Application of ZmHSP22 protein and coding gene thereof in regulating and controlling low-temperature stress tolerance of corn

Abstract

The invention relates to the technical field of gene engineering, in particular to application of ZmHSP22 protein and a coding gene thereof in regulation and control of low-temperature stress tolerance of plants. The nucleotide sequence of the ZmHSP22 gene is as shown in SEQ ID No. 1; the amino acid sequence of the protein is shown as SEQ ID No.3. After the ZmHSP22 gene is knocked out, compared with a control group plant, the cold resistance of a transgenic plant under a low-temperature stress treatment condition is improved. The invention verifies that the ZmHSP22 protein has a regulation function on low-temperature stress tolerance of plants (especially corn), the ZmHSP22 protein and related biological materials thereof can be applied to regulation of plant cold tolerance, excellent candidate gene resources are provided for cultivation and improvement of new varieties of cold-tolerant plants, and compared with a traditional breeding mode, a cold-tolerant plant breeding method provided by the invention has the advantages of strong purposiveness and low cost. According to the method, the cold-tolerant breeding period is remarkably shortened, the cold-tolerant breeding efficiency is improved, a theoretical foundation is laid for researching the stress signal response mechanism and the adverse environment tolerance molecular mechanism of plants, and the method has important theoretical and practical significance in promoting the corn breeding process.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of HSP22 protein and its encoding gene in regulating plant low temperature stress tolerance. Background Art

[0002] As the world's cultivated land area gradually decreases and the world's population increases, people's demand for food is increasing. Corn is one of the world's three major food crops, and it is also a crop that is relatively sensitive to temperature. Among all biological and environmental adversities, low temperature stress is one of the main limiting factors affecting the survival and yield of crops. Low temperature stress has an important impact on the germination of corn seedlings, the survival of seedlings, and the grain filling process at maturity. Cold stress affects the plasticity of plant enzymes and membranes, changes physiology and metabolism, and sometimes causes water shortage and dryness, causing stress conditions for plants, which has an adverse effect on plant growth, development and yield. Low temperature is also associated with protein dysfunction and denaturation, thereby inducing the accumulation of heat shock proteins. In Arabidopsis, tobacco, corn, rapeseed, chicory, poplar, wheat and barley, many heat shock proteins respond to cold stress and are upregulated. Under low temperature stress, heat shock proteins are induced and transported to various organelles to protect cells from low temperature stress.

[0003] Therefore, studying the effects of corn tolerance to low temperature stress is of great significance to corn production. Traditional breeding techniques are relatively difficult to cultivate and improve stress-resistant traits, and cannot quickly and efficiently obtain excellent cold-resistant varieties. However, with the development of molecular biology technology, in-depth research on the molecular mechanism of plant stress resistance, and significant progress in genetic engineering research, the use of genetic engineering methods such as transgenics to introduce stress-resistant exogenous genes into plants has become one of the new ways to improve plant stress resistance. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide an application of ZmHSP22 protein and its encoding gene in regulating plant tolerance to cold stress. By constructing CRISPR / Cas9 materials, the target gene ZmHSP22 is quickly knocked out in the wild type. Detection shows that the transgenic plants have improved cold stress tolerance compared with the wild type.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] Application of corn ZMHSP22 protein or its encoding gene, or biological material containing its encoding gene in any of the following aspects:

[0007] (1) Improve the cold tolerance of corn;

[0008] (2) Breeding transgenic corn with improved resistance to low temperature stress;

[0009] (3) Improve corn cold-resistant germplasm resources.

[0010] Preferably,

[0011] The cold tolerance of corn is improved by inhibiting or reducing the expression level and / or activity of ZMHSP22 protein in corn.

[0012] Preferably,

[0013] The amino acid sequence of the corn ZMHSP22 protein is any one of the following:

[0014] (A1) the amino acid sequence shown in SEQ ID No. 3;

[0015] (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID No. 3 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0016] Preferably,

[0017] The nucleotide sequence of the gene encoding the maize ZMHSP22 protein is any of the following:

[0018] (B1) the nucleotide sequence shown in SEQ ID No. 1;

[0019] (B2) the nucleotide sequence shown in SEQ ID No. 2;

[0020] (B3) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID No. 2 and the nucleotide sequence expresses a protein with the same function;

[0021] (B4) A nucleotide sequence completely complementary to the nucleotide sequence shown in SEQ ID No.2.

