CN118028349A

CN118028349A - Protein ZmHDT103 and application of coding gene thereof in regulation and control of drought resistance of plants

Info

Publication number: CN118028349A
Application number: CN202410049475.3A
Authority: CN
Inventors: 郑军; 王逸茹; 王小东
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-05-14

Abstract

The invention discloses an application of a protein ZmHDT103 and a coding gene thereof in regulating and controlling drought resistance of plants, belongs to the technical field of biology, and particularly relates to an application of a protein ZmHDT103 and a coding gene thereof in drought response of corn. The protein or the substance for regulating and controlling the expression of the gene or the substance for regulating and controlling the activity or the content of the protein can be applied to the aspect of regulating and controlling the drought resistance of plants, and the drought resistance of the plants can be improved by knocking out the coding gene of the protein ZmHDT, so that the protein or the substance for regulating and controlling the activity or the content of the protein has positive effects on accelerating the improvement of maize varieties.

Description

Protein ZmHDT103 and application of coding gene thereof in regulation and control of drought resistance of plants

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a protein ZmHDT103 and application of a coding gene thereof in regulating and controlling drought resistance of plants.

Background

Global warming leads to increased frequency and intensity of drought and other extreme climatic events. Corn (Zea mays l.) is the most widely planted dual-purpose crop in the world, and drought stress is the major abiotic stress factor affecting the growth and development of corn. Drought resistance of crops is a complex quantitative trait regulated by thousands of genes, and drought is the most severe environmental factor affecting crop yield, which is even considered as a "cancer" of plants due to the complexity and destructive nature of drought.

In plants, modifications of histones, including acetylation, methylation, phosphorylation, ubiquitination, etc., can cause dynamic changes in chromatin structure and gene expression, while drought stress typically induces changes in histone acetylation of "drought responsive" genes and other genes. Histone Deacetylases (HDACs) are an important class of enzymes widely distributed in plants, and are involved in regulating many biological processes.

In view of the role of HDACs in plant abiotic stress, the identification and cloning of HDT histone deacetylase in corn has important significance for crop stress resistance mechanism research and production application.

Disclosure of Invention

The invention aims to provide an application of a protein ZmHDT103 and a coding gene thereof in regulating and controlling drought resistance of plants.

To achieve the object of the present invention, in a first aspect, the present invention provides any one of the following uses of the protein ZmHDT103 or an expressed substance of a gene encoding or a gene regulating the activity or content of the protein:

1) For regulating drought resistance in plants;

2) For preparing products for regulating and controlling drought resistance of plants;

3) For growing plants with altered drought resistance;

4) For preparing a product for breeding plants with altered drought resistance;

5) Is used for plant breeding.

In the present invention, the protein ZmHDT is:

a1 Protein with the amino acid sequence shown as SEQ ID NO. 1;

a2 A protein which is derived from a 1) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1;

a3 A protein which has 80% or more identity with the sequence represented by a 1) or a 2) and has an equivalent function;

a4 A) a fusion protein obtained by ligating a tag to the end of any one of the sequences shown in a 1) to a 3).

Further, the application is negative regulation of drought resistance in plants.

Further, the substance regulating the expression of the gene or the substance regulating the activity or content of the protein is a biological material related to the protein ZmHDT103,103, and the biological material is any one of the following:

c1 A nucleic acid molecule encoding said protein;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

c3 A recombinant vector comprising c 1) said nucleic acid molecule, or a recombinant vector comprising c 2) said expression cassette;

c4 A recombinant microorganism comprising c 1) said nucleic acid molecule, or a recombinant microorganism comprising c 2) said expression cassette, or a recombinant microorganism comprising c 3) said recombinant vector;

c5 A transgenic plant cell line comprising c 1) said nucleic acid molecule, or a transgenic plant cell line comprising c 2) said expression cassette;

c6 A transgenic plant tissue comprising c 1) said nucleic acid molecule, or a transgenic plant tissue comprising c 2) said expression cassette;

c7 A transgenic plant organ comprising c 1) said nucleic acid molecule, or a transgenic plant organ comprising c 2) said expression cassette;

e1 A nucleic acid molecule that inhibits, reduces or silences expression of the protein-encoding gene;

e2 An expression cassette comprising e 1) said nucleic acid molecule;

e3 A recombinant vector comprising e 1) said nucleic acid molecule, or a recombinant vector comprising e 2) said expression cassette;

e4 A recombinant microorganism comprising e 1) said nucleic acid molecule, or a recombinant microorganism comprising e 2) said expression cassette, or a recombinant microorganism comprising e 3) said recombinant vector;

e5 A transgenic plant cell line comprising e 1) said nucleic acid molecule, or a transgenic plant cell line comprising e 2) said expression cassette;

e6 A transgenic plant tissue comprising e 1) said nucleic acid molecule, or a transgenic plant tissue comprising e 2) said expression cassette;

e7 A transgenic plant organ containing e 1) said nucleic acid molecule, or a transgenic plant organ containing e 2) said expression cassette.

Further, c 1) said nucleic acid molecule is a DNA molecule as shown in any one of the following:

d1 A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 3;

d2 A DNA molecule with a coding region sequence shown as SEQ ID NO. 2;

d3 A DNA molecule which has 90% or more identity to the sequence shown in d 1) or d 2) and which encodes the protein of claim 1;

d4 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in d 1) or d 2) and which codes for a protein according to claim 1.

In a second aspect, the present invention provides a method of increasing drought resistance in a plant, the method comprising: inhibiting, reducing, or silencing the activity and/or amount of protein ZmHDT103,103 in a plant of interest to increase drought resistance in the plant; and/or the number of the groups of groups,

Inhibiting, reducing or silencing the expression level of the coding gene of protein ZmHDT103,103 to increase drought resistance in plants.

