CN114752579B

CN114752579B - ZmMAPK protein and application of coding gene thereof in regulation and control of low-temperature stress tolerance of plants

Info

Publication number: CN114752579B
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: 2024-03-19
Anticipated expiration: 2040-12-25
Also published as: CN114752579A

Abstract

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

Description

ZmMAPK protein and application of 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 ZmMAPK protein and application of a coding gene thereof in regulating and controlling low-temperature stress tolerance of plants.

Background

Plants grown in fixation in nature inevitably suffer from various adverse conditions, and with the increasing shortage of water resources, low-temperature stress is used as one of main external environment factors, which not only affects the growth and development of plants and geographical distribution, but also seriously affects the yield of crops. Therefore, the physiological and biochemical changes and molecular mechanisms of the plants responding to the low-temperature stress are researched, and key genes for regulating and controlling the plants responding to the low-temperature stress are searched, so that valuable theoretical support can be provided for cultivating cold-resistant crop varieties, and the yield of crops is improved. Corn (Zea mays) belongs to the genus Zea of the family poaceae, is an important grain crop and feed crop, and has important significance for maintaining and improving the corn yield by improving the low-temperature stress tolerance of the corn through a genetic engineering method.

The MAPK (mitogen-activated protein kinase) protein kinase cascade is involved in a number of biological processes, a relatively clear class of signaling processes currently studied. After the receptor is activated by external stimulus signals such as biotic or abiotic stress, downstream MAPK cascade reaction can be activated. There have been reports in the literature that demonstrate studies of part of ZmMAPK proteins and plant stress tolerance, for example Zu Xiaofeng et al (2013) mention that the expression level of ZmMAPK4 gene in maize leaves is significantly increased within one week after inoculation of 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 expression of the ZmMAPK4 gene is positively regulated, and under SA induction, the relative expression level of the ZmMAPK4 gene is reduced in a negative regulation mode. Wu Liuji et al (2015) mention that overexpression of ZmMAPK-1 enhances the response of transgenic arabidopsis to drought and heat stress. However, there is currently no intensive study on ZmMAPK proteins related to plant low temperature stress tolerance. Meanwhile, the existing researches have focused on the GroupA and GroupB subfamilies, whereas the researches on the GroupC and GroupD subfamilies lacking CD domain (MAPKK recognition structure) at the C-terminal on the protein structure are currently less.

Disclosure of Invention

The present invention has found that ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein (Sun et al 2015) belonging to the subfamily of GroupC and GroupD and the genes encoding them have a significant effect on the low temperature stress tolerance of plants, especially maize. Based on the findings, the invention provides application of ZmMAPK protein and coding gene thereof in regulating low-temperature stress tolerance of plants.

Specifically, the invention firstly provides application of ZmMAPK protein, or a coding gene thereof, or an inhibitor thereof, or a biological material containing the coding gene or the inhibitor thereof in regulating low-temperature stress tolerance of plants, wherein the ZmMAPK protein is at least one protein of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein.

The invention also provides application of the ZmMAPK protein, or the coding gene thereof, or the inhibitor thereof, or the biological material containing the coding gene or the inhibitor thereof in breeding plants with improved low-temperature stress tolerance, wherein the ZmMAPK protein is at least one protein of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein.

Preferably, the low temperature stress tolerance of the plant is improved by inhibiting the expression of a gene encoding at least one of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein.

And the sensitivity of the plant to low temperature can be improved by expressing or over-expressing at least one of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein.

Preferably, the ZmMAPK8 protein has any one of the following amino acid sequences:

1) An amino acid sequence shown in SEQ ID NO. 1; or alternatively, the first and second heat exchangers may be,

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

Preferably, the ZmMAPK2 protein has any one of the following amino acid sequences:

1) An amino acid sequence shown in SEQ ID NO. 2; or alternatively, the first and second heat exchangers may be,

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

Preferably, the ZmMAPK17 protein has any one of the following amino acid sequences:

1) An amino acid sequence shown in SEQ ID NO. 3; or alternatively, the first and second heat exchangers may be,

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

It will be appreciated that, given the degeneracy of codons and the preference of codons of different species, one skilled in the art can use codons suitable for expression of a particular species as desired.