[0022] Preferably,

[0023] The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

[0024] A breeding method for cold-tolerant corn,

[0025] By inhibiting the expression amount and / or activity of the ZMHSP22 protein, a plant with improved cold tolerance is obtained; the amino acid sequence of the ZMHSP22 protein is any one of the following:

[0026] (A1) the amino acid sequence shown in SEQ ID No. 3;

[0027] (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID No. 3 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0028] The breeding method of cold-tolerant corn provided by the present invention comprises knocking out the ZMHSP22 gene in the receptor plant by CRISPR / Cas9 technology, or introducing a recombinant expression vector containing the coding gene target of the protein into the receptor B73-329 receptor corn to obtain a transgenic plant; the cold tolerance of the transgenic corn plant is improved.

[0029] The recombinant expression vector can be constructed using existing plant expression vectors, including binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derived plant expression vectors.

[0030] The above recombinant expression vector amplifies the target sequence of SEQ ID No.4 in the intermediate vector pCBC-MT1T2 by double primer amplification, and then connects to the pBUE411 vector through the Bsa1 restriction site. When using genes to construct recombinant expression vectors, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-induced promoter rd29A, etc., which can be used alone or in combination with other plant promoters; translation control signals and start codons can be natural or synthetic. The translation start region can come from the transcription start region or structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes, antibiotic markers with resistance, or chemical reagent resistance marker genes. It is also possible not to add any selective marker genes and directly screen transformed plants by adversity.

[0031] In the present invention, the promoter for initiating transcription of the protein encoding gene in the recombinant expression vector is a rice U6 promoter.

[0032] Preferably, the method comprises the following steps:

[0033] The gene encoding the ZMHSP22 protein is connected to the pBCXUN vector to obtain a recombinant vector pBCXUN-ZMHSP22 containing the ZMHSP22 coding sequence, the recombinant vector is transformed into Agrobacterium EHA105 to obtain a recombinant Agrobacterium EHA105 / pBCXUN-ZMHSP22 containing the recombinant vector pBCXUN-ZMHSP22, the recombinant Agrobacterium EHA105 / pBCXUN-ZMHSP22 is infected into corn, and introduced into a recipient plant to obtain a transgenic plant with increased cold tolerance;

[0034] The amino acid sequence of the ZMHSP22 protein is any one of the following:

[0035] (A1) the amino acid sequence shown in SEQ ID No. 2;

[0036] (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID No. 2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0037] Preferably,

[0038] The primers for detecting the ZMHSP22 gene are shown in SEQ ID No.5 and SEQ ID No.6.

[0039] Preferably,

[0040] This includes using DNA homologous recombination technology, Cre / Loxp technology, and Crispr / Gas9 technology to silence or reduce the expression of the corn ZMHSP22 gene to obtain transgenic plant strains.

[0041] The recombinant expression vector carrying the single target sequence of the ZmHSP22 gene is introduced into the recipient plant, including using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, conductivity, Agrobacterium-mediated method to introduce the recombinant expression vector containing the corn ZMHSP22 gene into corn to obtain a transgenic corn strain.

[0042] The use of a protein consisting of the amino acid sequence shown in SEQ ID No. 3 or its encoding gene in the following (a) or (b) or (c) also falls within the scope of protection of the present invention:

[0043] (a) A homozygous knockout strain of the ZmHSP22 gene was obtained;

[0044] (b) The knockout strain of ZmHSP22 gene can improve the ion leakage rate of corn under low temperature treatment;

[0045] (c) The knockout strain of ZmHSP22 gene can improve the osmotic potential of corn under low temperature treatment.

[0046] (d) The knockout strain of ZmHSP22 gene can improve the cold tolerance of maize seedlings;

[0047] In the above application or method, the plant can be a dicotyledonous plant or a monocotyledonous plant.

[0048] The beneficial effects of the present invention are:

[0049] The present invention verifies that the ZmHSP22 protein has a regulatory function on the cold stress tolerance of plants (especially corn), and the homozygous knockout strain of the ZmHSP22 gene has significantly improved tolerance to low temperature stress compared to wild-type control plants. The present invention is of great significance to the study of the molecular mechanism of cold tolerance of corn; it provides excellent candidate gene resources for cultivating and improving new varieties of cold-tolerant plants, and the method for cold-tolerant plant breeding provided is more purposeful than the traditional breeding method, significantly shortens the cycle of cold-tolerant breeding, improves the efficiency of cold-tolerant breeding, and lays a theoretical foundation for studying the mechanism of plant response to adverse signals and the molecular mechanism of tolerance to adverse environments, and has important theoretical and practical significance for promoting the process of corn breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0051] Figure 1 Schematic diagram of the detection of homozygous knockout strains of the ZmHSP22 gene.

