part).

[0086] The above-mentioned plant materials were subjected to freezing treatment at -7℃. After treatment, both WT and MIM319 showed softening of leaves and stems. After being moved to a greenhouse and restored to normal cultivation conditions for 21 days, the recovery of WT and MIM319 showed significant differences. Statistical analysis of regeneration rate revealed that the regeneration rate of WT reached approximately 80%, while the regeneration rate of MIM319 decreased from 70% to approximately 40% as the degree of miR319 inhibition increased. M2 and M1 showed significant differences compared to WT, with weaker regeneration ability. Figure 5 (Parts I and J); After freezing, WT and miR319-OX leaves softened, drooped, and lost water severely, but after 14 days of recovery, new buds appeared at the base and middle of the stem. Figure 5 As can be seen from the I and J sections, compared with WT, miR319-OX has more new shoots in each strain. Overall, the miR319-MsTCP3 module positively regulates bud regeneration after freezing and improves frost resistance.

[0087] Given that miR319 functions by targeting MsTCP3, this study further verified the positive regulation of cold hardiness of alfalfa by treating WT and MsTCP3-SRDX (SR) alfalfa at 4℃. Close-up images of leaf tips after 21 days of treatment showed that WT alfalfa exhibited significant chlorosis and whitening of leaf margins, while SR alfalfa only showed anthocyanin accumulation on the underside of leaves and reddening of the stem tips, with softened leaves but no chlorosis. Figure 6 (Parts A and B). At 0 days, the soluble sugar content of SR was slightly lower than that of WT, with no significant difference. However, after 21 days of low temperature, the accumulation of soluble sugar in all materials decreased. Among them, the soluble sugar content of SR alfalfa was higher than that of WT, with SR13 showing the most significant difference. Figure 6 (Part D); Low temperature stress reduced the chlorophyll content of leaves in all materials, but after 21 days of low temperature, the chlorophyll content of SR was significantly higher than that of WT. Figure 6 (Part C). After freezing, the leaves of WT and MsTCP3-SRDX softened, drooped, and lost water severely. However, after 7 days of recovery, new shoots appeared at the base and middle of the stem. A schematic diagram of the location of the new shoots was drawn, showing that compared with WT, the lines that inhibited MsTCP3 expression had more new shoots. Figure 6 (E and F sections). Overall, the miR319-MsTCP3 module positively modulates freeze resistance.

[0088] To further identify the target genes of MsTCP3 in alfalfa, this study detected the relative expression levels of the aforementioned candidate target genes in WT and MsTCP3-SRDX leaves. qRT-PCR results showed that MsXTH9 was the most heavily suppressed (…). Figure 7Part A). To confirm the regulatory relationship between MsTCP3 and MsXTH9, yeast single-hybrid assays, EMSA, and dual-luciferase assays were performed to identify and determine the TCP binding site on the MsXTH9 promoter ( ). Figure 7 (BD section).

[0089] This study further verified the positive regulatory function of miR319-MsTCP3-MsXTH9 on cold tolerance in alfalfa by treating WT and MsXTH9 alfalfa at 4℃. At 35 days, the terminal leaves of WT and OEXTH-5 wilted, and OEXTH-5 showed chlorosis. Figure 8 Parts A and B). Measurements of soluble sugar and chlorophyll content revealed that after 35 days of treatment at 4℃, the chlorophyll content of overexpressed MsXTH9 decreased, and the soluble sugar content was also significantly lower than that of the wild type. Figure 8 (Parts C and D). After treatment at -7℃ for 3 hours, and then restored to normal culture conditions in a greenhouse for 24 days, the recovery of WT and OEXTH-5 showed significant differences. Survival rate statistics revealed that the survival rate of WT was around 50%, while the survival rate of alfalfa overexpressing MsXTH9 was only 10%. Figure 8 (Parts E and F).

Claims

1. The application of a molecular module regulating alfalfa cold resistance in improving the cold resistance of alfalfa, characterized by, The molecular module for regulating alfalfa's cold resistance is MsmiR319-MsTCP3-MsXTH9.

2. The application according to claim 1, wherein the application improves the cold resistance of alfalfa by overexpressing MsmiR319, inhibiting MsTCP3 expression and / or inhibiting MsXTH9 expression.

3. The application according to claim 1 or 2, wherein the nucleotide sequence of MsmiR319 is SEQ ID NO.

7.

4. The application according to claim 3, wherein MsmiR319 is overexpressed by introducing an overexpression vector.

5. The application according to claim 1 or 2, wherein the nucleotide sequence of MsTCP3 is SEQ ID NO.

9.

6. The application according to claim 5, wherein MsTCP3SRDX with the nucleotide sequence SEQ ID NO.11 is introduced to inhibit MsTCP3 expression.

7. The application according to claim 1 or 2, wherein the nucleotide sequence of MsXTH9 is 13.

8. The application according to claim 1, wherein improving the cold resistance of alfalfa includes increasing the chlorophyll content and soluble sugar content in the plant, thereby increasing the plant survival rate.

9. The application according to any one of claims 1-8 in the breeding of cold-resistant varieties of alfalfa.

10. Cold-resistant alfalfa varieties, characterized by: The cold-resistant alfalfa varieties were overexpressed with MsmiR319, and MsTCP3 and / or MsXTH9 were suppressed.

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