CN114752579A

CN114752579A - Application of ZmMAPK protein and coding gene thereof in regulation and control of low-temperature stress tolerance of plants

Info

Publication number: CN114752579A
Application number: CN202011565861.6A
Authority: CN
Inventors: 施怡婷; 杨淑华; 李卓洋
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2022-07-15
Anticipated expiration: 2040-12-25
Also published as: CN114752579B

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to an application of ZmMAPK protein and a coding gene thereof in regulating and controlling low-temperature stress tolerance of plants. The invention discovers that the ZmMAPK8 protein, the ZmMAPK2 protein, the ZmMAPK17 protein and the coding genes thereof have obvious influence on the low-temperature stress tolerance of plants (particularly corns), provide good germplasm resources for cultivating new varieties of low-temperature-resistant plants, and lay a theoretical basis for researching the mechanism of plant response adversity signals and the molecular mechanism of adverse environment tolerance.

Description

Application of ZmMAPK protein and coding gene thereof in regulation and control of low-temperature stress tolerance of plants

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to an application of ZmMAPK protein and a coding gene thereof in regulating and controlling low-temperature stress tolerance of plants.

Background

Plants growing in a fixed manner in nature cannot be influenced by various adversity stresses, and with increasing shortage of water resources, low-temperature stress is taken as one of main external environmental factors, so that the growth, development and geographical distribution of the plants are influenced, and the yield of crops is also seriously influenced. Therefore, the research on the physiological and biochemical changes and molecular mechanisms of the plants responding to the low-temperature stress finds the key genes for regulating and controlling the plants responding to the low-temperature stress, and can provide valuable theoretical support for cultivating cold-resistant crop varieties so as to improve the yield of crops. Corn (Zea mays) belongs to plants of Zea of Gramineae, is an important food crop and feed crop, improves low-temperature stress tolerance of corn by a genetic engineering method, and has important significance for maintaining and improving the yield of corn.

MAPK (mitogen-activated protein kinase) protein kinase cascade reaction participates in a plurality of biological processes, and is a clear class of signal transmission process which is researched at present. After the receptor is activated by an external stimulation signal such as biotic or abiotic stress, a downstream MAPK cascade reaction can be activated. The research of partial ZmMAPK protein and plant adversity stress tolerance is proved by literature reports, for example, ZmMAPK protein and plant adversity stress tolerance research by ZmMAPK (ZmMAPK) and the like (2013) mentions that the expression level of ZmMAPK4 gene in corn leaves is obviously increased within one week after the inoculation of the biotic stress sugarcane mosaic virus; under abiotic stress and drought stress, the expression level of the ZmMAPK4 gene is increased, under high-temperature stress and drought stress, the expression level of the ZmMAPK4 gene is increased, and under salt stress induction, the relative expression level of the ZmMAPK4 gene is also obviously increased, which indicates that the ZmMAPK4 gene expression is positively regulated and controlled, and under SA induction, the relative expression level of the ZmMAPK4 gene is reduced to present a negative regulation and control mode. Wuliu et al (2015) suggested that overexpression of ZmMAPK-1 enhanced the response of transgenic Arabidopsis to drought and heat stress. However, there is no current intensive study on the association of ZmMAPK protein with low temperature stress tolerance in plants. Meanwhile, the existing research has been mostly focused on the group pa and group pb subfamilies, and less research is currently conducted on the group pc and group pd subfamilies lacking CD domains (MAPKK recognition structures) at the C-terminal of the protein structure.

Disclosure of Invention

The invention finds that ZmMAPK8 protein, ZmMAPK2 protein and ZmMAPK17 protein (Sun et al 2015) belonging to the GroupC and GroupD subfamilies and encoding genes thereof have obvious influence on the low-temperature stress tolerance of plants (particularly corns). Based on the discovery, the invention provides the ZmMAPK protein and the application of the coding gene thereof in regulating and controlling the low-temperature stress tolerance of plants.

Specifically, the invention firstly provides application of a ZmMAPK protein, or a coding gene thereof, or a suppressor thereof, or a biological material containing the coding gene or the suppressor thereof in regulating and controlling low-temperature stress tolerance of plants, wherein the ZmMAPK protein is at least one of a ZmMAPK8 protein, a ZmMAPK2 protein and a ZmMAPK17 protein.

The invention also provides application of the ZmMAPK protein, or a coding gene thereof, or a suppressor thereof, or a biological material containing the coding gene or the suppressor thereof in breeding plants with improved low-temperature stress tolerance, wherein the ZmMAPK protein is at least one protein of the ZmMAPK8 protein, the ZmMAPK2 protein and the ZmMAPK17 protein.

