CN105802932B

CN105802932B - Application of CRK4 protein and coding gene thereof in regulation and control of plant drought resistance

Info

Publication number: CN105802932B
Application number: CN201610384059.4A
Authority: CN
Inventors: 张大鹏; 路凯; 王小芳
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2016-06-01
Filing date: 2016-06-01
Publication date: 2020-02-07
Anticipated expiration: 2036-06-01
Also published as: CN105802932A

Abstract

The invention discloses an application of CRK4 protein and a coding gene thereof in regulating and controlling plant drought resistance. The application provided by the invention is specifically the application of the protein (namely CRK4 protein) consisting of the amino acid sequence shown in the sequence 3 in the sequence table in a1) or a2) as follows: a1) improving the drought resistance of the plants; a2) and breeding the plant variety with improved drought resistance. Compared with wild control plants, the CRK4 gene overexpression plants have obviously improved tolerance to drought stress. The invention has important significance for the research of the drought-resistant molecular mechanism of plants; in addition, the gene has an important function in cultivating drought-tolerant plant varieties, thereby providing important possibility for cultivating new species of stress-resistant crops and having great significance for agricultural production.

Description

Application of CRK4 protein and coding gene thereof in regulation and control of plant drought resistance

Technical Field

The invention belongs to the technical field of biology, and relates to application of CRK4 protein and a coding gene thereof in regulation and control of plant drought resistance.

Background

China is a large population country, and the problem of food supply is always a major strategic problem of harmonious and stable relationship and society. Among various natural disasters causing grain loss, drought disasters are main factors threatening grain production in China, and have the characteristics of high occurrence frequency, wide influence range, long duration and the like. Therefore, it is of great significance to improve or maintain grain yield by improving drought resistance of crops. Because the traditional breeding mode has the defects of long period, high blindness, large workload and the like, and the improvement of the grain yield by the traditional breeding has developed to a certain bottleneck. In recent years, with the development of the subjects such as plant molecular biology and genetics and the intensive research on the molecular mechanism of plant stress resistance, the improvement of the stress resistance of crops by introducing stress resistance-related genes into plants by genetic engineering methods has become increasingly mature.

Abscisic Acid (ABA) is a phytohormone synthesized in a plant body and having a wide range of physiological effects, and participates in the processes of regulating and controlling the growth and development of plants and responding to external environmental stress. In the aspect of regulating and controlling the growth and development of plants, the ABA can promote the maturation and dormancy processes of seeds and the accumulation of storage proteins in the maturation process of the seeds, inhibit the germination of the seeds and the growth of seedlings, inhibit the development of main roots, promote the development of lateral roots, promote the senescence and abscission of leaves and the like. In terms of regulating plant response to biotic stress, ABA inhibits invasion of pathogenic bacteria from stomata into plants by promoting stomata closure, and regulates plant defense response to pathogenic microorganisms by regulating other signal transduction processes. In the aspect of regulating and controlling the response of plants to abiotic stress, ABA enhances the drought resistance of plants through the processes of promoting stomata closing, inhibiting stomata opening, regulating the accumulation of drought-resistant substances and the like. In addition, the ABA can also enhance the adaptability of plants to low temperature, high salt and other adversities.