[0052] Figure 2 This is a low temperature treatment experiment for ZmHSP22 mutant strains and wild type control in the same batch at 4°C. Maize seedlings were grown at 23°C for 13 days, treated at 4°C for 3 days, and recovered at 23°C for 24 hours.

[0053] Figure 3 The responses of ZmHSP22 mutant strains and wild type control strains in the ion permeation experiment after 4℃ low temperature treatment and the ion leakage rate after 4℃ treatment. * indicates that the difference is extremely significant compared with Col-0 group (P<0.05). Figure 4 The results of the ion permeation test and the freezing point osmotic pressure measurement after 4℃ low temperature treatment of ZmHSP22 mutant strains and wild type from the same batch. * indicates that the difference is extremely significant compared with Col-0 group (P<0.05). DETAILED DESCRIPTION

[0054] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0057] The % in the following examples, unless otherwise specified, are all mass percentages. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0058] pBUE411 vector intermediate vector pCBC-MT1T2 was provided by China Agricultural University (document: Xing, HL#, Dong, L.#, Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, and Chen, QJ* (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14, 327.). The upstream promoter in the pBUE411 vector is the rice U6-26p promoter. Maize wild type: B73-329 was provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0059] Agrobacterium tumefaciens: Agrobacterium tumefaciens strain GV3101, provided by our laboratory (reference: R. Berres, L. Otten, B. Tinland et al. Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T_6b gene. Plant Cell Reports, 1992(11):192-195.).

[0060] Escherichia coli strain DH5α (DE3) competent cell: a product of Quanshijin Biotechnology Co., Ltd.

[0061] Example 1 Acquisition and identification of ZmHSP22 transgenic plants

[0062] The ZmHSP22 gene involved in this embodiment is derived from corn (Zea may), and its sequence in the corn genome is shown in SEQ ID No.1, which consists of 1270 nucleotides, of which the 438th to 568th positions are intron sequences; the cDNA sequence of the ZmHSP22 gene is shown in SEQ ID No.2, which consists of 1139 nucleotides, of which the 185th to 841st positions are coding sequences (ORF); SEQ ID No.1 and SEQ ID No.2 both encode the protein shown in SEQ ID No.3, which consists of 219 amino acid residues. SEQ ID No.10 (TAGCGTCCGCGAGTACGAT) is a gRNA sequence located on the first exon.

[0063] 1. Construction of the recombinant expression vector pBUE411-pCBC-MTaTb

[0064] She is Figure 1 The sequence shown was constructed, and the Ta single target site was amplified on pCBC-T1T2 by double primer amplification using pCBC-T1T2 plasmid as a template, and the connection fragment was connected to the pBUE411 vector to obtain the pBUE411-pCBC-MT1T2 vector.

[0065] BsF: 5'-ATATATGGTCTCGATTGATCGTACTCGCGGACGCTA-3' (SEQ ID No. 4)

[0066] F0:5'-TGATCGTACTCGCGGACGCTAGTTTTAGAGCTAGAAATAGC-3'(SEQ ID No.5)

[0067] (The large font is the recognition site of Bsa1, and the bold font is the Ta sequence, which is the reverse complementary sequence of positions 370-388 of SEQ ID No. 2);

[0068] R0:5'-AACTAGCGTCCGCGAGTACGATCAATCTCTTAGTCGACTCTAC-3'(SEQ ID No.6)

[0069] BsR:5'-ATTATTGGTCTCGAAACTAGCGTCCCGCGAGTACGATC-3'

[0070] (SEQ ID No.7)

[0071] (The large font is the recognition site of Bsa1, and the bold font is the Ta sequence, which is the sequence from positions 370 to 388 of SEQ ID No. 2).

[0072] The pBUE411 vector was obtained by digestion with restriction endonuclease Bsa1, and the above two pairs of primers were used to perform four-primer amplification with pCBC-MT1T2 as the template. The concentrations of F0 and R0 primers were diluted ten times based on the working concentrations. After PCR amplification, the connection fragment pCBC-MTaTb was obtained by gel excision and recovery, and then Soluton1 ligase was used to connect to obtain the recombinant plasmid. The recombinant plasmid was sent for sequencing, and the recombinant plasmid with the DNA fragment of pCBC-MTaTb inserted between the restriction site Bsa1 of the pBUE411 vector was named pBUE411-pCBC-MTaTb.