Preferably, the low temperature stress tolerance of the plant is increased by inhibiting the expression of a gene encoding at least one of the ZmMAPK8 protein, the ZmMAPK2 protein, and the ZmMAPK17 protein.

And the sensitivity of the plant to low temperature can also be improved by expressing or over-expressing at least one protein of the ZmMAPK8 protein, the ZmMAPK2 protein and the ZmMAPK17 protein.

Preferably, the ZmMAPK8 protein has an amino acid sequence of any one of:

1) an amino acid sequence shown as SEQ ID NO. 1; or the like, or a combination thereof,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the ZmMAPK2 protein has an amino acid sequence of any one of:

1) an amino acid sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

Preferably, the ZmMAPK17 protein has an amino acid sequence of any one of:

1) an amino acid sequence shown as SEQ ID NO. 3; or the like, or, alternatively,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 3.

It is understood that, considering the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

Preferably, the gene encoding the ZmMAPK8 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.4, or,

(2) the nucleotide sequence shown in SEQ ID NO.4 is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence to obtain a coding nucleotide sequence of the protein with the same function; or the like, or, alternatively,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.4 under strict conditions.

Preferably, the gene encoding the ZmMAPK2 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.5, or,

(2) the nucleotide sequence shown in SEQ ID NO.5 is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence to obtain a coding nucleotide sequence of the protein with the same function; or the like, or, alternatively,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.5 under strict conditions.

Preferably, the gene encoding the ZmMAPK17 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.6, or,

(2) the coding nucleotide sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 6; or the like, or a combination thereof,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.6 under strict conditions.

The maize ZmMAPK8 gene is numbered GRMZM2G048455 in a maize genome database and consists of 1110 bases, and the reading frame of the gene is from 456 th to 1566 th bases of the 5' end. The reading frame of the gene only consists of two exons and one intron sequence.

The maize ZmMAPK2 gene is numbered GRMZM2G062914 in the maize genomic database and consists of 1110 bases in frame from base 166 to 1269 of the 5' end. The reading frame of the gene only consists of two exons and one intron sequence.

The maize ZmMAPK17 gene is numbered GRMZM2G178822 in the maize genomic database and consists of 1380 bases in frame from base 843 to base 2223 from the 5' end. The reading frame of the gene only consists of four exons and three intron sequences.

In some embodiments, the biological material is an expression cassette, a vector, a host cell, or a recombinant bacterium.

Preferably, the plant is a monocotyledonous plant; more preferably corn.

The invention further provides a method for constructing transgenic corn with improved low-temperature stress tolerance, which inhibits the expression of at least one protein coding gene of ZmMAPK8 protein, ZmMAPK2 protein and ZmMAPK17 protein in the corn.

In some embodiments, a gene encoding at least one of the ZmMAPK8 protein, the ZmMAPK2 protein, and the ZmMAPK17 protein is edited in maize by CRISPR/Cas9 technology, inhibiting expression of the encoded gene.

Accordingly, if transgenic maize with improved low temperature sensitivity is required to be constructed, genes encoding at least one of the ZmMAPK8 protein, the ZmMAPK2 protein and the ZmMAPK17 protein in maize can be expressed or overexpressed.

In practice, the expression or overexpression may be achieved by transgene, hybridization, backcross, selfing or asexual propagation, wherein the transgene comprises introducing a recombinant expression vector comprising a gene encoding at least one of ZmMAPK8 protein, ZmMAPK2 protein and ZmMAPK17 protein into maize using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance, agrobacterium-mediated method to obtain transgenic maize lines.

Based on the scheme, the invention has the beneficial effects that:

the invention discovers that the ZmMAPK8 protein, the ZmMAPK2 protein, the ZmMAPK17 protein and coding genes thereof have obvious influence on the low-temperature stress tolerance of plants (particularly corns), provides good germplasm resources for cultivating new varieties of low-temperature-resistant plants, and lays a theoretical basis for researching a mechanism of plant response stress signals and a molecular mechanism of adverse environment tolerance.

Drawings

FIG. 1 is a gene overexpression profile of the overexpression lines of example 2 of the present invention.

FIG. 2 is a photograph of the growth of plants before and after restoration of the low temperature treatment of the lines overexpressing the ZmMAPK2 gene in example 3 of the present invention.

FIG. 3 is a photograph of the growth of plants before and after restoration of the low temperature treatment of the lines overexpressing the ZmMAPK8 gene in example 3 of the present invention.

FIG. 4 is a photograph of the plant growth before and after restoration of the low temperature treatment of the line overexpressing ZmMAPK17 gene in example 3 of the present invention.

FIG. 5 is a statistical chart of the ion leakage rate of the overexpression lines in example 3 of the present invention.