The Receptor-like protein kinase RLKs (Receptor-like kinases) family is the largest membrane Receptor family of plants, and the number of members of the Receptor-like protein kinase RLKs in Arabidopsis thaliana and rice exceeds 610 and 1100 respectively. Typical receptor-like protein kinase structures include an Extracellular domain (Extracellular domain), a single Transmembrane domain (Transmembrane domain), and an intracellular kinase domain (Cytoplasmic kinase domain). Extracellular domains vary widely, primarily for sensing signals or external stimuli; intracellular kinase domains are conserved and signal by phosphorylation of downstream substrates. Based on the structure of the extracellular domain and the differences in the amino acid sequence of the intracellular kinase domain, researchers have grouped receptor-like protein kinases into different families: leucine-rich receptor-like protein kinases LRR-RLKs (leucine-rich repeat RLKs), cysteine-rich receptor-like protein kinases CRKs (cysteine-rich repeat RLKs), S-domain receptor-like protein kinases (S-domain RLKs), lectin-like receptor protein kinases LecRLK (lectin receptor kinase), and the like. Cysteine-rich receptor-like protein kinases CRKs (Cysteine-rich receptor-like kinases) are a subfamily of RLKs, which have a total of 46 members in arabidopsis thaliana. CRKs are widely involved in plant response to biotic and abiotic stresses. The gene number of the CRK4 gene on the site of Arabidopsis tair is AT3G45860(https:// www.arabidopsis.org /).

Disclosure of Invention

The invention aims to provide application of CRK4 protein and a coding gene thereof in regulation and control of plant drought resistance.

The application provided by the invention is specifically the following A or B:

A. the application of protein (CRK4 protein) consisting of amino acid sequence shown in sequence 3 in a sequence table in any one of a1) -a2) as follows:

a1) improving the drought resistance of the plants;

a2) and breeding the plant variety with improved drought resistance.

B. The application of the coding gene of protein (CRK4 protein) composed of amino acid sequence shown in sequence 3 in the sequence table in any one of a1) -a2) as follows:

a1) improving the drought resistance of the plants;

a2) and breeding the plant variety with improved drought resistance.

In the present invention, the method for breeding a plant variety with improved drought resistance in a2) above may specifically include a step of crossing a plant with a high CRK4 protein expression level as a parent.

It is another object of the present invention to provide a method for breeding transgenic plants with improved drought resistance.

The method for cultivating the transgenic plant with improved drought resistance provided by the invention specifically comprises the following steps: introducing a coding gene of a protein (CRK4 protein) consisting of an amino acid sequence shown as a sequence 3 in a sequence table into a receptor plant to obtain a transgenic plant; the transgenic plant has increased drought resistance compared to the recipient plant.

In the above application or method, the gene encoding the protein consisting of the amino acid sequence shown in sequence 3 in the sequence table (i.e., CRK4 gene) is a DNA molecule as described in any one of the following 1) to 4):

1) DNA molecule shown in sequence 2 in the sequence table;

2) DNA molecule shown in sequence 1 in the sequence table;

3) a DNA molecule which is hybridized with the DNA molecule defined in 1) or 2) under strict conditions and codes protein consisting of an amino acid sequence shown in a sequence 3 in a sequence table;

4) a DNA molecule which has more than 90 percent of homology with the DNA molecule defined by any one of 1) to 3) and codes protein consisting of an amino acid sequence shown as a sequence 3 in a sequence table.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

Wherein, the sequence 1 consists of 2641 nucleotides and is the sequence of the CRK4 gene in an Arabidopsis genome, wherein the 845-934, 1070-1151, 1274-1446, 1658-1747, 1986-2061 and 2219-2317 sites are intron sequences; the sequence 2 consists of 2031 nucleotides and is a cDNA sequence of the CRK4 gene, wherein, the 1 st to 2031 st position is a coding sequence (ORF); the sequence 1 and the sequence 2 both encode the protein shown in the sequence 3 in the sequence table, and the sequence 3 consists of 676 amino acid residues.

In the method, the coding gene of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table is introduced into the recipient plant through a recombinant expression vector containing the coding gene of the protein.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide, and 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 recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

In the present invention, the promoter for promoting transcription of the gene encoding the protein in the recombinant expression vector is a 35S promoter.

More specifically, the recombinant expression vector is a recombinant plasmid obtained by inserting the CRK4 gene into the multiple cloning sites Sma I and Kpn I of the pCAMBIA-1300-221 vector.

