CN114561420B

CN114561420B - Plant drought resistance related protein AGL27 and application of coding gene thereof

Info

Publication number: CN114561420B
Application number: CN202011355612.4A
Authority: CN
Inventors: 向成斌; 赵娉霞
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2024-02-23
Anticipated expiration: 2040-11-27
Also published as: CN114561420A

Abstract

The invention relates to a plant stress resistance related protein from arabidopsis thaliana, a coding gene thereof and application thereof in regulating plant stress resistance. The protein provided by the invention has an amino acid sequence shown as SEQ ID NO.1, and has negative correlation with drought resistance of plants. The protein or the functional deletion or reduction of the encoding gene can improve drought resistance of plants, so that the plants have longer and developed root systems under stress conditions, and have relatively less pore density and smaller pore diameter.

Description

Plant drought resistance related protein AGL27 and application of coding gene thereof

Technical Field

The invention belongs to the technical field of biology, and mainly relates to application of a protein and a coding gene thereof in improving drought resistance of plants.

Background

Drought has become one of the abiotic stress factors severely limiting agricultural production in the world, and is at the first place in all abiotic stress at present, and the yield reduction of crops caused by drought in the world is more than 50% each year, and the harm of the drought is equivalent to the sum of other natural disasters. The drought area occupies more than 1/2 of the territory area, which severely restricts the agricultural production of China. In arid and semiarid regions, which occupy 52.5% of the total area of the domestic soil, the annual rainfall is only 250-500mm or less, and the soil water is seriously deficient, so that the osmotic potential in plants is changed, the cell turgor pressure is reduced, the cell membrane fluidity, the membrane protein conformation and activity are changed, the cell metabolism disorder is caused, a large amount of active oxygen is generated to poison plants, the growth and the yield of the plants are seriously influenced, and the forestry development and the ecological environment are also influenced.

At present, drought stress is a major problem limiting the rise in demand of food crops, and is one of the most severe environmental pressures worldwide affecting crop yield. With the growing population worldwide, the pressure on crop yield presents a significant challenge. However, due to the rapid changes in global warming and climate, crops are severely reduced in yield, seriously compromising global food safety. Therefore, the management of crop yield reduction caused by drought stress is an unprecedented important problem.

Drought stress causes changes in the expression of various genes involved in stress response. The change of the genes can depend on Abscisic Acid (ABA) or not on the signal path of ABA to regulate drought-resistant mechanism.

A large number of research works prove that ABA is a key medium for controlling plant response to abiotic stress, and ABA regulates the movement of stomata and closes and activates a plurality of genes related to abiotic stress through being combined with PYR/PYL/RCAR receptor proteins, so that the expression of stress tolerance ABA regulation related stress resistance genes of plants to stress is improved. First, ABA binding to the receptor (PP 2C) activates downstream signaling, and AREB/ABF protein binding to the cis-acting element of ABRE activates expression of downstream ABA-associated genes, affecting stomatal response.

At present, new researches show that CLE25 peptide expressed in roots of arabidopsis CLE family can move from roots to leaves, stimulate the synthesis capability of ABA to induce stomatal closure, reduce water loss, and further enhance drought resistance of plants. In the ABA independent signaling pathway, transcription factors can bind DRE/CRT cis-acting elements to activate expression of downstream stress-resistance genes.

For the management of crop yield reduction caused by drought stress, plant varieties with improved drought resistance are required, and characteristics of such plants can include, for example, drought-related phenotypes such as having longer and developed root systems, relatively lower stomatal density and smaller stomatal pore diameter, and plants exhibiting significant drought resistance, such as high survival rates under water-deficient conditions.

In order to obtain plant varieties of plants with increased drought resistance, it is necessary to find genetic loci in the plants that are associated with the modulation of a variety of drought-related phenotypes not limited to those described above.