In a third aspect, the present invention provides a method of reducing drought resistance in a plant, the method comprising: enhancing, increasing or upregulating the activity and/or content of protein ZmHDT103,103 in a plant of interest to reduce drought resistance in the plant; and/or the number of the groups of groups,

Enhancing, increasing or upregulating the expression level of the coding gene of protein ZmHDT, to reduce drought resistance in plants.

In a fourth aspect, the present invention provides a method of breeding a plant with increased drought resistance, the method comprising: modifying the plant of interest to obtain a plant with increased drought resistance by inhibiting, reducing or silencing the activity and/or content of protein ZmHDT103,103 in the plant of interest; and/or the number of the groups of groups,

Obtaining a plant with increased drought resistance by inhibiting, reducing or silencing the expression level of the gene encoding the protein ZmHDT103,103;

Wherein the drought resistance of the plant with increased drought resistance is higher than that of an unmodified plant.

Further, the method comprises the steps of:

(1) Constructing a recombinant expression vector of a coding gene of the inhibition, reduction or silencing protein ZmHDT;

(2) Transferring the recombinant expression vector constructed in the step (1) into a target plant (receptor plant) to obtain a plant with improved drought resistance.

In one embodiment of the invention, the method comprises: and taking the coding gene of the protein ZmHDT as a target, designing an sgRNA sequence based on CRISPR-Cas9, connecting a DNA fragment containing the coding sgRNA sequence into a carrier carrying the CRISPR-Cas9, and transforming plants to obtain transgenic plants with the gene function deletion.

Preferably, the nucleotide sequences of the sgRNA sites of action are 5'-GTTAAGTGTGAGCCTGGATATGG-3' (SEQ ID NO: 6) and 5'-GATGATCAGAAACTTGCCATTGG-3' (SEQ ID NO: 7).

The plant is any one of the following:

N1) monocotyledonous plants:

N2) gramineae plants;

n3) a gramineous plant;

n4) zea plants;

N5) corn.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention identifies a corn ZmHDT (Zm 00001d 038832) protein and a coding gene thereof, the protein ZmHDT belongs to a member of HDAC family, and the identification and cloning of HDAC histone deacetylase in corn has important significance for crop stress resistance mechanism research and production application. By constructing PBUE411-sgRNA recombinant plasmid to knock-out ZmHDT103,103 genes, three different editing plants of ZmHDT103,103 genes are obtained, drought resistance of seedling stage is identified in a greenhouse, and drought resistance of the editing plants is found to be improved. Provides new materials for drought-resistant corn variety breeding, and has positive effects on accelerating the improvement of corn varieties.

Drawings

FIG. 1 is a schematic diagram of the structure of a PBUE411-sgRNA recombinant vector according to a preferred embodiment of the present invention.

FIG. 2 is a diagram illustrating the identification of ZmHDT's 103 knockout mutant in a preferred embodiment of the present invention. Wherein A is a structural schematic diagram of ZmHDT gene, and gRNA1 and gRNA2 are two target sites of PBUE 411-gRNA. B is 3 mutation sites of ZmHDT editing strains, compared with a wild type, zmHDT103-1 is inserted into a first gRNA1 by 1bp, and a second gRNA2 by 1bp; zmHDT103-2 deleted 3bp and replaced 11bp in the first gRNA1 and 1bp insert in the second gRNA 2; zmHDT.sup.103-3 was inserted 1bp in the first gRNA1 and 6bp in the second gRNA 2.

FIG. 3 is a phenotypic identification of CRISPR/Cas9 knockout ZmHDT103,103 mutants in a preferred embodiment of the invention. Wherein A is the leaf phenotype of the inbred lines Z31 and ZmHDT gene knockout mutant lines; b is the relative expression quantity of ZmHDT103 genes of the inbred lines Z31 and ZmHDT103 gene knockout mutants; c is the survival rate of the knockout mutants of inbred lines Z31 and ZmHDT.

FIG. 4 shows the physiological changes of ZmHDT A103 knockout mutant in a preferred embodiment of the present invention. A is relative conductivity; b is the relative water content; c is hydrogen peroxide content; d is the content of malondialdehyde; e is the peroxidase content; f is the proline content.

Detailed Description

The invention aims to improve drought resistance of plants.

In order to solve the technical problems, the invention provides application of protein or an expression substance of a regulatory gene or a substance for regulating the activity or the content of the protein in regulating drought resistance of plants.

The invention adopts the following technical scheme:

the invention provides application of protein or expression substance of regulatory genes or substance for regulating activity or content of the protein in any one of the following:

1) Use of a protein or an expression substance of a regulatory gene or a substance regulating the activity or content of said protein for regulating drought resistance in a plant;

2) The application of protein or the expression substance of regulating gene or the substance regulating the activity or content of the protein in preparing the product regulating the drought resistance of plants;

3) Use of a protein or an expression substance of a regulatory gene or a substance regulating the activity or content of said protein for growing plants with altered drought resistance;

4) Use of a protein or an expression substance of a regulatory gene or a substance regulating the activity or content of said protein for the preparation of a product for breeding plants with altered drought resistance;

5) Use of a protein or an expression substance of a regulatory gene or a substance regulating the activity or content of said protein in plant breeding;

the protein is any one of the following proteins:

a1 Protein with the amino acid sequence of SEQ ID NO. 1;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1 and has the same function;

a3 A protein having an amino acid sequence defined in any one of a 1) to a 2) and 80% or more identity and the same function;

a4 A fusion protein obtained by ligating a tag to the end of the protein defined in any one of a 1) to a 3).

The protein of a 1) is ZmHDT103,103.

In order to facilitate purification or detection of the protein of a 1), a tag protein may be attached at the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO. 1.

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence encoding protein ZmHDT of the present invention may be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein ZmHDT103,103 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein ZmHDT103,103 and have the function of the protein ZmHDT103,103.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the present invention, identity refers to identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.