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

(1) The nucleotide sequence shown as SEQ ID NO.4, or,

(2) The nucleotide sequence shown in SEQ ID NO.4 is a coded nucleotide sequence of the protein with the same function obtained by replacing, deleting or inserting one or more nucleotides; or alternatively, the first and second heat exchangers may be,

(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO. 4.

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

(1) The nucleotide sequence shown as SEQ ID NO.5, or,

(2) The nucleotide sequence shown in SEQ ID NO.5 is a coded nucleotide sequence of the protein with the same function obtained by replacing, deleting or inserting one or more nucleotides; or alternatively, the first and second heat exchangers may be,

(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID No. 5.

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

(1) The nucleotide sequence shown as SEQ ID NO.6, or,

(2) The nucleotide sequence shown in SEQ ID NO.6 is a coded nucleotide sequence of the protein with the same function obtained by replacing, deleting or inserting one or more nucleotides; or alternatively, the first and second heat exchangers may be,

(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO. 6.

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

The corn ZmMAPK2 gene has the number GRMZM2G062914 in a corn genome database and consists of 1110 bases, and the reading frame of the gene is from 166 th base to 1269 th base of the 5' end. The gene reading frame consists of only two exons, one intron sequence.

The maize ZmMAPK17 gene is numbered GRMZM2G178822 in the maize genome database and consists of 1380 bases, and the reading frame of the gene is from 843 rd to 2223 rd bases of the 5' end. The gene reading frame consists of four exons and three intronic 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 monocot; more preferably corn.

The invention further provides a method for constructing transgenic corn with improved low-temperature stress tolerance, which can inhibit the expression of at least one of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein coding genes in corn.

In some embodiments, the expression of the coding gene is inhibited by editing the coding gene for at least one of the ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein in maize by CRISPR/Cas9 technology.

Accordingly, if it is desired to construct transgenic maize with increased low temperature sensitivity, the coding gene for at least one of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein in maize may be expressed or overexpressed.

In practical implementation, the expression or over-expression mode may be transgenic, hybrid, backcross, selfing or asexual reproduction, wherein the transgenic includes introducing a recombinant expression vector containing a coding gene of at least one of ZmMAPK8 protein, zmMAPK2 protein and ZmMAPK17 protein into corn by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, conductance and agrobacterium-mediated method to obtain transgenic corn strain.

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

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

Drawings

FIG. 1 is a graph showing the gene overexpression of the overexpression line in example 2 of the invention.

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

FIG. 3 is a photograph showing the growth of plants before and after recovery from the low temperature treatment of the strain overexpressing ZmMAPK8 gene in example 3 of the present invention.

FIG. 4 is a photograph showing the growth of plants before and after recovery from the low temperature treatment of the strain overexpressing ZmMAPK17 gene in example 3 of the present invention.

FIG. 5 is a graph showing the statistics of ion leakage rate of the over-expressed strain in example 3 of the present invention.

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

FIG. 7 is a photograph showing the growth of plants before and after the recovery of the low temperature treatment of the ZmMAPK17 gene knockout strain in example 5 according to the present invention.

FIG. 8 is a graph showing the mutant knockout pattern and ion permeability statistics of the strain in which ZmMAPK17 gene was knocked out in example 5 of the present invention.

FIG. 9 shows the specific sequencing alignment of the strain knocked out ZmMAPK17 gene in example 5 of the present invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The pBSK vector in the examples below is a commonly used cloning vector and is commercially available; the pBCXUN vector (the pBCXUN vector takes a commercial vector pCAMBIA1300 as a framework, replaces hygromycin resistance gene hpt therein with herbicide resistance gene barM, and simultaneously clones a promoter of a corn ubiquitin gene Ubi onto the vector in a way of enzyme digestion connection to drive transcription of a downstream over-expressed gene), and drives the expression of a CRR1 gene by using the Ubi promoter; agrobacterium EHA105 strain was purchased from Shanghai Biotechnology Inc. (cat# AC 1010S).

The main reagents in the following examples were: various restriction enzymes, taq DNA polymerase, T4 ligase, pyrobest Taq enzyme, KOD from NEB, toyobo and other biological companies; dNTPs are available from Genestar; the plasmid miniprep kit and the agarose gel recovery kit are purchased from Shanghai Jierui bioengineering company; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), and rifampicin (Rif), and the like, and companies such as Glucose, BSA, LB Medium, and the like are purchased from Sigma, bio-Rad, and the like; the reagents used for real-time quantitative PCR were purchased from TaKaRa, and the various other chemical reagents used in the examples were all imported or custom analytical pure reagents.