FIG. 6 shows the gene expression levels of three ZmMAPK genes detected by real-time quantitative PCR after low-temperature treatment.

FIG. 7 is a photograph showing the growth of the plant before and after restoration of the low temperature treatment of the ZmMAPK17 gene knockout line in example 5 of the present invention.

FIG. 8 is a statistical chart of the mutant knockout pattern and the ion leakage rate of the ZmMAPK17 gene knockout strain in example 5 of the present invention.

FIG. 9 shows the result of sequencing alignment of the ZmMAPK17 gene knock-out strain in example 5 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The pBSK vector in the following examples is a commonly used cloning vector, and is commercially available; pBCXUN vector (the pBCXUN vector takes a commercial vector pCAMBIA1300 as a framework, replaces the hygromycin resistance gene hpt in the pBCXUN vector with a herbicide resistance gene barM, clones the promoter of the maize ubiquitin gene Ubi to the vector in an enzyme digestion connection mode, drives the transcription of downstream overexpression gene), and drives the expression of CRR1 gene by the Ubi promoter; agrobacterium EHA105 strain is available from Shanghai Diego Biotechnology Ltd (cat. No.: AC 1010S).

The main reagents in the following examples are: various restriction enzymes, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, KOD from NEB, Toyobo, etc.; dNTPs were purchased from Genestar; the plasmid miniextraction kit and the agarose gel recovery kit are purchased from Shanghai Czeri bioengineering company; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), and rifampicin (Rif), and Glucose, BSA, and LB Medium were purchased from Sigma, Bio-Rad, and the like; reagents for real-time quantitative PCR were purchased from TaKaRa, and various other chemical reagents used in the examples were imported or domestic analytical reagents.

The primers used in the examples were synthesized from Hexa Huada and subjected to related sequencing.

The examples are carried out according to techniques or conditions described in the literature in the field, for example in the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: analytical manual,21), or according to the product instructions. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1 construction and detection of ZmMAPK8 Gene overexpression vector

In order to understand the molecular mechanism of plant response low temperature, 3000 more maize over-expression strains provided by the Chinese agricultural university crop breeding and function center are used for low-temperature treatment screening to obtain three over-expression strains ZmMAPK8, ZmMAPK2 and ZmMAPK17, wherein the cold resistance of the three over-expression strains under low-temperature treatment is obviously lower than that of the wild type. The three belong to MAPK families, namely, GroupC subfamilies and GroupD subfamilies respectively, but the study on the participation of the two subfamilies in plant low-temperature response is less at present, and meanwhile, the function of the ZmMAPK gene in cold resistance of corn is not clearly and deeply studied. Thus, the ZmMAPK2, ZmMAPK8, ZmMAPK17 genes were cloned from the maize B73 genome. Based on the sequence analysis of the coding region, primers F and R were designed, and the coding region of the gene was amplified and ligated to the overexpression vector pBCXUN with UBI promoter.

The primers used were:

ZmMAPK2：

an upstream primer F: 5'-ATGGCGATGATGGTGGATCCTC-3' (Seq ID No.7)

ZmMAPK8：

An upstream primer F: 5'-ATGGCCATGGCGATGATGGTGGATCC-3' (Seq ID No.8)

ZmMAPK17：

An upstream primer F: 5'-ATGGATAGATTCAAGTTGATTA-3' (Seq ID No.9)

ZmMAPK2：

A downstream primer R: 5'-TCATGACATGCTTATTGCTGTGGC-3' (Seq ID No.10)

ZmMAPK8：

A downstream primer R: 5'-GGTACCTCACATGCTGATTCTCGTGA-3' (Seq ID No.11)

ZmMAPK17：

A downstream primer R: 5'-TTAGCTGACCAATTTTCTCGT-3' (Seq ID No.12)

The specific method for connecting the ZmMAPK8 gene to the vector PCUN with the UBI promoter is as follows: firstly, taking cDNA as a template, amplifying ZmMAPK2, ZmMAPK8 and ZmMAPK17 by using upstream and downstream primers, connecting a PCR product with a pBSK vector, and naming a connection product as ZmMAPK 8-pBSK; the ZmMAPK8 is cut from the correctly sequenced ZmMAPK8-pBSK by utilizing SalI and KpnI and then is recycled and connected into a pBCXUN vector, and a connection product is named as pBCXUN-ZmMAPK 8.

Carrying out enzyme digestion on the plasmid obtained in the last step, and carrying out electrophoresis detection, wherein the specific method comprises the following steps: three pBCXUN-ZmMAPKs were digested with SalI and KpnI, and scanned and imaged by a UVP Gel Documentation Gel analysis system after electrophoresis with 1% agarose Gel, 120V, 50 mA.