In the present invention, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In the present invention, the 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In the above application, the protein is derived from corn (Zea mays l.).

In the present invention, the substance that regulates the activity and/or content of the protein may be a substance that regulates the expression of a gene encoding the protein ZmHDT103.

Further, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

In the present invention, the modulation may be up-regulation or enhancement or improvement. The modulation may also be down-regulation or reduced or lowered.

In the above application, the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein may be a biological material related to the protein as described above, and the biological material may be any of the following:

c1 A nucleic acid molecule encoding a protein as described above;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

e1 A nucleic acid molecule that inhibits or reduces or silences the expression of a gene encoding a protein as described above;

e2 An expression cassette comprising e 1) said nucleic acid molecule;

In the above application, the nucleic acid molecule of c 1) may be a DNA molecule as shown in any one of the following,

D1 A DNA molecule with a nucleotide sequence shown in SEQ ID NO. 3;

d2 A DNA molecule with a coding region sequence shown in SEQ ID NO. 2;

d3 A DNA molecule which has 90% or more identity to the nucleotide sequence defined in d 1) or d 2) and which encodes a protein as described above;

d4 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined under d 1) or d 2) and which codes for a protein as described above.

In the above application, the nucleic acid molecule of e 1) may be a DNA molecule whose nucleotide sequence is shown in SEQ ID NO. 3.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or an antisense RNA.

Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. Specifically, the carrier PBUE411 carrier may be used.

Recombinant expression vectors containing ZmHDT103,103 genes can be constructed using existing plant expression vectors. Such plant expression vectors include, but are not limited to, vectors such as binary Agrobacterium vectors and vectors useful for microprojectile bombardment of plants, and the like. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to untranslated regions transcribed from the 3' end of plant genes including, but not limited to, agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase Nos genes), plant genes (e.g., soybean storage protein genes).

When the ZmHDT gene is used to construct a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter can be added before the transcription initiation nucleotide, including, but not limited to, a cauliflower mosaic virus (CAMV) 35S promoter, a ubiquitin promoter (ubiquitin) of corn, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, such as by adding genes encoding enzymes or luminescent compounds that produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

By using any vector capable of guiding exogenous gene to express in plant, the ZmHDT gene or gene fragment provided by the invention is introduced into plant cell or acceptor plant to obtain transgenic cell line with altered drought resistance and transgenic plant. Expression vectors carrying ZmHDT103,103 genes can be transformed into plant cells or tissues by conventional biological methods using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium-mediated, etc., and the transformed plant tissues are grown into plants.

Alternatively, the expression cassette of e 2) is an expression cassette having the DNA molecule shown in positions 1-16731 of SEQ ID NO. 4.

In a specific embodiment, e 3) the nucleotide sequence of the recombinant plasmid PBUE411-sgRNA is shown in SEQ ID NO. 4 and 5 (the complete sequence of the recombinant plasmid is formed by tandem sequences shown in SEQ ID NO. 4 and 5). PBUE411-sgRNA expression targets sgRNA1 and sgRNA2 of ZmHDT genes, and the target sequence of the sgRNA1 is: 5'-GTTAAGTGTGAGCCTGGATATGG-3' (SEQ ID NO: 6), the target of the sgRNA1 being located at the first exon of the ZmHDT gene, the nucleotide sequence of the target of the sgRNA1 being positions 802-824 of SEQ ID NO:3 or positions 40-62 of SEQ ID NO: 2; the sgRNA2 target sequence is: 5'-GATGATCAGAAACTTGCCATTGG-3' (SEQ ID NO: 7), the target of the sgRNA2 is located at the second exon of the ZmHDT gene, and the nucleotide sequence of the target of the sgRNA is the 1026-1049 th position of SEQ ID NO:3 or the 139-161 th position corresponding to SEQ ID NO: 2.

The invention also provides a method for improving drought resistance of plants, which comprises the step M, wherein the step M is used for inhibiting or reducing or silencing the activity and/or content of the protein in the target plants, or/and inhibiting or reducing or silencing the expression level of the encoding gene of the protein to improve the drought resistance of the plants.

The invention also provides a method for reducing drought resistance of plants, which comprises the step P, wherein the step P is used for enhancing, increasing or up-regulating the activity and/or content of the protein in the target plants, or/and enhancing, increasing or up-regulating the expression level of the encoding gene of the protein to reduce the drought resistance of the plants.

In the above method, the inhibiting or reducing or silencing the expression level and/or activity of the gene encoding the protein ZmHDT103,103 in the plant of interest may be by reducing or inactivating the activity of the gene encoding the protein ZmHDT103,103 in the genome of the plant of interest using a gene mutation, gene knockout, gene editing or gene knockdown technique.

The gene knockout (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by a gene editing technique. Gene knockout is the inactivation of a particular target gene by a change in DNA sequence.

The gene silencing refers to the phenomenon that the gene is not expressed or expressed under the condition of not damaging the original DNA. Gene silencing is premised on the fact that the DNA sequence is not altered, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level due to DNA methylation, heterochromatin, and positional effects, and the other is post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of a target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression (co-suppression), gene suppression (quelling), RNA interference (RNAi), and microrna (miRNA) -mediated translational inhibition, among others.

The present invention provides a method for growing plants with increased drought resistance, comprising inhibiting or reducing or silencing the expression of the gene encoding the protein and/or the content and/or activity of the protein in a plant of interest, or/and inhibiting or reducing or silencing the activity and/or content of the gene encoding the protein, to obtain plants with increased drought resistance.

In one embodiment of the present invention, the breeding method for growing drought resistance-improving plants comprises the steps of:

(1) Constructing a recombinant expression vector for inhibiting or reducing or silencing the encoding gene of the protein;

(2) Transferring the recombinant expression vector constructed in the step (1) into a receptor plant to obtain a plant with drought resistance lower than that of the receptor plant.