[0073] 2. Obtaining and identifying ZmHSP22 gene knockout maize

[0074] 1) The recombinant expression vector pBUE411-pCBC-MTaTb of ZmHSP22 transgenic corn and corn plants transformed with the pBUE411-pCBC-MTaTb empty vector were introduced into Agrobacterium GV3101 competent cells by freeze-thaw method to obtain recombinant Agrobacterium. The transformed recombinant Agrobacterium was identified by PCR using a primer pair consisting of primer F0 and primer R0. The Agrobacterium GV3101 identified as containing pCBC-MTaTb (the PCR target band size was about 1000 bp) was named pBUE411-pCBC-MTaTb-1.

[0075] The recombinant Agrobacterium pBUE411-pCBC-MTaTb-1 obtained above was transformed into wild-type maize (ecotype B73-329) by Agrobacterium inflorescence infection method (SJ Clough, AF Bent. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 1998, 16 (6): 735-743.) The transformation and infection of maize callus (screening was performed using bar resistance, completed by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University).

[0076] 2) Identification of ZmHSP22 gene CRISPR knockout strains

[0077] The pBUE411 vector can produce homozygous stable knockout strains in the T0 generation, and then the homozygous knockout strains with the CRISPR / Cas9 background removed can be obtained by selfing the F2 generation.

[0078] 3. CRISPR sequencing and identification of ZmHSP22 gene strains

[0079] Total DNA was extracted from wild-type maize (ecotype B73-329) and knockout plants (I1 and D4), and PCR was used to detect the DNA sequence differences of the ZmHSP22 gene in each material.

[0080] The details are as follows:

[0081] The PCR amplification method is as follows:

[0082] Among them, the primer sequence for amplifying the ZmHSP22 gene is:

[0083] HSP22RT-F1: 5'-GCAGGATCTTCGTGAACCTT-3' (SEQ ID No. 8) (positions 2-21 of SEQ ID No. 2);

[0084] HSP22RT-R1: 5'-CTTGTCCATCCTGTAAACCTCTG-3' (SEQ ID No. 9) (the reverse complementary sequence of positions 726 to 748 of SEQ ID No. 2).

[0085] The reaction conditions of the above primers are as follows:

[0086] (1) Establishment of reaction system

[0087] PCR reaction system

[0088]

[0089] (2) Repeat three times, shake gently to mix, and perform the experiment using a Bio-Rad PCR instrument.

[0090] (3) Setting of reaction program:

[0091] PCR reaction procedure

[0092]

[0093] The band size was about 750 bp using 1% agarose gel electrophoresis at 157 V, and was sent to a sequencing company for sequencing.

[0094] The results of PCR detection and sequencing of ZmHSP22 gene are as follows Figure 1 As shown, there are two different knockout forms of the ZmHSP22 gene, I1 is an insertion of a base after the 385th position of the sequence shown in SEQ ID No. 2, and D4 is a deletion of the 383rd-386th positions shown in SEQ ID No. 2. Both knockout forms lead to frameshift mutations.

[0095] Example 2 Low temperature treatment test of ZmHSP22 gene knockout strain

[0096] Low temperature stress can cause leaf damage and also cause leaf dehydration and shrinkage. Figure 2 As shown, 14-day corn seedlings (including 3-4 days of germination) grown under normal light conditions at 23°C were placed in a 4°C incubator for 3 days, and the treated ZmHSP22 gene CRISPR / Cas9 knockout strains I1 and D4 and the same batch of wild-type (B73-329 ecotype) were placed at 23°C for 24 hours for recovery, and then phenotype observation and image collection were performed. It can be seen that compared with wild-type corn plants, the ability of I1 and D4 to tolerate low temperature stress was significantly improved after low temperature treatment, and the ability of corn to tolerate low temperature stress can be significantly improved after knocking out the ZmHSP22 gene (verified by more than three low temperature treatments in this experiment).