The primers used in the examples were synthesized by Hexakuda and subjected to related sequencing.

The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the field, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: alaboratory manual, 21), or according to the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.

EXAMPLE 1 construction and detection of ZmMAPK8 Gene overexpression vector

In order to understand the molecular mechanism of plant response to low temperature, more than 3000 corn over-expression lines provided by crop breeding and function centers of China agricultural university are used for low temperature treatment screening, and three over-expression lines ZmMAPK8, zmMAPK2 and ZmMAPK17 with cold resistance obviously lower than that of a wild type under low temperature treatment are obtained. The three are respectively belonging to MAPK families GroupC and GroupD subfamilies, but the research on the low-temperature response of plants involving the two subfamilies is less at present, and meanwhile, the effect of ZmMAPK genes in cold resistance of corns is not clearly and deeply studied. Thus, the ZmMAPK2, zmMAPK8, zmMAPK17 genes were cloned from the maize B73 genome. Primers F and R were designed based on sequence analysis of the coding region, and the coding region of the gene was amplified and ligated to the overexpression vector pBCXUN having the UBI promoter.

The primers used were:

ZmMAPK2：

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

ZmMAPK8：

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

ZmMAPK17：

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

ZmMAPK2：

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

ZmMAPK8：

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

ZmMAPK17：

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

The specific method for ligating ZmMAPK8 gene to vector PCUN with UBI promoter is: firstly, using cDNA as a template, amplifying ZmMAPK2, zmMAPK8 and ZmMAPK17 by using an upstream primer and a downstream primer, connecting a PCR product with a pBSK vector, and naming the connection product as ZmMAPK8-pBSK; zmMAPK8 was digested with SalI and KpnI from the well-sequenced ZmMAPK8-pBSK and recovered and ligated into the pBCXUN vector, the ligation product was named pBCXUN-ZmMAPK8.

And (3) carrying out electrophoresis detection after enzyme digestion of the plasmid obtained in the last step, wherein the specific method comprises the following steps: three pBCXUN-ZmMAPKs were digested with SalI and KpnI, and visualized by a 1% agarose gel, 120V,50mA, and scanned by a UVP Gel Documentation gel analysis system.

Example 2 construction and detection of ZmMAPK2, zmMAPK8, zmMAPK17 Gene overexpressing plants

The pBCXUN vector containing the ZmMAPK2, zmMAPK8 and ZmMAPK17 genes described in example 1 was transformed into Agrobacterium EHA105 strain, and maize callus was infected to obtain transgenic seedlings. The specific method comprises the following steps: inoculating agrobacterium containing target vector into 100mL LB three-antibody liquid culture solution (Kan 50 μg/mL, rif 50 μg/mL, gen 50 μg/mL), shake culturing at 28deg.C overnight, centrifuging at room temperature for 15min at 50g until OD6 value is 1.0-2.0, and collecting thallus; 2mL of the transformant (1/2 MS,5% sucrose, 40. Mu.L Silwet L-77) was used to suspend the cells; soaking corn callus in agrobacterium transformation liquid, and sealing. And (5) placing the plants back to the illumination culture rack for normal growth until the plants grow out. And then carrying out a low-temperature stress treatment experiment on the seeds obtained by screening.

In this example, the overexpression lines were isolated and the gene expression of ZmMAPK2, zmMAPK8, zmMAPK17 in the obtained overexpression lines was detected by real-time quantitative PCR.

1) Extracting total RNA of plants, and carrying out reverse transcription to obtain cDNA.

2) After 5-fold dilution of the cDNA obtained by reverse transcription, real-time quantitative PCR was performed using a Takara kit, using a reaction system comprising: 2X SYBR Premix ExTaq buffer, 0.2. Mu.L DyII, 0.4. Mu.L LPrimer (F/R), 2. Mu.L cDNA template, and finally ddH ₂ O is filled to 20 mu L, and the mixture is put into an ABI PRISM 75 real-time quantitative PCR instrument for PCR amplification by a two-step method after being fully and evenly mixed, and the reaction conditions are as follows: 95 ℃ for 30s;95 ℃ for 5s; 40s at 60 ℃;40cycles. After completion of the PCR reaction according to 2 ^-Δ(ΔCt) The relative expression level between wild type and over-expression was calculated and plotted. Simultaneously with the amplification of the identified genes, each sample was amplified simultaneously with UBI as an internal reference. The results are shown in FIG. 1.