Example 2 construction and testing of ZmMAPK2, ZmMAPK8, ZmMAPK17 Gene overexpressing plants

The pBCXUN vector containing ZmMAPK2, ZmMAPK8 and ZmMAPK17 genes described in example 1 was transformed into Agrobacterium EHA105 strain and infected with maize callus to obtain transgenic seedlings. The specific method comprises the following steps: inoculating Agrobacterium containing the target vector into 100mL LB three-antibody liquid culture medium (Kan 50. mu.g/mL, Rif 50. mu.g/mL, Gen 50. mu.g/mL), shaking and culturing at 28 deg.C overnight until OD6 value is 1.0-2.0, centrifuging at 50g at room temperature for 15min, and collecting thallus; the cells were suspended in 2mL of transformation medium (1/2MS, 5% sucrose, 40. mu.L Silwet L-77); soaking the corn callus in agrobacterium transformation liquid, and sealing. And putting the seeds back to the illumination culture shelf to grow normally until plants grow. And then, carrying out a low-temperature stress treatment experiment on the screened seeds.

The overexpression strain obtained by separation in this example adopts real-time quantitative PCR to detect the gene expression of ZmMAPK2, ZmMAPK8 and ZmMAPK17 in the overexpression strain.

1) Extracting total plant RNA and reverse transcribing to obtain cDNA.

2) After the cDNA obtained by reverse transcription was diluted 5 times, real-time quantitative PCR was carried out using a Takara kit, and the reaction system used included: 2 × SYBR Premix ExTaq buffer, 0.2. mu.L DyII, 0.4. mu.L LPrimer (F/R), 2. mu.L cDNA template, and finally ddH₂And (3) supplementing the oxygen to 20 mu L, fully and uniformly mixing, and then putting the mixture into an ABI PRISM 75 real-time quantitative PCR instrument for carrying out PCR amplification by a two-step method, wherein the reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃; 40s at 60 ℃; 40 cycles. After completion of PCR reaction according to 2^-Δ(ΔCt)The relative expression between wild type and overexpression was calculated and mapped. While amplifying the identified genes, each sample was simultaneously amplified with UBI as an internal control. The results are shown in FIG. 1.

Example 3 detection of Effect of overexpression of ZmMAPK2, ZmMAPK8 and ZmMAPK17 genes on Low temperature response capability of plants

Firstly, sowing seeds of CK and overexpression strains in black soil, small pots which are 10cm in length, 10cm in width and 10cm in height and contain imported soil and vermiculite (1:1:1), placing 5 seeds in each pot, covering 2cm of soil, placing the pots in a tray, watering until the soil is completely wet, placing the trays in a 23-degree culture room, and illuminating for 16 hours and being dark for 8 hours. After the leaves grow for 14 days, carrying out low-temperature treatment at 4 ℃ until the second leaf shrinks and wilts, taking out the leaves, putting the leaves into a 23-degree culture room, recovering for two days, then taking pictures, taking materials and carrying out statistics on ion leakage rate.

The ion leakage rate in this example was calculated by taking the relative conductivity L of the test vane (S1-S0)/(S2-S0). Putting the whole plant of the corn subjected to low-temperature treatment into a 15ml centrifuge tube filled with 10ml of distilled water, exhausting air for 30min by using a vacuum pump, then placing the centrifuge tube in a shaking table, shaking for 1h at room temperature, measuring the initial conductance value of the whole plant by using a conductance meter to be S1, then placing the sample in boiling water for 15min, taking out the sample, placing the sample in the shaking table, shaking for 2h, and measuring the conductance value to be S2. S0 is the conductivity of the blank control distilled water.

FIG. 2 is a photograph of plant growth before and after restoration of the low temperature treatment of a line overexpressing the ZmMAPK2 gene. FIG. 3 is a photograph of plant growth before and after restoration of the low temperature treatment of a line overexpressing the ZmMAPK8 gene. FIG. 4 is a photograph of plant growth before and after restoration of the low temperature treatment of a line overexpressing the ZmMAPK17 gene. FIG. 5 is a statistical graph of the ion leakage rate of the overexpression lines.

The results show (FIGS. 2-5) that the over-expression strains all show a low-temperature sensitive phenotype. The over-expression of the ZmMAPK8 gene can weaken the freezing resistance of the corn.

Example 4 maize ZmMAPK family genes cryogenically treated Gene expression detection

After the ecotype LH244 is treated at 4 ℃, materials are removed in 0h, 6h, 12h and 24h respectively, Trizol reagent is used for RNA extraction, and then Realtime-PCR is used for detecting the expression level of the ZmMAPK family gene.