In the above method, the introduction of the recombinant expression vector carrying the CRK4 gene into the recipient plant may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In addition, the application of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table or the coding gene thereof in the following (a) or (b) also belongs to the protection scope of the invention:

(a) promoting stomatal closure of plants by cooperating with ABA;

(b) synergistic ABA inhibits stomatal opening in plants.

In the application, the ABA is used at a concentration of 30 μ M.

In the above application or method, the plant may be either a dicotyledonous plant or a monocotyledonous plant.

Further, the dicotyledonous plant may be a crucifer. In one embodiment of the invention, the plant is specifically Arabidopsis thaliana, more specifically Arabidopsis thaliana wild type (Col-0 ecotype).

In the present invention, all the above-mentioned proteins consisting of the amino acid sequence shown in sequence 3 in the sequence table can be replaced by fusion proteins formed by the protein shown in sequence 3 and the tag protein, such as fusion proteins expressed by recombinant plasmids obtained by inserting the DNA fragment shown in positions 1-2028 of sequence 2 between the restriction sites Sma I and Kpn I of the pCAMBIA-1300-221 vector.

Experiments prove that the tolerance of the CRK4 gene over-expression plant to drought stress is obviously improved compared with a wild control plant. The invention has important significance for the research of the drought-resistant molecular mechanism of plants; in addition, the gene has an important function in cultivating drought-tolerant plant varieties, thereby providing important possibility for cultivating new species of stress-resistant crops and having great significance for agricultural production.

Drawings

FIG. 1 shows the real-time fluorescent quantitative PCR detection of CRK4mRNA expression in CRK4 overexpression material.

FIG. 2 shows the reaction of CRK4 overexpression strain in the experiment that ABA promotes stomatal closure and ABA inhibits stomatal opening. Error bars indicate Standard Error (SE), with different letters representing significant differences between treatments at the same ABA concentration (P < 0.05). Wherein, A is ABA pore opening inhibition experiment; b is ABA promoted stomatal closure assay.

FIG. 3 shows the reaction of the CRK4 overexpression strain in the drought test and the survival rate after rehydration. A represents the rehydration experiments of wild Col-0 and CRK4 gene high expression strains C4OE-1 and C4OE-2 after normal watering, drought treatment and drought treatment respectively. And B is the statistical analysis of the survival rate of each genotype plant in A. Error bars indicate Standard Error (SE), with different letters representing significant differences between treatments at the same ABA concentration (P < 0.05).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples,% is by mass unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

pCAMBIA-1300-221 vector: supplied by the university of Qinghua (written article: Living Liu, Yiyue Zhuang, Sanyuan Tang, et al. an effect system to detect protein ubiquitination by growth in biochemical and Plant Journal,2010(61): 893-903.). In the pCAMBIA-1300-221 vector, the promoter located upstream of the Multiple Cloning Site (MCS) was the 35S promoter. The pCAMBIA-1300-221 vector contains the GFP gene. pCAMBIA-1300-221 vector-related information: http:// www.cambia.org/day/cambia/materials/vectors/585. html.

Arabidopsis wild type (Col-0 ecotype): arabidopsis thaliana wild-type seed (Arabidopsis thaliana, ecotype Columbia-0), is a product of Arabidopsis thaliana biological research center (ABRC, https:// www.arabidopsis.org /).

Agrobacterium tumefaciens (Agrobacterium tumefaciens): agrobacterium tumefaciens strain GV3101, supplied by the university of Qinghua (described in R. Berres, L. otten, B. tinland et al. transformation of vitamins tissue by differential strains of Agrobacterium tumefaciens T-6 gene. plant Cell Reports,1992(11): 192-.

Escherichia coli (Escherichia coli) strain DH5 α (DE3) is competent, a product of all-grass gold organisms, Inc.