Example 3 detection of the Cold-response ability of plants affected by overexpression of the ZmMAPK2, zmMAPK8, zmMAPK17 genes

Seeds of CK and over-expressed strains are sown in black soil, imported soil and vermiculite (1:1:1) in small basins 10cm long, 10cm wide and 10cm high, 5 grains are placed in each basin, 2cm of soil is covered in a tray, watering is carried out until the soil is completely wet, the seeds are placed in a culture chamber at 23 ℃, and the seeds are illuminated for 16 hours and dark for 8 hours. After 14 days of growth, 4 ℃ low temperature treatment is carried out until the second leaf is wilted, the second leaf is taken out to be placed in a 23 ℃ culture room for two days of recovery, and then photographing and taking materials are carried out for statistics of ion leakage rate.

In this example, the relative conductivity l= (S1-S0)/(S2-S0) of the measuring blade was used for the ion leakage rate statistics. All the whole plants of the corn subjected to low-temperature treatment are placed into a 15ml centrifuge tube filled with 10ml of distilled water, a vacuum pump is used for pumping air for 30min, then the whole plants are placed into a shaking table for shaking at room temperature for 1h, then a conductivity meter is used for measuring the initial conductivity value of the whole plants to be S1, then a sample is placed into boiling water for water bath for 15min, the whole plants are taken out and placed into the shaking table for shaking for 2h, and then the conductivity is measured and recorded as S2. S0 is the conductivity of the control distilled water.

FIG. 2 is a photograph showing plant growth before and after recovery from low temperature treatment of a strain overexpressing the ZmMAPK2 gene. FIG. 3 is a photograph showing plant growth before and after recovery from low temperature treatment of a strain overexpressing the ZmMAPK8 gene. FIG. 4 is a photograph showing plant growth before and after recovery from low temperature treatment of a strain overexpressing the ZmMAPK17 gene. FIG. 5 is a statistical plot of ion leakage rate for over-expressed strains.

The results show (FIGS. 2-5) that the over-expressed lines all exhibited a low temperature sensitive phenotype. Overexpression of the ZmMAPK8 gene can attenuate maize freezing tolerance.

Example 4 corn ZmMAPK family Gene expression detection

Ecotype LH244 was treated at 4℃and then subjected to material removal at 0h, 6h, 12h, 24h, respectively, and then RNA extraction was performed using Trizol reagent and then expression levels of ZmMAPK family genes were detected using Realtime-PCR.

The expression of the ZmMAPK2, zmMAPK8 and ZmMAPK17 genes is shown in fig. 6. In nature, plants evolved a series of adversity response genes that deal with the effects of external environmental stress, whereas plants need to regulate the expression of positive regulatory genes to change some biological metabolic processes in plants to deal with the effects of stress. In this process, plants often increase the response to adverse environments at the cost of stopping or weakening the normal growth and development, but the process requires negative regulatory factors such as ZmMAPK related genes in the invention to weaken the effect of positive regulatory genes to maintain balance, prevent the influence of excessive stress response of plants to external stimuli on the normal growth and development, and further ensure the production of crops. The experimental results of the ZmMAPK2, zmMAPK8 and ZmMAPK17 gene expression induced by low temperature are shown in FIG. 6, and the results show that the expression of the ZmMAPK2, the ZmMAPK8 and the ZmMAPK17 has obvious gene expression rise after 12 hours of low temperature treatment, and the results prove that the three are the balance of the corn which is induced to be expressed after the corn is subjected to low temperature stress for a period of time and the corn is kept against the low temperature stress.

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

Example 5 corn ZmMAPK8 mutant phenotype validation

1.ZmMAPK 8 mutant construction

1. Construction of recombinant expression vectors

gRNA sequence: AATGATGCCTGCGCATAGA (Seq ID No. 13)

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

The primer sequences are 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 using Soluton1 ligase to obtain a recombinant plasmid. The recombinant plasmid was subjected to sample sequencing, and the sequencing showed that the recombinant plasmid was named pBUE411-gmapk8 between cleavage sites Bsa1 of pBUE411 vector.