As shown in FIG. 6, the expression of the ZmMAPK2, ZmMAPK8 and ZmMAPK17 genes was observed. In nature, plants have evolved a series of stress response genes which respond to the influence of external environmental stress, and the plants need to regulate the expression of the up-regulation genes to change a series of biological metabolic processes in plants to respond to the influence of the stress. In the process, the response to the adverse environment is enhanced by the plant at the cost of stopping or weakening the normal growth and development, but the process needs some negative regulatory factors such as the ZmMAPK related gene in the invention to weaken the action of the positive regulatory gene to maintain balance, so that the influence of the excessive stress response of the plant to the external stimulus on the normal growth and development is prevented, and the production of crops is further ensured. The experimental results of the low-temperature induction of the ZmMAPK2, ZmMAPK8 and ZmMAPK17 gene expression are shown in figure 6, and the gene expression is obviously increased after the low-temperature treatment for 12 hours, so that the three are proved to be induced to express and maintain the balance of the low-temperature stress of the corn after the corn is subjected to the low-temperature stress for a period of time.

Therefore, after the maize low-temperature response gene ZmMAPK family gene is over-expressed, the tolerance capability of the plant to low temperature can be obviously influenced.

Example 5 maize ZmMAPK8 mutant phenotype validation

Construction of ZmMAPK8 mutant

1. Construction of recombinant expression vectors

gRNA sequence: AATGATGCCTGCGCATAGA (Seq ID No.13)

The designed target spot is synthesized into a double-chain form from a single-chain form through touch down PCR and is connected to a pBUE411 carrier through the connecting segment.

The primer sequence is as follows:

F：5’-GGTCTAATGATGCCTGCGCATAGA-3’(Seq ID No.14)

R:5’-GAAATCTATGCGCAGGCATCATT-3’(Seq ID No.15)

the pBUE411 vector obtained above was digested with restriction enzyme Bsa1, and then ligated with Soluton1 ligase to obtain a recombinant plasmid. The recombinant plasmid is sent for sequencing, and the recombinant plasmid which is shown by the sequencing to be positioned between the enzyme cutting sites Bsa1 of the pBUE411 vector is named pBUE411-gmapk 8.

2. Obtaining and identifying ZmMAPK8 gene knockout corn

(1) ZmMAPK8 transgenic maize and the obtainment of maize plants transformed with pBUE411-gmapk8

The recombinant expression vector pBUE411-gmapk8 constructed in 1 was introduced into Agrobacterium GV3101 competent by freeze-thaw method. The obtained recombinant Agrobacterium pBUE411-gmapk8 (wild type (LH244 ecotype) was transformed into infected maize callus (using bar resistance for selection, the process was completed by the research center for functional genomics and molecular breeding of Chinese university of agriculture)

(2) CRISPR/Cas9 knock-out strain identification of ZmMAPK8

The pBUE411 vector can obtain a homozygous stable knockout strain at the T0 generation, and then obtain a homozygous knockout strain with the CRISPR/Cas9 background removed by selfing the F2 generation.

3. Sequencing and identification of ZmMAPK8 CRISPR strain

Total DNA of maize wild type (LH244 ecotype) and knockout plants (478 and 664) was extracted and DNA sequence differences of the ZmMAPK8 gene in the material were detected using PCR. The method comprises the following specific steps:

the PCR amplification method is as follows:

wherein, the primer sequence for amplifying the ZmMAPK8 gene is as follows:

ZmMAPK8RT-F1：5’-TGTGGCAGACACTGTTTGAG-3’(Seq ID No.16)

ZmMAPK8RT-R1：5’-TGACTGATGACAGATTACTGAACA-3’(Seq ID No.17)

the reaction conditions of the above primers were as follows:

(1) establishing a reaction system:

the PCR reaction system is shown in Table 1 below:

TABLE 1

(2) The three were repeated, gently thrown and mixed, and subjected to the experiment using a Bio-Rad PCR instrument.

(3) Setting a reaction program:

the PCR reaction procedure is shown in table 2 below:

TABLE 2

The band size was about 500bp as detected by 1% agarose gel 157V electrophoresis, and the sequence was sequenced by sequencer.

The sequencing result of the ZmMAPK8 PCR detection is shown in a schematic diagram in FIG. 8, and two different knockout forms 478 of the ZmMAPK8 gene insert a T at the position of the ZmMAPK8 mRNA sequence 306 shown in the following in a target spot, and 664 are deletions at the positions 306 and 307 shown in the ZmMAPK8 mRNA sequence. Both knockout forms result in frameshift mutations. The specific sequencing alignment is shown in figure 9.

Secondly, knocking out ZmMAPK8 gene to influence low-temperature response capability detection of plants

The constructed mutants were tested for their ability to cope with low temperature by the same method as in example 3.