Example 1 acquisition and characterization of CRK4 transgenic plants

The CRK4 gene involved in the embodiment is derived from Arabidopsis thaliana (Arabidopsis thaliana), the sequence of the CRK4 gene in the Arabidopsis thaliana genome is shown as the sequence 1 in the sequence table, the sequence 1 consists of 2641 nucleotides, and the CRK4 gene in the Arabidopsis thaliana genome, wherein the sites 845, 934, 1070, 1151, 1274, 1446, 1658, 1747, 1986, 2061 and 2219, 2317 are intron sequences; the cDNA sequence of the CRK4 gene is shown as a sequence 2 in a sequence table, the sequence 2 consists of 2031 nucleotides and is the cDNA sequence of the CRK4 gene, wherein the 1 st to 2031 th position is an encoding sequence (ORF); the sequence 1 and the sequence 2 both encode the protein shown in the sequence 3 in the sequence table, and the sequence 3 consists of 676 amino acid residues.

First, construction of recombinant expression vector pCAMBIA-1300-221-CRK4

Extracting total RNA of arabidopsis wild type (Col ecotype), and obtaining cDNA after reverse transcription. And (3) carrying out PCR amplification by using the obtained cDNA as a template through a primer 1 and a primer 2, purifying a product after the reaction is finished, indicating that about 2000bp fragments are obtained by amplification, and sequencing indicates that the fragments have 1-2028 th nucleotide sequences from the 5' end of a sequence 2 in a sequence list.

Primer 1: 5' -TCCCCCGGGATGTCTTTCTTCTGGCTTTTTC-3' (underlined is the recognition site for Sma I, positions 10-31 of this sequence are positions 1-22 of sequence 2);

primer 2: 5' -GGGGTACCACGAGGAGTTACATTAGTAAT-3' (the underlined part is the recognition site for Kpn I, and positions 9-29 of the sequence are the reverse complement of position 2008-2028 of sequence 2).

The PCR product obtained by the double enzyme digestion of restriction enzymes Sma I and Kpn I is used, the enzyme digestion fragment is recovered by glue and is connected with the pCAMBIA-1300-221 vector skeleton which is subjected to the same double enzyme digestion, and the recombinant plasmid is obtained. The recombinant plasmid was subjected to sample sequencing, and the recombinant plasmid in which the DNA fragment shown in the 1 st to 2028 th positions of the sequence 2 was inserted between the restriction sites Sma I and Kpn I of the pCAMBIA-1300-221-vector was designated as pCAMBIA-1300-CRK 4. In the recombinant expression vector pCAMBIA-1300-221-CRK4, the promoter for promoting the transcription of the CRK4 gene is 35S promoter.

In the construction process of the recombinant expression vector pCAMBIA-1300-221-CRK4, the CRK4 gene shown in the sequence 2 of the artificially synthesized sequence table can also be used as a template.

II, obtaining and identifying CRK4 transgenic arabidopsis

1. Acquisition of CRK4 transgenic Arabidopsis plants

The recombinant expression vector pCAMBIA-1300-221-CRK4 constructed in step one was introduced into Agrobacterium GV3101 competent. The transformed recombinant Agrobacterium was identified by PCR using a primer pair consisting of primer 1 and primer 2 (supra). The Agrobacterium GV3101 identified as containing the CRK4 gene (the size of the PCR band is about 2000 bp) was designated GV3101/pCAMBIA-1300-221-CRK 4.

The recombinant Agrobacterium GV 3101/pCAMBIA-1300-.

After transformation, hygromycin resistance screening is carried out, the seeds of the transgenic arabidopsis with hygromycin resistance are collected after being cultured on an MS culture medium containing 40mg/L of hygromycin, and a transgenic seedling with hygromycin resistance, namely an arabidopsis plant (T) transformed into pCAMBIA-1300-221-CRK4 is obtained₁Generation).

In previous researches, transgenic lines obtained by transferring pCAMBIA-1300-221-empty vectors into Arabidopsis thaliana wild type (Col ecotype) have no influence on the growth and development of plants, the hormone response and the drought resistance of plants, and the phenotype of the transgenic lines is consistent with that of the wild type, so that wild type Col-0 is used as a control part of the experiment.