In the present invention, the object of plant breeding may include growing plants with increased drought resistance.

In the above method, the coding gene of the protein of the target corn can be obtained by mutating the coding gene of the protein shown in SEQ ID NO.3 in the corn genome:

1) Replacing 5'-GTTAAGTGTGAGCCTGGATATGG-3' in the gene encoding the protein in the maize genomic DNA with 5'-GTTAAGTGTGAGCCTGGGATATGG-3', thereby knocking out the gene encoding the ZmHDT103 protein; replacing 5'-GATGATCAGAAACTTGCCATTGG-3' in the gene encoding the protein in the maize genomic DNA with 5'-ATGATCAGAAACTTGCCCATTGG-3', thereby knocking out the gene encoding the ZmHDT103 protein;

2) Replacing 5'-GTTAAGTGTGAGCCTGGATATGG-3' in the gene encoding the protein in the maize genomic DNA with 5'-GTTACCTTTCCCAGGTAAAAC-3', thereby knocking out the gene encoding the ZmHDT103 protein; replacing 5'-GATGATCAGAAACTTGCCATTGG-3' in the gene encoding the protein in the maize genomic DNA with 5'-ATGATCAGAAACTTGCACATTGG-3', thereby knocking out the gene encoding the ZmHDT103 protein;

3) 5'-GTTAAGTGTGAGCCTGGATATGG-3' of the gene encoding the protein in the maize genomic DNA was replaced with 5'-GTTAAGTGTGAGCCTGGGATATGG-3', so that the gene encoding the ZmHDT103 protein was knocked out. Replacing 5'-GATGATCAGAAACTTGCCATTGG-3' in the gene encoding the protein in the maize genomic DNA with 5'-GATGATCAGAAACATCAAAAACTACCAG-3', thereby knocking out the gene encoding the ZmHDT103 protein;

In the invention, the plant is any one of the following:

N1) monocotyledonous plants:

N2) gramineae plants;

n3) a gramineous plant;

n4) zea plants;

N5) corn.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated. The data were processed using EXCEL statistical software, and experimental results were expressed as mean ± standard deviation, with P < 0.05 (x) indicating significant differences, P < 0.01 (x) indicating significant differences, and P < 0.001 (x) indicating significant differences using the T-Test.

The PBUE411 vector used in the following examples (professor Chen Jijun by chinese agricultural university, see Xing HL,Dong L,Wang ZP,Zhang HY,Han CY,Liu B,Wang XC,Chen QJ.ACRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol.2014Nov29;14:327)、PCBC-MT1T2 vector (professor Chen Jijun by chinese agricultural university, see Xing HL,Dong L,Wang ZP,Zhang HY,Han CY,Liu B,Wang XC,Chen QJ.A CRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol.2014Nov 29;14:327)、 maize inbred B73 and maize inbred Z31, both publicly available from applicant) was used only for repeated experiments of the invention, and not for other uses.

Example 1ZmHDT Gene mutant acquisition

1. Cloning of ZmHDT103,103 Gene

Leaves of maize inbred line B73 seedlings grown to two leaves and one heart were immediately snap frozen with liquid nitrogen, RNA extracted using RNA EASY FAST PLANT Tissue kit (TIANGEN) kit, reverse transcribed using HISCRIPT III 1st Strand cDNA Synthesis Kit (Vazyme) kit, and first strand cDNA was synthesized. Designing a homologous recombination primer HDT103-CDS-F/HDT103-CDS-R according to the cDNA sequence of ZmHDT genes by using a cDNA template to carry out PCR amplification, wherein the amplification primer has the following sequence:

HDT103-CDS-F：5’-ATGGAGTTCTGGGGTCTCGA-3’

HDT103-CDS-R：5’-TCACTTGCCCCCATGCTTCG-3’

the PCR amplification reaction system is shown in Table 1.PCR amplification procedure: 94 ℃ for 2min;98 ℃ for 10s; 30s at 60 ℃;68 ℃ for 1min;68 ℃ for 7min;35 cycles.

TABLE 1 PCR amplification reaction System

The obtained PCR product is sent to be sequenced, the CDS sequence length of ZmHDT103,103 is 903bp, and the PCR product codes for protein of 300 amino acids, and belongs to HDAC family members. The coding sequence (CDS) of ZmHDT.sup.103 gene in maize B73 is SEQ ID NO.2 and the coding amino acid sequence is ZmHDT protein of SEQ ID NO. 1. In the genome DNA of the corn B73, the genome gene of the ZmHDT protein is shown as SEQ ID NO. 3.

2. Extraction of corn leaf DNA

Extracting corn leaf DNA by using a biliquid plant DNA extraction kit. 2-10mg of seedling leaf blade is taken and put into a sterilized 2mL centrifuge tube containing steel balls, the liquid nitrogen is frozen and is crushed into powder on a plant tissue crusher, 100 mu L of PDA is added into the centrifuge tube for uniform mixing, water bath is carried out at 50 ℃ for 5-15min, or the centrifuge is swayed, 100 mu L of PDB is added for gentle overturning and uniform mixing, standing is carried out at room temperature for 5min, and centrifugation is carried out at 12000rpm for 10min. The pipette sucks 80-100 mu L of supernatant, transfers the supernatant into a new 1.5mL centrifuge tube, adds equal volume of absolute ethyl alcohol, mixes uniformly, stands for 10min, centrifiges for 2min at 12000rpm, discards the supernatant, adds 800 mu L of 70% ethyl alcohol to wash precipitate, centrifiges for 2min at 12000rpm to precipitate DNA, discards the supernatant, cools the supernatant to exert residual ethyl alcohol, and adds 20-100 mu L of sterilizing water to obtain corn leaf DNA.

3. ZmHDT103 acquisition of CRISPR/Cas9 knockout mutant of 103

Two target sequences with low off-target rate and high targeting rate are selected on a first exon and a second exon of ZmHDT gene respectively to be used as target sequences of a construction vector, and are connected with a PBUE411 vector, and then the maize inbred line Z31 is transformed.