As shown in fig. 7, mutant strains obtained by CRISPR/Cas9 gene editing techniques in two independent, different knockout forms exhibited distinct low temperature tolerant phenotypes after low temperature treatment compared to wild-type. Mutant knockout patterns and ion leakage profiles are shown in fig. 8, demonstrating that the ZmMAPK8 mutation is beneficial for improving tolerance to low temperatures in maize.

Meanwhile, because the over-expressed strains of ZmMAPK2 and ZmMAPK17 have obvious low temperature sensitive phenotype, the mutant of the over-expressed strains is supposed to have the same low temperature tolerant phenotype as the ZmMAPK8 mutant strain.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Asp Arg Phe Lys Leu Ile Lys Glu Val Gly Asp Gly Thr Phe Gly

1 5 10 15

Ser Val Trp Arg Ala Met Asn Lys Gln Asn Gly Glu Val Val Ala Val

20 25 30

Lys Lys Met Lys Lys Lys Tyr Tyr Ser Phe Glu Glu Cys Met Ser Leu

35 40 45

Arg Glu Val Lys Ser Leu Arg Arg Met Asn His Pro Asn Ile Val Lys

50 55 60

Leu Lys Glu Val Ile Arg Glu Asn Asp Ile Leu Tyr Phe Ile Met Glu

65 70 75 80

Tyr Met Glu Cys Asn Leu Tyr Gln Leu Met Lys Glu Arg Val Lys Pro

85 90 95

Phe Ser Glu Ser Glu Val Arg Asn Trp Cys Phe Gln Ile Phe Gln Ala

100 105 110

Leu Ala Tyr Met His Gln Arg Gly Tyr Phe His Arg Asp Leu Lys Pro

115 120 125

Glu Asn Leu Leu Val Ser Lys Gly Val Ile Lys Leu Ala Asp Phe Gly

130 135 140

Leu Ala Arg Glu Val Ser Ser Leu Pro Pro Tyr Thr Glu Tyr Val Ser

145 150 155 160

Thr Arg Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Ser Ala Tyr

165 170 175

Asp Ser Ala Val Asp Met Trp Ala Met Gly Ala Ile Met Ala Glu Leu

180 185 190

Leu Thr Leu His Pro Leu Phe Pro Gly Thr Ser Glu Pro Asp Glu Ile

195 200 205

His Lys Ile Cys Asn Val Ile Gly Ser Pro Asp Glu Gln Ser Trp Pro

210 215 220

Gln Gly Leu Ser Leu Ala Glu Ala Met Lys Tyr Gln Phe Pro Gln Thr

225 230 235 240

Lys Gly Ser Gln Leu Ser Glu Val Met Thr Thr Ala Ser Ser Glu Ala

245 250 255

Ile Asp Leu Ile Ser Ser Leu Cys Ser Trp Asp Pro Ser Lys Arg Pro

260 265 270

Lys Ala Thr Glu Val Leu Gln His Thr Phe Phe Gln Gly Cys Thr Cys

275 280 285

Val Pro Leu Pro Val Arg Arg Lys Ala Ser Ser Leu Pro Lys Thr Pro

290 295 300

Pro Cys Val Gly Ser Lys Arg Ile Ser Glu Asn Ser Val Ala Arg Arg

305 310 315 320

Phe Ser Thr Gly Thr Leu Ser Thr Met Lys Ser His Ser Asn Ala Pro

325 330 335

Ala Lys Ser Asn Gly Leu Ser Arg Thr Gly Val Gln Arg Lys Leu His

340 345 350

Leu Asp Arg Gln Pro Pro Gln Lys Ser Thr Lys Pro Thr Glu Asn Ser

355 360 365

Asn Lys Leu Ala Thr Asn Arg Val Pro Ala Arg Asn Ser Pro Gly Asn

370 375 380

Pro Val Leu Arg His Ser Arg Ser Leu Pro Glu Thr Gly Arg Arg Ala

385 390 395 400

Val Gln Lys Val Ser Ser Ile Thr Glu Lys Leu Ser Gln Met Ser Val

405 410 415

Thr Ser Arg Thr Arg Ser Ala Val Lys Pro Ala Ala Pro Thr Met Lys

420 425 430

Ala Gly His Gly Lys Ser Asp Phe Leu Gly Lys Ser Asp Asp Ile Pro

435 440 445

Pro Ala Lys Arg Leu Thr Arg Lys Leu Val Ser

450 455

<210> 4

<211> 1110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggcgatga tggtggatcc ccccaatggc atggcaagtc aaggaaagca ttactatact 60