2. CRK4 transgenic Arabidopsis identification

(1) Genetic segregation ratio method for identifying insert copy number

According to genetic principles, selfed progeny will yield a segregation ratio of 3:1 after single copy insertion. And (4) counting the number of resistant seedlings and non-resistant seedlings on the antibiotic culture medium by combining a statistical method. The segregation ratio method was used to identify transgenic plants as single copy inserted lines (single copy CRK4 transgenic Arabidopsis) for selection of homozygotes.

(2) Screening of transgenic Arabidopsis C4OE-1 and C4OE-2 homozygous lines

After the identification and analysis, two single-copy CRK4 transgenic Arabidopsis strains are randomly selected from the two single-copy CRK4 transgenic Arabidopsis strains, which are respectively marked as C4OE-1 and C4OE-2 (T)₁Generation). Sowing the seeds on a MS culture medium containing 40mg/L of hygromycin, and taking all parent plants which can normally grow (namely all the offspring have hygromycin resistance) as homozygous lines through continuous 2-generation screening to finally obtain T₃Transgenic Arabidopsis C4OE-1 and C4OE-2 homozygous line plants were used as experimental material for subsequent experimental analysis.

Thirdly, CRK4 gene expression analysis in transgenic arabidopsis C4OE-1 and C4OE-2 homozygous lines

Extracting total RNA of arabidopsis wild type (Col-0 ecotype) and over-expressed plants (C4OE-1 and C4OE-2), and detecting the expression condition of CRK4 gene in the material on the transcription level by using real-time fluorescent quantitative PCR. The method comprises the following specific steps:

1. analysis of transcript level (RNA expression level)

The obtained transgenic Arabidopsis plants (C4OE-1 and C4OE-2) and Arabidopsis wild type (Col-0 ecotype) are used as experimental materials. Taking arabidopsis thaliana seedlings growing for about 4 weeks, extracting RNA and carrying out reverse transcription on cDNA, and then analyzing the expression condition of CRK4 gene in each experimental material by a real-time fluorescent quantitative PCR method.

Wherein, the primer sequence for amplifying the CRK4 gene is as follows:

CRK4 RT-F1: 5'-TCTACAATGAAACCGCCACT-3' (737-756 th site of SEQ ID NO: 2);

CRK4 RT-R1: 5'-CCCGGAGACTAAAGAAAGCT-3' (reverse complement of sequence 2 at position 915-934).

The primer sequence for amplifying the internal reference Actin takes Actin2/8 as an internal reference gene as follows:

Actin-F：5’-GGTAACATTGTGCTCAGTGGTGG-3’；

Actin-R：5’-AACGACCTTAATCTTCATGCTGC-3’。

the reaction conditions of the above primers were as follows:

(1) establishment of reaction System

Real-time fluorescent quantitative PCR reaction system

(2) The three were repeated, gently shaken and mixed, and subjected to the experiment using a Bio-Rad CFX96 fluorescent quantitative PCR instrument.

(3) Setting a reaction program:

real-time fluorescent quantitative PCR reaction program

(4) Numerical analysis, in2^-ΔCtAs a measure of the relative difference in gene transcription levels, the expression of the CRK4 gene in each strain was analyzed and compared. The Ct value is the cycle number when the PCR reaction fluorescence signal reaches a set threshold value, and the delta Ct value is the difference between the Ct value of the specific primer and the Ct value of the Actin primer.

The real-time fluorescent quantitative PCR detection result of the CRK4 related genetic material is shown in figure 1, the expression of CRK4 gene is relative value, and the expression of CRK4 gene in Arabidopsis wild type (Col-0) is 1. As can be seen from the figure, the CRK4mRNA expression level in the transgenic Arabidopsis thaliana C4OE-1 and C4OE-2 is significantly higher than that in the wild type (Col-0) (P < 0.05).