1) ZmHDT103 acquisition of the target sequence

The coding region of ZmHDT was knocked out using the CRISPR/Cas9 system. Specific target sequences sgRNA1 (SEQ ID NO: 6) and sgRNA2 (SEQ ID NO: 7) of ZmHDT103 were designed using Guide Design Resources (https:// zlab. Bio/guide-design-resources) and primers containing the target sequences were designed with the following information:

ZmHDT103-MT1-BsF:5’-ATATATGGTCTCTGGCGGTTAAGTGTGAGCCTGGATA-3’

ZmHDT103-MT1-F0:5’-GGTTAAGTGTGAGCCTGGATAGTTTTAGAGCTAGAAATAGC-3’

ZmHDT103-MT2-R0:5’-ATGGCAAGTTTCTGATCATCCGCTTCTTGGTGCC-3’

ZmHDT103-MT2-BsR:5’-ATTATTGGTCTCTAAACATGGCAAGTTTCTGATCATC-3’

PCR was performed using the intermediate vector PCBC-MT1T2 (given benefit from the teaching of China agricultural university Chen Jijun) as a template, zmHDT103-MT1-F0 and ZmHDT103-MT2-R0 as primers, the first round of PCR products as templates, zmHDT-MT 1-BsF and ZmHDT103-MT2-BsR as primers, and the second round of PCR amplification as follows: KOD Buffer 15 μl; dNTP 5. Mu.L; ddH ₂ O7.4. Mu.L; KOD FOX 0.6. Mu.L; first round PCR amplification primers ZmHDT-MT 1-F0.75. Mu.L, zmHDT103-MT 2-R0.75. Mu.L; second round PCR amplification primers ZmHDT-MT 1-BsF 0.75. Mu.L, zmHDT103-MT2-BsR 0.75. Mu.L; PCBC-MT1T20.5. Mu.L.

PCR amplification procedure: 94 ℃ for 2min;98 ℃ for 10s; 30s at 60 ℃;68 ℃ for 1min;68 ℃ for 7min;35 cycles, the target sequence containing the cleavage site was obtained.

2) Construction of PBUE411-sgRNA recombinant plasmid

The PBUE411 vector is taught by chinese university of agriculture Chen Jijun for benefit and the vector structure is as in fig. 1. PBUE411 vector and the second round PCR product were digested separately with BsaI-HF in a 37℃water bath for 2 hours, the digestion system was as follows: 10X Cutsmart. Mu.L; bsaI-HF 1. Mu.L; 10. Mu.L of PBUE411/PCR product; ddH ₂ O34. Mu.L. And then, performing homologous recombination on the PBUE411 and the PCR product by using a ligase Ligation Mix enzyme, wherein a Ligation system is as follows: PBUE 411. Mu.L, PCR product 1. Mu.L, ligation Mix 10. Mu.L.

After the above reaction was carried out at room temperature for 30 minutes, the reaction system was immediately added to E.coli TOP10, ice-bathed for 30 minutes, immediately after the water bath at 42℃for 90 seconds, ice-bathed for 2 minutes, 500. Mu.L of LB medium was added to a super clean bench, shaking was carried out at 37℃for 1 hour, centrifugation was carried out at 5000rpm for 2 minutes, and the cells were uniformly spread on the LB medium at a super clean bench, and cultured in an incubator at 37℃for 12 hours. The monoclonal is picked up on an ultra-clean workbench for PCR amplification and sequencing, and the PCR amplification primers are as follows:

OsU3-FD3:5’-GACAGGCGTCTTCTACTGGTGCTAC-3’

TaU3-RD:5’-CTCACAAATTATCAGCACGCTAGTC-3’

The method comprises the steps of carrying out propagation on a monoclonal transformed agrobacterium LBA4404 with correct sequencing (see Liu Y,Zhang Y,Liu Y,Lu W,Wang G.Metabolic effects of glyphosate on transgenic maize expressing a G2-EPSPS gene from Pseudomonas fluorescens.J Plant Biochem Biot.2015:24(2):233–241).. Mu.L of the monoclonal transformed agrobacterium LBA4404, firstly, carrying out propagation on the monoclonal with correct sequencing in a YEB liquid medium, extracting recombinant plasmids by using a plasmid extraction kit, adding the plasmids into the agrobacterium LBA4404, carrying out ice bath for 30 minutes, quickly freezing with liquid nitrogen for 5 minutes, carrying out water bath at 37 ℃ for 2 minutes, then carrying out ice bath for 5 minutes, adding 800 mu.L of the YEB medium into an ultra-clean bench, shaking the bacteria for 5 hours by a shaking table at 28 ℃, centrifuging for 2 minutes at 5000rpm, carrying out bacterial cell precipitation, uniformly coating the bacterial cells on the YEB medium on the ultra-clean bench, carrying out propagation on the bacterial cells in the YEB liquid medium by picking up the monoclonal, and extracting positive cloned plasmids by using a plasmid extraction kit (Jin Lianbao Biotechnology limited company, product number DP 105-03), thereby obtaining the recombinant vector PBUE411-sgRNA.

The structure of the recombinant plasmid PBUE411-sgRNA is schematically shown in FIG. 1. The nucleotide sequence of the recombinant plasmid PBUE411-sgRNA is shown in SEQ ID NO. 4 and SEQ ID NO. 5. PBUE411-sgRNA expression targets sgRNA1 and sgRNA2 of ZmHDT genes, and the target sequence of the sgRNA1 is: 5'-GTTAAGTGTGAGCCTGGATATGG-3' (SEQ ID NO: 6), the target of the sgRNA being located at the first exon of the ZmHDT gene, the nucleotide sequence of the target of the sgRNA1 being positions 802-824 of SEQ ID NO:3 or positions 40-62 corresponding to SEQ ID NO: 2; the sgRNA2 target sequence is: 5'-ATCATGGACGGCGCCGACGGCGG-3' (SEQ ID NO: 7), the target of the sgRNA2 is located at the second exon of the ZmHDT gene, and the nucleotide sequence of the target of the sgRNA is the 1026-1049 th or 139-161 th positions of SEQ ID NO:3 or corresponding to SEQ ID NO: 2.