atgtggcaga cactgtttga gatcgacacc aagtacgtgc cgatcaagcc catcggaaga 120

ggagcttacg gaatcgtttg ctcatctgtt aaccgcgaga ccaatgaaaa agtcgcgata 180

aaaaagataa acaacgtctt tgacaaccgt gtggatgcgc taaggacgct gagagagctg 240

aaactccttc ggcacctgag gcacgagaat gttattgctc tcaaggacat aatgatgcct 300

gcgcatagaa ggagcttcaa ggacgtttac ttggtttatg agctcatgga cactgatctg 360

catcaaataa ttaagtcatc tcaaccacta tccaatgacc actgccagta tttccttttt 420

cagctgctcc gaggcctgaa gtacctccat tcagccggga tactccacag agacctaaag 480

ccagggaacc tcctggtcaa cgcaaactgt gacctgaaga tatgcgactt cgggctcgcc 540

cgcacgaaca acaccaaggg ccagttcatg acggagtacg tggtgacccg ctggtacagg 600

gcacccgagc tgctgctctg ctgcgacaac tacggcacgt ccattgacgt ctggtctgtg 660

gggtgcatat tcgcggagct gcttggccgc aagccgatct tcccaggaac cgagtgcctg 720

aaccagctca agctcatcgt caacgtcctc ggcaccatgg gcgaggccga cctcgcgttc 780

atcgacaacc cgaaggcccg caagtacatc aagtcccttc cgtacgcccc gggcgccccc 840

ttcaccggca tgtaccctca ggcgcaccct ctcgccatcg acctgctgca gaagatgctc 900

gtgttcgacc cgtccaagag gatcagcgtc accgaggcgc tggagcaccc gtacatgtct 960

ccgctctatg acccgagcgc gaaccctccc gcgcaggtgc cgatcgacct cgacatcgac 1020

gagaacctcg gcgtcgacat gatcagggag atgatgtggc aggagatgat ccactaccac 1080

cccgaggtcc tcacgagaat cagcatgtga 1110

<210> 5

<211> 1032

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atagacacca aatatgtacc gatcaagccc attggtcgag gagcttatgg gatagtttgt 60

tcatccatta atcgtgaaac aaatgagaaa gtagcaataa agaagataca caacgttttc 120

gacaaccgtg tggatgcact acggaccttg cgggagctga aactccttcg ccatctccgg 180

catgagaatg tcattgcttt gaaggatata atgatgccaa tacacaggag aagctttaag 240

gatgtgtact tggtatacga actcatggat actgatttgc accagataat caaatcacct 300

cagggccttt ccaatgacca ctgccagtat tttctttttc agttgctccg aggactcaaa 360

tatctccatt cagcagaaat actccacaga gacctaaaac ctggaaacct gctggtgaat 420

gcaaattgtg atctgaagat atgtgatttt ggtctcgcac gtacaaacag tagcaaaggc 480

cagttcatga ctgaatacgt cgtcacccgc tggtacagag ctcctgagct gctcctctgc 540

tgcgacaact acggcacatc catagacgtc tggtctgttg ggtgcatctt tgctgagctc 600

cttggccgca agccaatatt tccaggaact gaatgcctga atcaactcaa gctcatagtg 660

aacgtcctcg gcaccatgag tgaggctgac ctagagttca tcgacaaccc aaaggctcgg 720

agatacatca agtcccttcc ctatacccct ggtgttcccc tcgtaagtat gtacccacat 780

gcgcaccctc ttgccattga tctgttgcag aagatgctca tcttcgaccc caccaaaagg 840

atcagtgtca ccgaggctct cgagcaccct tacatgtccc ctctgtatga tccaagcgca 900

aatcccccag cccaagtgcc catcgatctg gacatagacg aaaacatcag ctcagagatg 960

atccgggaaa tgatgtggca ggagatgctt cactaccacc ctgaagttgc cacagcaata 1020

agcatgtcat ga 1032

<210> 6

<211> 1380

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggatagat tcaagttgat taaggaagtt ggcgatggga cttttgggag tgtatggcgt 60