Disclosure of Invention

The present inventors found that an AGL27 gene deletion mutant of arabidopsis obtained by inserting T-DNA into the first exon of the arabidopsis AT1G77080 gene, whereby plants possess longer and developed root systems under stress conditions, relatively less stomatal density and smaller stomatal pore diameter, exhibited significantly improved drought resistance. In this mutant, the DNA sequence shown in SEQ ID NO. 2 is functionally deleted.

Specifically, the inventor conducts experiments of root elongation, soil drought, stomatal density, stomatal pore diameter statistics and the like of the deletion mutant in arabidopsis, and takes WT (wild type) as a control, and discovers that the deletion mutant of the gene AGL27 has certain growth advantage (longer main root) under the condition of Mannitol (Mannitol) treatment in the root elongation stage of plants; under the drought treatment condition of the soil, the deletion mutant has obvious drought resistance function (higher survival rate). Therefore, the AGL27 gene from arabidopsis thaliana has the function of negatively regulating drought resistance of plants.

One embodiment of the invention provides application of the gene AGL27 and the encoded protein thereof in regulating drought resistance of plants.

Further embodiments relate to methods of obtaining gene editing mutants that enhance plant stress resistance using the drought resistance-related genes and their encoded proteins, and methods of breeding and/or breeding drought-resistant transgenic plants, plant varieties that enhance drought resistance.

The present invention specifically includes the following.

1. Stress resistance-related protein AGL27, wherein the stress resistance-related protein is:

1) A protein consisting of an amino acid sequence shown as SEQ ID NO. 1; or (b)

2) Consists of more than one amino acid sequence which has conservative substitution, deletion or addition compared with the amino acid sequence shown as SEQ ID NO.1, and has the same activity as the protein with the amino acid sequence shown as SEQ ID NO. 1.

In some preferred embodiments of the invention, the derivative protein has an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1.

In other preferred embodiments of the invention, the derived protein has an amino acid sequence having at least 95% homology with the amino acid sequence as shown in SEQ ID NO. 1.

The amino acid sequence shown as SEQ ID NO.1 consists of 196 amino acid residues.

2. The DNA sequence of the gene AGL27 encoding the stress resistance related protein is selected from the following:

1) A DNA sequence shown in SEQ ID NO. 2;

2) A DNA sequence encoding an amino acid sequence shown in SEQ ID NO. 1;

3) A DNA molecule which has more than one base conservatively substituted, deleted or added compared with the DNA sequence shown as SEQ ID NO. 2, and has the same function as the DNA sequence shown as SEQ ID NO. 2.

In some preferred embodiments of the invention, the DNA sequence of the DNA molecule having the same function as the DNA sequence shown in SEQ ID NO. 2 has at least 90% homology with the DNA sequence shown in SEQ ID NO. 2.

In other preferred embodiments of the invention, the DNA sequence of the DNA molecule having the same function as the DNA sequence shown in SEQ ID NO. 2 has at least 95% homology with the DNA sequence shown in SEQ ID NO. 2.

In other embodiments of the present invention, the DNA sequence of the gene encoding the stress-resistance related protein may be a DNA sequence capable of hybridizing with the DNA sequence shown in SEQ ID NO. 2 of the sequence Listing under high stringency conditions;

the high stringency conditions are, for example: hybridization was performed at 65℃in a solution containing 6 XSSC, 0.5% SDS, and then the membranes were washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the present invention, the DNA sequence shown as SEQ ID NO. 2 consists of 591 nucleotides.

3. A method of increasing stress resistance in a plant comprising: the protein related to stress resistance in plants, or the gene encoding the protein is deleted or reduced in function,

in some embodiments of the invention, the stress resistance is drought resistance and/or permeation resistance.

The loss of function or the reduction of function is achieved by gene editing, preferably by means of T-DNA insertion, more preferably by means of a DNA sequence flanking the T-DNA insertion site as shown in SEQ ID NO. 3, thereby obtaining a loss-of-function mutant,

the stress resistance is drought resistance,

the plant is selected from Arabidopsis thaliana, tobacco, rice, wheat, maize, cotton, canola or soybean,

more preferably from Arabidopsis thaliana and rice.