PBUE411-sgRNA contains a sgRNA gene expression cassette with a nucleotide sequence of SEQ ID NO:4 at 1 st-1781 st, the sgRNA1 gene is shown as 465 th-487 th nucleotide of SEQ ID NO:4, the 84 th-463 th nucleotide is a promoter for starting the transcription of the sgRNA1 gene, the 564 th-854 th nucleotide is a terminator for stopping the transcription of the sgRNA1 gene, the sgRNA2 gene is shown as 1380 th-1402 th nucleotide of SEQ ID NO:4, the 855 th-1378 th nucleotide is a promoter for starting the transcription of the sgRNA2 gene, and the 1479 th-1769 th nucleotide is a terminator for stopping the transcription of the sgRNA2 gene.

The nucleotide sequence of the Cas9 protein gene expression cassette is 1782-8699 of SEQ ID NO. 4, 3909-8009 of SEQ ID NO. 4 codes Cas9 protein, 1782-3773 is promoter for starting Cas9 protein gene transcription, 8605-8699 is terminator for stopping Cas9 protein gene transcription.

3) Obtaining transgenic plants of T _O generations

Taking corn pollinated for about 10 days, soaking the corn in 75% alcohol for 3-5 minutes, and then washing the corn with distilled water. Picking out embryo in the seed grains by using a dissecting knife at an ultra clean bench, washing the embryo twice by using embryo washing liquid, sucking out embryo washing liquid by using a pipetting gun, adding bacterium liquid containing recombinant plasmid PBUE411-sgRNA infection, uniformly mixing, standing for 5 minutes in the dark in a water bath at 42 ℃, sucking out the bacterium liquid, pouring a culture medium, leading the embryo concave surface to be downward, airing the culture medium, and placing the culture medium in a culture box at 23 ℃ for 3 days. Transferring the embryo cultured for 3 days to a new culture medium, culturing in dark at 28deg.C for 7 days, cutting bud, transferring to the new culture medium, culturing in dim light at 28deg.C for 14 days, transferring the seedling cultured for 14 days to a culture bottle, culturing in normal light at 28deg.C until complete root and leaf tissue grows, transplanting to flowerpot, and growing to obtain T0 generation transgenic seedling

And culturing the obtained T0 generation transgenic ZmHDT103,103 gene knockout corn to T3 generation, carrying out PCR and sequencing identification on each generation of cultured corn after selfing, and screening the homozygous strain. 3 homozygous mutant plants (i.e., the mutations of the two homologous chromosomes are identical) are obtained by screening and are named ZmHDT103-1, zmHDT103-2 and ZmHDT103-3 strains respectively.

The DNA of the leaf of the plant of the T3 generation is extracted by a biliquid method, the specific sequencing primer ZmHDT-F/ZmHDT-103-R is used for amplification, the DNA of the wild type maize inbred line Z31 is used as a reference, and the PCR product is sent to a company for sequencing. The sequencing primers were as follows:

ZmHDT103-F：5’-CATGCATGAACTTGTTGGTCTT-3’

ZmHDT103-R：5’-TCCTCGTCTTCATCTTCAGAATC-3’

the PCR amplification system is shown in Table 2.PCR amplification procedure: 3min at 95 ℃;95 ℃ for 15s; 15s at 60 ℃; 80s at 72 ℃;72 ℃ for 5min;35 cycles.

TABLE 2 reaction System for PCR amplification of sequencing primers

2×Es Taq Master Mix	12.5μL
		Forward Primer	1μL
Reverse Primer	1μL
		DNA	2μL
DMSO	0.5μL
		ddH₂O	13μL

The results are shown in FIG. 2: a total of 3 different editing lines were identified: zmHDT103-1, zmHDT103-2, zmHDT103-3.

Sequencing and identifying that compared with the genome DNA of the maize inbred line Z31, the two homologous chromosomes of ZmHDT-1 plants (expressed by ZmHDT 103-1) have the following mutation in the gene encoding ZmHDT103 protein, the first target sgRNA is mutated, 5'-GTTAAGTGTGAGCCTGGATATGG-3' is mutated to 5'-GTTAAGTGTGAGCCTGGGATATGG-3', and the mutation site corresponds to 818-819 of SEQ ID NO. 3 and 56-57 of SEQ ID NO. 2; the sgRNA of the second target spot is mutated to 5'-GATGATCAGAAACTTGCCCATTGG-3', 5'-GATGATCAGAAACTTGCCATTGG-3' is mutated to 5'-GATGATCAGAAACTTGCCCATTGG-3', and the mutation site corresponds to positions 1044-1045 of SEQ ID NO. 3 and 156-157 of SEQ ID NO. 2. When the first sgRNA1 was mutated, its ZmHDT103,103 protein sequence had a frame shift mutation at position 20, so that the gene encoding ZmHDT103,103 protein was knocked out, and the sequencing results of the mutation site and its surrounding nucleotides are shown in FIG. 2.