gctatgaata aacagaatgg cgaagttgtt gctgttaaga aaatgaagaa aaaatattat 120

tcttttgagg aatgtatgag tctacgtgaa gtaaagtctt tgcggcgcat gaatcatcct 180

aacattgtga agctcaaaga ggttatcagg gaaaatgata tattatactt cataatggaa 240

tacatggagt gtaatctcta tcaacttatg aaagaaaggg tcaagccttt ctccgagtct 300

gaagtccgca actggtgttt tcagatattt caggctcttg catacatgca ccagaggggc 360

tactttcatc gtgacctcaa acctgagaat ctgttggtta gcaaaggtgt cataaagcta 420

gcagactttg gtcttgcaag ggaagtctca tcattgccac catatacaga atatgtctca 480

actcgctggt atcgggcacc agaagtcttg ctccagtcat ctgcttatga ttctgcagtt 540

gatatgtggg caatgggtgc cataatggct gagctgttga cactccatcc tctctttcct 600

ggaaccagtg aaccagatga gattcacaag atatgcaatg tcatcggtag tccagatgag 660

caatcttggc ctcaaggatt gtctcttgca gaagcaatga agtatcagtt cccacagacc 720

aaaggcagtc aattgtctga ggtgatgaca acagctagta gcgaggcaat tgacctcatc 780

tcatcactat gctcatggga tcctagcaag agaccaaagg ccacagaagt cctccagcat 840

accttcttcc agggttgtac atgtgttccg cttcctgtcc gtcggaaagc ttcatcgctt 900

cctaaaacac ctccatgtgt tggatcaaag agaatttccg agaacagtgt tgctagaaga 960

ttctcgaccg ggactctatc tacgatgaaa tctcatagca atgcacctgc aaaatcaaac 1020

ggtttatcta ggactggtgt acaaagaaaa cttcacttgg atcgtcagcc accgcagaag 1080

agtacaaaac cgaccgagaa cagcaataag ctagctacaa atcgggtccc agcccggaac 1140

agcccaggga atcctgtact taggcattca cgcagcttgc ctgaaactgg tcgacgagca 1200

gtacagaagg tctcatcaat cacagagaaa ctgtcgcaaa tgtcggtgac ctccagaaca 1260

cgaagcgccg tgaagcctgc cgccccgacg atgaaggccg gacatggcaa gtcagacttc 1320

ctggggaagt ctgacgatat ccctccggca aagaggctga cgagaaaatt ggtcagctaa 1380

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggcgatga tggtggatcc tc 22

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggccatgg cgatgatggt ggatcc 26

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggatagat tcaagttgat ta 22

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcatgacatg cttattgctg tggc 24

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtacctcac atgctgattc tcgtga 26

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttagctgacc aattttctcg t 21

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aatgatgcct gcgcataga 19

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggtctaatga tgcctgcgca taga 24

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gaaatctatg cgcaggcatc att 23

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgtggcagac actgtttgag 20

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tgactgatga cagattactg aaca 24

Claims

The use of the ZmMAPK protein, or a gene encoding it, or an inhibitor of it, or a biological material containing a gene encoding it or an inhibitor, in any of the following:

(1) regulating and controlling the low-temperature stress tolerance of the plant;

(2) breeding plants with improved low temperature stress tolerance;

the ZmMAPK protein is at least one of a ZmMAPK8 protein, a ZmMAPK2 protein and a ZmMAPK17 protein.
2. The use according to claim 1, wherein the low temperature stress tolerance of a plant is increased by inhibiting the expression of a gene encoding at least one of the ZmMAPK8 protein, the ZmMAPK2 protein, and the ZmMAPK17 protein.
3. The use according to claim 1 or 2, wherein the ZmMAPK8 protein has the amino acid sequence of any one of seq id no:

1) an amino acid sequence shown as SEQ ID NO. 1; or the like, or, alternatively,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.
4. The use according to claim 1 or 2, wherein the ZmMAPK2 protein has the amino acid sequence of any one of seq id no:

1) an amino acid sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.
5. The use of claim 1 or 2, wherein the ZmMAPK17 protein has the amino acid sequence of any one of seq id no:

1) an amino acid sequence shown as SEQ ID NO. 3; or the like, or a combination thereof,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 3.
6. The use according to claim 1 or 2, wherein the gene encoding the ZmMAPK8 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.4, or,

(2) the nucleotide sequence shown in SEQ ID NO.4 is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence to obtain a coding nucleotide sequence of the protein with the same function; or the like, or, alternatively,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.4 under strict conditions.
7. The use according to claim 1 or 2, wherein the gene encoding the ZmMAPK2 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.5, or,

(2) the nucleotide sequence shown in SEQ ID NO.5 is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence to obtain a coding nucleotide sequence of the protein with the same function; or the like, or, alternatively,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.5 under strict conditions.
8. The use according to claim 1 or 2, wherein the gene encoding the ZmMAPK17 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.6, or,

(2) the nucleotide sequence shown in SEQ ID NO.6 is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence to obtain a coding nucleotide sequence of the protein with the same function; or the like, or, alternatively,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.6 under strict conditions.
9. Use according to claim 1 or 2, wherein the plant is a monocotyledonous plant; preferably corn.
10. A method for constructing a transgenic maize having improved tolerance to low temperature stress, characterized by inhibiting the expression of a gene encoding at least one protein selected from the group consisting of ZmMAPK8 protein, ZmMAPK2 protein and ZmMAPK17 protein in maize.