In one embodiment, the loss-of-function mutant is Salk_072872.

4. The cultivation method of the plant with improved drought resistance comprises the steps of breeding a plant variety with improved stress resistance, especially good drought resistance, by using the stress resistance related protein and the coding gene thereof.

5. A method of growing plants with improved drought resistance comprising:

selecting a gene with highest sequence consistency with an amino acid sequence shown as SEQ ID NO.1 in the plant as a homologous gene, and constructing a coding sequence of the homologous gene into a gene knockout vector;

the gene knockout vector is used for transforming cells or tissues of target plants to obtain transgenic plants;

and identifying the obtained transgenic plant, and determining the functional deficiency or reduction of the homologous gene, thereby obtaining a functional deficiency strain.

6. The cultivation method as claimed in item 5, comprising

Screening the loss-of-function strain based on stress resistance, preferably drought resistance or osmotic stress resistance.

7. The cultivation method according to item 6, wherein the screening comprises:

plant plants obtained using the method of claim 5 or 6 are selected as plants with increased drought resistance for which the survival rate of rehydration after soil drought treatment at a specific time is higher than the corresponding unedited control plants.

8. The cultivation method according to item 6, wherein the screening comprises:

selecting a plant obtained using the method of claim 5 or 6 as a plant with increased drought resistance after soil drought treatment at a specific time, as compared to a corresponding unedited control plant, a plant line that meets at least one of the following drought-related phenotypes:

the root phenotype is more vigorous, for example, the length of the main root is longer,

the pore phenotype is more drought tolerant, e.g., less pore density and smaller pore size.

9. A protein consisting of an amino acid sequence shown in SEQ ID NO. 1; or the application of a DNA molecule consisting of a DNA sequence shown as SEQ ID NO. 2 in changing plant stress resistance.

In the manner of simulating stress conditions, mannitol treatment is used in the subsequent embodiments, but PEG (10%), ABA (1 μm), naCl (150 mM) and the like may be used, and the dosage thereof may be adjusted according to actual needs.

It will be appreciated by those skilled in the art that plants with deleted or reduced AGL27 gene function may be obtained by gene editing, not limited to T-DNA insertion, but by, for example, knock-out (knock-out) techniques.

The treatment time for simulating the stress condition is determined according to factors such as the temperature and humidity of air, the moisture content of soil, the size of plants and the like, and generally about 15 to 20 days are required. Plants suitable for use in the method include, but are not limited to, arabidopsis, tobacco, rice, wheat, corn, cotton, canola, or soybean, and the like, preferably Arabidopsis and rice.

Drawings

The invention will be described below with reference to the accompanying drawings.

FIG. 1 (A) schematic representation of the T-DNA insertion site of mutant agl 27;

(B) Identification of mutant agl27 at genomic level;

(C) Identification of mutant agl27 at the RNA level;

FIG. 2. (A) root growth of mutant agl27 strain and WT seedlings on MS medium with 0, 250mM mannitol respectively;

(B) Primary root length statistics for mutant agl27 and WT strains under different treatment conditions.

FIG. 3. (A) soil drought experiments with mutant agl27 and WT material;

(B) The survival rate statistics of different strains under the soil drought experimental treatment condition;

FIG. 4. (A) leaf stomata density of mutant agl27 and WT material;

(B) Counting the densities of the air holes of the blades of different strains;

FIG. 5. (A) leaf pore size of mutant agl27 and WT material;

(B) And (6) counting pore diameters of leaves of different strains.

Detailed Description

The invention will be described in further detail with reference to specific examples. These examples are for illustrative purposes only and are not meant to limit the scope of the invention thereto.

The experimental methods in the following examples are conventional experimental methods unless otherwise specified. The experimental instruments of the kit used in the experiment can be purchased from biological instruments and reagent companies unless otherwise specified.

In the present invention, the background of the plants used was Arabidopsis thaliana (Arabidopsis thaliana) of the Columbia type.