Sequencing and identifying that compared with the genome DNA of the maize inbred line Z31, the two homologous chromosomes of the ZmHDT103-2 plant (shown by ZmHDT 103-2) have the following mutation in the gene for coding ZmHDT103 protein, wherein the first target sgRNA is mutated to 5'-GTTACCTTTCCCAGGTAAAAC-3', the 5'-GTTAAGTGTGAGCCTGGATATGG-3' mutation site corresponds to the 805-807, 807-809, 809-814, 815-818, 819-821, 821-823, 43-46, 46-47, 47-51, 52-55 and 55-58 of SEQ ID NO: 2; the sgRNA of the second target spot is mutated to 5'-GATGATCAGAAACTTGCCCATTGG-3', 5'-GATGATCAGAAACTTGCCATTGG-3' is mutated to 5'-GATGATCAGAAACTTGCCCATTGG-3', and the mutation site corresponds to positions 1043-1044 of SEQ ID NO. 3 and 155-156 of SEQ ID NO. 2. When the first sgRNA1 was mutated, its ZmHDT103,103 protein sequence had a frame shift mutation at position 14, so that the gene encoding ZmHDT103,103 protein was knocked out, and the sequencing results of the mutation site and its peripheral nucleotides are shown in FIG. 2.

Sequencing and identifying that compared with the genome DNA of the maize inbred line Z31, the two homologous chromosomes of ZmHDT-3 plants (denoted by ZmHDT 103-3) have the following mutation in the gene encoding ZmHDT103 protein, the first target sgRNA is mutated, 5'-GTTAAGTGTGAGCCTGGATATGG-3' is mutated to 5'-GTTAAGTGTGAGCCTGGGATATGG-3', and the mutation site corresponds to 818-819 of SEQ ID NO.3 and 56-57 of SEQ ID NO. 2; the sgRNA of the second target spot is mutated, 5'-GATGATCAGAAACTTGCCATTGG-3' is mutated into 5'-GATGATCAGAAACATCAAAAACTACCAG-3', and mutation sites correspond to 1039-1041, 1041-1042, 1042-1043, 1045-1049 of SEQ ID NO.3, 151-153, 153-154, 154-155 and 155-159 of SEQ ID NO. 2. When the first sgRNA1 was mutated, its ZmHDT103,103 protein sequence had a frame shift mutation at position 20, so that the gene encoding ZmHDT103,103 protein was knocked out, and the sequencing results of the mutation site and its surrounding nucleotides are shown in FIG. 2.

4. Extraction of corn leaf RNA and fluorescent quantitative RT-PCR quantitative analysis

And (3) measuring plants: gene editing plants ZmHDT-1, zmHDT-103-2, zmHDT-103-3, wild type control maize inbred Z31.

The leaf RNA of the plant was isolated using RNA EASY FAST PLANT tissue kit (TIANGEN) and genomic DNA was removed from the sample according to the instructions of RNase-FREE DNASE I (Lany), and the RNA concentration and purity were determined using spectrophotometry (Nanodrop 2000C) and agarose gel electrophoresis, and first strand cDNA was synthesized from DNase l-treated 1. Mu.g RNA using HISCRIPT III 1st Strand cDNA Synthesis Kit (Vazyme). RT-qPCR was performed using SYBR GREEN FAST QPCR Mix (Vazyme) and Bio-Rad CFX96 instrument with GAPDH as the reference gene. qRT-PCR primers were as follows:

ZmHDT103-Q-F：5’-CTGAAGAAGGCGATGATGATTC-3’

ZmHDT103-Q-R：5’-AGATAGAGGAGTTTTCAGCACG-3’

GAPDH-F：5’-AGGATATCAAGAAAGCTATTAAGGC-3’

GAPDH-R：5’-GTAGCCCCACTCGTTGTCG-3’

the qRT-PCR amplification reaction system is shown in Table 3.qRT-PCR amplification procedure: 30S at 95 ℃;95 ℃ for 10s; 30s at 60 ℃;35 cycles; 95 ℃ for 15s;60 s at 60 ℃;95℃for 15s.

TABLE 3 qRT-PCR amplification reaction System

2×ChamQ SYBR qPCR	10μL
		Forward Primer	0.4μL
Reverse Primer	0.4μL
		Template cDNA	2μL
ddH₂O	7.2μL

CRISPR/Cas9 knockout mutant seedling drought tolerance identification of example 2ZmHDT103

Maize inbred line Z31 and transgenic lines ZmHDT103, zmHDT103,103-2, zmHDT103,103-3 were planted in one pot (length x width x height = 60 x 25 x 15 cm), each line was sown with 32 grains, 2L of water was evenly watered after sowing, 20 days after water control, inbred line Z31 and transgene exhibited significant phenotypic differences, and 2L of water was evenly watered again, and CRISPR/Cas9 knockout mutants of ZmHDT were identified for drought tolerance. All plant materials were grown in a greenhouse with a 28 ℃ light cycle of 16 hours light/8 hours darkness, and three biological replicates were set.

Detecting the expression of ZmHDT gene in the knockout line by qRT-PCR, wherein the expression level of ZmHDT103 in the knockout lines ZmHDT103-1, zmHDT103-2 and ZmHDT103-3 is obviously reduced (B in FIG. 3); after 20 days of water control, the leaves of the inbred line Z31 were completely desiccated, whereas the leaves of the transgenic line of ZmHDT103,103 gene exhibited a curled or wilted phenotype (a in fig. 3); after 3 days of drought rehydration, the numbers of transgenic survival strains of the inbred lines Z31 and ZmHDT103,103 genes were counted, and the survival rates of the transgenic strains ZmHDT103,103-1, zmHDT103,103-2 and ZmHDT103,103-3 were significantly higher than that of the inbred line Z31 (C in FIG. 3).

Example 3ZmHDT evaluation of drought resistance of CRISPR/Cas9 knockout mutants

Maize inbred line Z31 and transgenic lines ZmHDT103, zmHDT103,103-2, zmHDT103,103-3 were planted in one pot (length x width x height = 60 x 25 x 15 cm), each line was sown with 32 grains, and planted in a greenhouse with a 28 ℃ light cycle of 16 hours light/8 hours darkness. Two treatment modes are set in the study, the normal treatment group (WW) is normally watered after corn sowing, and the experimental group (Drought) is only watered once after corn sowing. After the maize plants grow to 15-16 days, sampling is carried out when obvious phenotype differences appear in the inbred lines and the knockout mutants of the experimental group, and the drought-related physiological indexes are determined. Three biological replicates were set for each treatment regimen.