[0097] 1) Plant ion leakage measurement experiment

[0098] When plants are subjected to low temperature stress, low temperature can damage plant cells, causing the cell membrane to lose or partially lose its selective permeability to ions. Therefore, measuring ion leakage can reflect the tolerance of plants to various stresses such as low temperature stress. Place the above-ground part of the corn seedlings in a 15ml centrifuge tube, add 10ml of deionized water, and immerse the plants completely below the water surface. Shake at 23℃ at 120rpm for 1h, and use a particle leakage detector to measure and record it as S0. Then place all samples in a 100℃ water bath for 1h, shake at 23℃ at 120rpm for 1h, and then measure the samples again and record them as S1 (Note: Before measuring each sample, rinse the instrument probe and then measure the value of the rinse solution (deionized water) and record it as S2 and S3. The final particle leakage rate is calculated as follows:

[0099] Ion leakage(%)=S0-S2 / S1-S3

[0100] In this experiment, at least three corn seedlings of the same strain were measured and the average value was taken (three experiments, * indicates significant difference, P < 0.05)

[0101] like Figure 3 As shown, the leakage rates of I1 and D4 particles were significantly lower than that of the wild type (B73-329 ecotype), proving that their ability to tolerate low temperature stress under low temperature stress treatment was significantly improved compared with the wild type.

[0102] 2) Osmotic test

[0103] Maintaining osmotic pressure is an important part of maintaining the homeostasis of the plant's internal environment. When plants are stimulated by external stress, they can accumulate protective substances in their cells, thereby increasing their freezing point osmotic pressure. After 14-day-old corn seedlings were treated at 4°C for 12 hours, the above-ground part of the corn seedlings was squeezed with a syringe and the juice was collected in a 1.5ml centrifuge tube at 12,000 rpm for 5 minutes to remove the supernatant, and then measured using a freezing point osmotic pressure meter. Figure 4 As shown, the freezing point osmotic pressure of I1 and D4 was higher than that of the wild type (B73-329 ecotype), indicating that the ability to tolerate low temperature stress under low temperature stress treatment was significantly improved compared with the wild type.

[0104] The above experiments have proved that knocking out the ZmHSP22 gene can improve the cold tolerance of corn.

[0105] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0106] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of maize ZMHSP22 protein or its encoding gene, or biological materials containing its encoding gene in any of the following aspects: (1) Improve the cold tolerance of corn; (2) Breeding transgenic corn with improved resistance to low temperature stress; (3) Improve corn cold-resistant germplasm resources.

2. The use according to claim 1, characterized in that: The cold tolerance of corn is improved by inhibiting or reducing the expression level and / or activity of ZMHSP22 protein in corn.

3. The use according to claim 1 or 2, characterized in that: The amino acid sequence of the corn ZMHSP22 protein is any one of the following: (A1) the amino acid sequence shown in SEQ ID No. 3; (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID No. 3 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

4. The use according to claim 1 or 2, characterized in that: The nucleotide sequence of the gene encoding the maize ZMHSP22 protein is any of the following: (B1) the nucleotide sequence shown in SEQ ID No. 1; (B2) the nucleotide sequence shown in SEQ ID No. 2; (B3) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID No. 2 and the nucleotide sequence expresses a protein with the same function; (B4) A nucleotide sequence completely complementary to the nucleotide sequence shown in SEQ ID No.

2.

5. The use according to claim 1 or 2, characterized in that: The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

6. A method for breeding cold-tolerant corn, characterized in that: By inhibiting the expression amount and / or activity of the ZMHSP22 protein, a plant with improved cold tolerance is obtained; the amino acid sequence of the ZMHSP22 protein is any one of the following: (A1) the amino acid sequence shown in SEQ ID No. 3; (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID No. 3 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

7. The breeding method of cold-tolerant corn according to claim 6, comprising the following steps: The gene encoding the ZMHSP22 protein is connected to the pBCXUN vector to obtain a recombinant vector pBCXUN-ZMHSP22 containing the ZMHSP22 coding sequence, the recombinant vector is transformed into Agrobacterium EHA105 to obtain a recombinant Agrobacterium EHA105 / pBCXUN-ZMHSP22 containing the recombinant vector pBCXUN-ZMHSP22, the recombinant Agrobacterium EHA105 / pBCXUN-ZMHSP22 is infected into corn, and introduced into a recipient plant to obtain a transgenic plant with increased cold tolerance; The amino acid sequence of the ZMHSP22 protein is any one of the following: (A1) the amino acid sequence shown in SEQ ID No. 2; (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID No. 2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

8. The breeding method for cold-tolerant corn according to claim 7, characterized in that: The primers for detecting the ZmMYB121 gene are shown in SEQ ID No.5 and SEQ ID No.

6.

9. The method for breeding cold-tolerant corn according to claim 6, characterized in that: It includes using DNA homologous recombination technology, Cre / Loxp technology, and Crispr / Gas9 technology to silence the expression or reduce the expression level of the corn ZMHSP22 gene to obtain a transgenic plant strain.

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