Example 2 CRK4 transgenic plants drought resistance assay

ABA is an important signal molecule for resisting external stress of plants. Under drought conditions, ABA can promote stomatal closure by modulating the ion channels of stomatal guard cells to reduce water loss. Calcium ions, protein kinase, phosphatase and the like are all involved in ABA-mediated guard cell signal transduction and signal regulation of ABA-related stress-resistant genes. Therefore, whether CRK4 plays a role in the process of adjusting plant drought stress response by ABA is detected by using ABA induced stomatal movement experiments and drought experiments.

Experiment for inhibiting stomatal opening by ABA and promoting stomatal closing by ABA

(1) Experiment for inhibiting stomatal opening by ABA

Two T's obtained in example 1 in Arabidopsis thaliana wild type (Col-0 ecotype)₃The generation-homozygous CRK4 transgenic lines C4OE-1 and C4OE-2 are experimental materials. Seeds of each test material were sown on the MS medium (80-100 seeds were sown for each test material). After being laminated at low temperature of 4 ℃ for 3 days, the obtained product is transferred into a light incubator. Placing each genotype plant growing for about 4 weeks in the dark for 24h to ensure the stomata to be closed; then soaking the leaves in the same state in skin strip buffer solution (formula: 50mM KCl, 10mM MES-KOH, pH 6.15) without ABA and containing 30 μ M ABA; finally, the pore diameter was observed and recorded after 2h of illumination under a cold light source. The experiment was repeated 5 times with consistent results, about 60 pore diameters were recorded for each sample, the error bars represent Standard Error (SE), and different letters represent significant differences between treatments at the same ABA concentration (P)<0.05)。

The results are shown in A in FIG. 2, and it can be seen from the graph that the degrees of stomatal closure of the wild type Col-0 and CRK4 transgenic lines (C4OE-1 and C4OE-2) are substantially consistent (P >0.05) after dark treatment for 24 hours; after 2 hours of illumination, the pore diameters of C4OE-1 and C4OE-2 stomata of CRK4 transgenic lines are basically consistent with that of wild type Col-0(P >0.05) in an ABA-free group, but the pore diameters of C4OE-1 and C4OE-2 of CRK4 transgenic lines are smaller relative to that of wild type Col-0(P <0.05) in an ABA-treated group, namely C4OE-1 and C4OE-2 stomata opening is remarkably inhibited compared with Col-0, and the mutant is more sensitive to ABA inhibition stomata opening process and shows a hypersensitive phenotype to ABA.

(2) ABA-promoted stomatal closure assay

Two T's obtained in example 1 in Arabidopsis thaliana wild type (Col-0 ecotype)₃The generation-homozygous CRK4 transgenic lines C4OE-1 and C4OE-2 are experimental materials. Seeds of each test material were sown on the MS medium (80-100 seeds were sown for each test material). Low temperature lamination at 4 deg.CAfter 3 days, the cells were transferred to a light incubator. Soaking leaves of each genotype plant growing for about 4 weeks in epidermal strip buffer solution (formula: 50mM KCl, 10mM MES-KOH, pH 6.15), and irradiating for 3 hr under cold light source to completely open pores; then treating the leaves for 2h by 0 mu M ABA and 30 mu M ABA respectively; finally, the pore size was observed and recorded under a cold light source. The experiment was repeated 5 times with consistent results, about 60 pore diameters were recorded for each sample, the error bars represent Standard Error (SE), and different letters represent significant differences between treatments at the same ABA concentration (P)<0.05)。

Results as shown in fig. 2B, after 3h of cold light source irradiation, the initial pore sizes of C4OE-1 and C4OE-2 stomata of CRK4 transgenic lines were substantially consistent with that of wild type Col-0(P >0.05), but after 30 μ M ABA treatment was continued for 2 hours, the pore sizes of C4OE-1 and C4OE-2 of CRK4 transgenic lines were smaller relative to wild type Col-0, i.e., the degree of closure of C4OE-1 and C4OE-2 stomata was greater than that of Col-0(P <0.05), which was more sensitive to ABA-induced stomata closure, showing a hypersensitive phenotype; CRK4 was shown to have a positive regulatory effect in ABA-induced stomatal closure.