2. Obtaining and identifying ZmMAPK8 gene knockout corn

(1) Obtaining ZmMAPK8 transgenic maize and maize plants transformed into pBUE411-gmapk8

The recombinant expression vector pBUE411-gmapk8 constructed in 1 was introduced into Agrobacterium GV3101 by freeze thawing. The recombinant Agrobacterium pBUE411-gmapk8 obtained above (transformed maize wild type (LH 244 ecology)) was transformed into maize callus by the method of Agrobacterium inflorescence (SJ Clough, AF bent. Flora dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thiana. The Plant Journal,1998,16 (6): 735-743.). The procedure was completed by the China agricultural university crop functional genome and molecular breeding research center using bar resistance for selection

(2) CRISPR/Cas9 knockout strain identification of ZmMAPK8

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

3. Sequencing identification of ZmMAPK8 CRISPR strain

Total DNA of maize wild type (LH 244 ecotype) and knockout plants (478 and 664) was extracted and the DNA sequence differences of ZmMAPK8 gene in the material were detected by PCR. The method comprises the following steps:

the PCR amplification method is as follows:

wherein, the primer sequences for amplifying ZmMAPK8 gene are 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 are as follows:

(1) And (3) establishing a reaction system:

the PCR reaction system is shown in Table 1 below:

TABLE 1

(2) Three replicates were gently mixed and tested using a Bio-Rad PCR instrument.

(3) Setting a reaction program:

the PCR reaction procedure is shown in Table 2 below:

TABLE 2

The size of the detection band was about 500bp using 1% agarose gel 157V voltage electrophoresis, and was sent to sequencing company for sequencing.

The sequencing results of ZmMAPK8 PCR detection are shown in the schematic diagram of FIG. 8, and the presence of two different knockdown forms 478 of the ZmMAPK8 gene inserts a T at the 306 position of the ZmMAPK8 mRNA sequence shown below in the target site, and 664 is the deletion at the 306 and 307 positions shown in the ZmMAPK8 mRNA sequence. Both knockdown forms resulted in frameshift mutations. The specific sequencing alignment is shown in FIG. 9.

2. Detection of low-temperature response capability of plant affected by knockout of ZmMAPK8 gene

The low temperature coping ability of the constructed mutants was examined by the same method as in example 3.

As shown in fig. 7, two independent mutant lines of different knockout forms obtained by CRISPR/Cas9 gene editing techniques exhibited a distinct low temperature tolerance phenotype after low temperature treatment compared to the wild type. Mutant knockout patterns and ion leakage patterns are shown in fig. 8, demonstrating that ZmMAPK8 mutations are beneficial for improving low temperature tolerance in maize.

Meanwhile, zmMAPK2 and ZmMAPK17 have obvious low-temperature sensitive phenotype because of the over-expression strain, and the mutants of the ZmMAPK2 and the ZmMAPK17 are presumed to have the same low-temperature tolerance phenotype as the ZmMAPK8 mutant strain.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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370

<210> 3

<211> 459

<212> PRT

<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

Use of a zmmapk protein, or a coding gene thereof, or an inhibitor thereof, or a biomaterial comprising a coding gene or inhibitor thereof, in any of the following aspects:

(1) Regulating and controlling low-temperature stress tolerance of corn;

(2) Selecting corn with improved low-temperature stress tolerance;

the ZmMAPK protein is ZmMAPK8 protein; the amino acid sequence of the ZmMAPK8 protein is shown as SEQ ID NO.1.
2. The use according to claim 1, wherein the low temperature stress tolerance of maize is increased by inhibiting the expression of the gene encoding the ZmMAPK8 protein.
3. The use according to claim 1 or 2, wherein the ZmMAPK8 protein encoding gene has any of the following nucleotide sequences:

(1) The nucleotide sequence shown as SEQ ID NO.4, or,

(2) The nucleotide sequence shown in SEQ ID NO.4 is a coded nucleotide sequence of the protein with the same function obtained by replacing, deleting or inserting one or more nucleotides; or alternatively, the first and second heat exchangers may be,

(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO. 4.
4. A method of constructing transgenic maize with increased low temperature stress tolerance, characterized by inhibiting expression of a gene encoding a ZmMAPK8 protein in maize; the amino acid sequence of the ZmMAPK8 protein is shown as SEQ ID NO.1.