In the present invention, "WT" or control plants each refer to a non-transgenic wild type strain of arabidopsis thaliana columbia.

In the present invention, the Arabidopsis thaliana stress-resistant mutant used is Salk_072872 from the Arabidopsis thaliana seed pool ABRC, also referred to as mutant agl27 in the present invention.

EXAMPLE 1 identification of Arabidopsis agl27 loss-of-function mutants at the DNA and RNA levels

The inventors obtained Arabidopsis seeds named Salk_072872 from the Arabidopsis seed pool ABRC (Arabidopsis Biological Resource Center, the Ohio State University Rightmire Hall 1060 Carmack Road,Columbus,OH 43210 USA) and used the following experiments.

Arabidopsis thaliana Salk_072872 is a deletion mutant in which T-DNA is inserted AT the first exon of the AT1G77080 gene (FIG. 1A), which is also referred to as an agl27 mutant hereinafter. The flanking sequences of the T-DNA insertion position are shown in SEQ ID NO. 3.

The following parts use the following method steps and materials.

Extraction of genomic DNA

(1) About 0.1g of fresh Arabidopsis leaves was weighed into a mortar, liquid nitrogen was added thereto, the material was rapidly ground into powder, and 400uL of DNA extraction buffer was added thereto for further grinding. After completion of the grinding, the liquid in the mortar was rapidly transferred to a 1.5mL EP tube, 5uL of 10mg/mL RNase was added to the tube, mixed upside down, and placed in a warm bath at 65℃for 15 minutes, and the mixture was taken out at 3 minute intervals and mixed upside down to allow full reaction.

(2) The sample was removed, equal volumes of unsaturated phenol and chloroform were added to each tube, mixed upside down and the excess protein was removed.

(3) Centrifuging at 12000rpm for 10 min, transferring the supernatant to a new 1.5mL EP tube, adding 0.7 times volume of isopropanol and 0.1 times volume of 3M sodium acetate into the tube, mixing, standing at room temperature for 10 min, and promoting DNA precipitation.

(4) Centrifugation was performed at 12000rpm for 10 minutes, the supernatant was discarded, and 1mL of 75% alcohol was added to the tube to wash the DNA pellet, and the procedure was repeated twice.

(5) The precipitate was dried at room temperature and 20uL ddH was added to the tube ₂ O dissolves the DNA pellet as a genomic template and stores it at-20 ℃.

PCR amplification

The primers used were as follows:

upstream primer LP: as shown in SEQ ID NO. 4.

Downstream primer RP: as shown in SEQ ID NO. 5.

Intermediate primer lbb1.3: shown in SEQ ID NO. 6.

The PCR amplification system was as follows (20 uL):

wherein the 2x Taq Master mix enzyme is purchased from Nanjinouzan biotechnology Co., ltd

2x Taq Master mix enzyme	10uL
		10uM LP	0.5uL
10uM RP	0.5uL
		10uM LBb1.3	0.5uL
DNA	0.5uL
		ddH ₂ O	8uL

The PCR procedure was:

(1) Pre-denaturation at 95 ℃ for 3 min;

(2) Denaturation at 95℃for 30 sec;

(3) The annealing temperature is 56 ℃;

(4) Extension time at 72℃for 30 seconds;

(5) Finally, the temperature is kept at 72 ℃ for fully extending for 10 minutes;

(6) The cycle number was set at 40 and stored at 25℃after completion.

RNA extraction

(1) About 0.1g of fresh Arabidopsis seedlings was weighed into a mortar, the material was ground to a powder by adding liquid nitrogen thereto a small number of times, 1mL TRIZOL reagent (Beijing full gold Biotechnology Co., ltd.) was rapidly added, grinding was continued at low temperature, and the ground tissue fluid was transferred to a sterile 1.5mL EP tube.

(2) 10uL of beta-mercaptoethanol was added to the tube to help remove proteins from the tissue fluid, and the tube was allowed to stand at room temperature for 5 minutes for complete reaction.