Taken together, the results above show that T obtained in example 1 is comparable to that obtained in wild-type Col-0₃The degrees of stomatal closure of the transgenic lines (C4OE-1 and C4OE-2) of the generation homozygote CRK4 in the processes of promoting stomatal closure by ABA and inhibiting stomatal opening by ABA are higher than those of wild Col-0, and the transgenic lines show a hypersensitive phenotype to ABA, which indicates that CRK4 has a positive regulation effect in the processes of inducing stomatal closure by ABA and inhibiting stomatal opening by ABA.

Second, drought experiment

Two T's obtained from Arabidopsis thaliana wild type (Col-0 ecotype) and example 1₃The generation-homozygous CRK4 transgenic lines (C4OE-1 and C4OE-2) are experimental materials. After seedlings of each genotype plant that grew for two weeks were transferred from the petri dish to soil, the drought experimental group remained unwatered and the control group watered normally. And after phenotype appears, photographing to record the experimental result, and then rehydrating. After 24h of rehydration, the experimental results were recorded by photographing and the survival rate was counted. The experiment was repeated 5 times, at least 30 plants were used for each genotype, the results were consistent, the error bars represent Standard Error (SE), different letters represent the same ABA concentrationThe difference between the treatments at different degrees is significant (P)<0.05)。

The results are shown in FIG. 3, A, where there was no significant difference in growth among the plants in the control group with normal watering. In the drought group, CRK4 transgenic lines C4OE-1 and C4OE-2 grow relatively normally compared with Col-0, the sensitivity to drought stress is reduced, and the drought resistance is shown. As shown in B in figure 3, after 24 hours of rehydration in the drought group, the survival rates of the CRK4 transgenic lines C4OE-1 and C4OE-2 were significantly higher than that of the wild type Col-0(P < 0.05). Therefore, high expression of CRK4 enhances drought resistance of plants.

Claims

1. The application of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table in any one of the following a1) -a 2):

a1) improving the drought resistance of the plants;

a2) breeding plant varieties with improved drought resistance;

the plant is Arabidopsis thaliana.

2. The application of the coding gene of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table in any one of the following a1) -a 2):

a1) improving the drought resistance of the plants;

a2) breeding plant varieties with improved drought resistance;

the plant is Arabidopsis thaliana.

3. Use according to claim 2, characterized in that: the coding gene of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table is the DNA molecule as shown in 1) or 2):

1) DNA molecule shown in sequence 2 in the sequence table;

2) DNA molecule shown in sequence 1 in the sequence table.

4. A method for breeding a transgenic plant with improved drought resistance, comprising the steps of: introducing a coding gene of a protein consisting of an amino acid sequence shown as a sequence 3 in a sequence table into a receptor plant to obtain a transgenic plant; the transgenic plant has increased drought resistance compared to the recipient plant;

the plant is Arabidopsis thaliana.

5. The use or method according to claim 4, wherein: the coding gene of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table is the DNA molecule as shown in 1) or 2):

1) DNA molecule shown in sequence 2 in the sequence table;

2) DNA molecule shown in sequence 1 in the sequence table.

6. The method according to claim 4 or 5, characterized in that: in the method, the gene coding for the protein consisting of the amino acid sequence shown as the sequence 3 in the sequence table is introduced into the recipient plant through a recombinant expression vector containing the gene coding for the protein.

7. The method of claim 6, wherein: the promoter for promoting the protein coding gene to be transcribed in the recombinant expression vector is a 35S promoter.