0.5G of the leaf was placed in 15mL of deionized water and after 12h of immersion the relative conductivity (Relative electrolytic leakage) was measured, the conductivity of the extract R1 was measured by a conductivity meter, and then heated in boiling water for 30min, cooled to room temperature, the extract conductivity R2 was measured, relative conductivity = R1/r2×100%.

The results show that: under normal treatment, there was no significant difference in the relative conductivities of wild type Z31 and ZmHDT103-1, zmHDT103-2, zmHDT103-3, whereas under drought treatment the relative conductivities of wild type Z31 were significantly higher than for the knockout strain (a in fig. 4).

Washing corn seedling leaves with deionized water, weighing W1, placing in distilled water for 24 hours, wiping off the surface moisture of the leaves, weighing W2, placing in an oven for drying at 80 ℃ for 48 hours until the weight is unchanged, weighing W3, and measuring the relative moisture content (RELATIVE WATER content) = (W1-W3)/(W2-W3) multiplied by 100%.

The results show that: under normal treatment, there was no significant difference in the relative moisture content of wild type Z31 and ZmHDT103-1, zmHDT103-2, zmHDT103-3, whereas under drought treatment the relative moisture content of wild type Z31 was significantly lower than that of the knockout strain (B in FIG. 4).

The hydrogen peroxide content (H ₂O₂ content), malondialdehyde content (MDA content), peroxide content (POD content), proline content (Pro content) were measured using the kit. 0.1g of the leaf blade was weighed and placed in a 2.0mL centrifuge tube containing steel balls, and the enzyme content was measured according to the kit instructions (Comin Assay Kit).

Under normal treatment, the levels of H ₂O₂, MDA, POD, pro of wild-type inbred line Z31 and knockout lines ZmHDT-103-1, zmHDT-103-2, zmHDT-103-3 were not significantly changed, while under drought stress, the levels of H ₂O₂ and MDA of wild-type Z31 were significantly higher than those of knockout lines, and the levels of POD and Pro were significantly lower than those of knockout lines (C-F in FIG. 4), indicating that the knockout mutant of ZmHDT103 was more drought-resistant than the wild-type control.

In conclusion, the drought resistance of the maize seedling stage can be improved by knocking out the maize ZmHDT103 gene by using CRISPR/Cas 9.

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.

Claims

1. Protein ZmHDT103 or any one of the following applications of the coding gene or the expression substance of the regulatory gene or the substance regulating the activity or the content of the protein:

1) For regulating drought resistance in plants;

3) For growing plants with altered drought resistance;

4) For preparing a product for breeding plants with altered drought resistance;

5) Used for plant breeding;

The protein ZmHDT is:

a1 Protein with the amino acid sequence shown as SEQ ID NO. 1;

2. The use according to claim 1, wherein the use is negative regulation of drought resistance in plants.

3. The use according to claim 1 or 2, wherein the substance regulating the expression of a gene or the substance regulating the activity or content of the protein is a biological material related to the protein in the use according to claim 1 or 2, the biological material being any of the following:

c1 A nucleic acid molecule encoding said protein;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

e2 An expression cassette comprising e 1) said nucleic acid molecule;

e4 A recombinant microorganism comprising e 1) said nucleic acid molecule, or a recombinant microorganism comprising e 2) said expression cassette, or a recombinant microorganism comprising e 3) said recombinant vector.

4. The use according to claim 3, wherein c 1) said nucleic acid molecule is a DNA molecule as set forth in any one of the following:

d1 A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 3;

d2 A DNA molecule with a coding region sequence shown as SEQ ID NO. 2;

5. A method of increasing drought resistance in a plant, the method comprising: inhibiting, reducing or silencing the activity and/or amount of a protein in the use of claim 1 or 2 in a plant of interest to increase drought resistance in the plant; and/or the number of the groups of groups,

Inhibiting, reducing or silencing the expression level of a gene encoding a protein for use according to claim 1 or 2, to increase drought resistance in plants.

6. A method of reducing drought resistance in a plant, the method comprising: enhancing, increasing or upregulating the activity and/or content of a protein in the use of claim 1 or 2 in a plant of interest, to reduce drought resistance in the plant; and/or the number of the groups of groups,

Enhancing, increasing or upregulating the expression of a gene encoding a protein in the use according to claim 1 or 2, in order to reduce drought resistance in plants.

7. A method of breeding plants with increased drought resistance, the method comprising: modifying a plant of interest to obtain a plant with increased drought resistance by inhibiting, reducing or silencing the activity and/or amount of a protein in the use of claim 1 or 2 in the plant of interest; and/or the number of the groups of groups,

Obtaining a plant with increased drought resistance by inhibiting, reducing or silencing the expression level of a gene encoding a protein in the use according to claim 1 or 2;

8. The method according to claim 7, comprising the steps of:

(1) Constructing a recombinant expression vector that inhibits, reduces or silences the gene encoding the protein in the use of claim 1 or 2;

9. The method according to claim 8, characterized in that the method comprises: designing a sgRNA sequence based on CRISPR-Cas9 by taking a coding gene of protein ZmHDT as a target, connecting a DNA fragment containing the sgRNA sequence to a carrier carrying CRISPR-Cas9, and transforming plants to obtain transgenic plants with the gene function deletion;

preferably, the nucleotide sequences of the sgRNA sites of action are 5'-GTTAAGTGTGAGCCTGGATATGG-3' and 5'-GATGATCAGAAACTTGCCATTGG-3'.

10. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9, wherein the plant is any one of the following:

N1) monocotyledonous plants:

N2) gramineae plants;

n3) a gramineous plant;

n4) zea plants;

N5) corn.