(3) 200uL of chloroform was added to the tube, mixed by gently inverting, left to stand at room temperature for 5 minutes, and centrifuged at 12000rpm for 15 minutes at 4 ℃.

(4) 200uL of the supernatant was transferred to a fresh 1.5mL EP tube, and after adding an equal volume of isopropanol and mixing, the mixture was left at-20℃for 30 minutes to promote precipitation.

(5) Centrifugation was performed at 12000rpm for 15 minutes at 4℃to remove the supernatant, and 1mL of 75% glacial alcohol was added to the tube to wash RNA, and the procedure was repeated twice.

(6) Centrifugation was performed at 12000rpm for 10 minutes at 4℃and the supernatant was discarded, the RNA was dried, and 20uL of DEPC-treated deionized water was added to dissolve the RNA.

(7) The extracted RNA was stored at-20 ℃.

RNA reverse transcription

Using easy script One-Step gDNA Removal and cDNA SynthesisSuperMix reverse transcription kit (Beijing full gold Biotechnology Co., ltd.), appropriate amount of RNA template was used for reverse transcription to form cDNA for qRT-PCR detection.

The reverse transcription system is as follows (20 uL):

2×ES Reaction Mix	10μL
		Anchored Oligo(dT) ₁₈ Primer	1μL
EasyScript RT/RI Enzyme Mix	5μL
		gDNA remover	1μL
RNase-free water	9μL
		Total RNA	25ng-2.5μg
Total volume of	20μL

The prepared reaction system is placed in a constant temperature water bath at 42 ℃ for incubation for 30 minutes, then placed in a heating device at 85 ℃ for 5 seconds to inactivate easy script RT/RI enzymes and gDNA, and finally the reacted cDNA is stored at-20 ℃ for subsequent qRT-PCR detection.

-qRT-PCR

Wherein the Ubiqutin5 gene is used as an internal reference gene.

The apparatus used in qRT-PCR technology is a StepOne real-time PCR system in which SYBR Green reagent is available from Beijing full gold Biotechnology Co., ltd

The qRT-PCR primers used were as follows:

AGL27 Gene

The upstream primer P1: SEQ ID NO. 7;

downstream primer P2: SEQ ID NO. 8.

Reference gene Ubiqutin5

Upstream primer P3: SEQ ID NO. 9;

downstream primer P4: SEQ ID NO. 10.

Specific reaction system (10 uL):

SYBR green	5uL
		10uM P1 (or P3)	0.25uL
10uM P2 (or P4)	0.25uL
		Template	0.25uL
ddH ₂ O	4.75uL

The amplification conditions for qRT-PCR were as follows:

(1) Pre-denaturation at 95 ℃ for 20 seconds; (2) denaturation at 95℃for 10 sec; (3) annealing at 60 ℃; (4) extending for 30 seconds; (5) 40 cycles.

First, agl27 mutant and WT (wild type) seeds were aseptically washed, placed at 4℃for vernalization for 2 days, germinated on MS dishes, and after 5-7 days of growth, seedlings were transferred to soil. Seedling leaves grown in soil for 3 weeks were extracted with plant genomic DNA and PCR amplified as described above, and the expression of AGL27 gene was detected, and the electrophoresed pattern was shown in FIG. 1B.

The result of PCR amplification of genomic DNA showed that the WT group had a band around 1200bp in molecular weight, which was a band of 1215bp for the AGL27 gene, and the AGL27 mutant group had a band around 850bp in molecular weight, which was consistent with the increase in molecular weight caused by the insertion of T-DNA.

That is, by genome-level identification, an agl27 mutant strain in which the strain Salk_072872 was homozygous was identified.

Then, seedlings of AGL27 mutant and WT (wild type) were subjected to RNA extraction and reverse transcription respectively to obtain cDNA templates, and the expression level of AGL27 gene in different materials was detected by qRT-PCR technique, and the results are shown in FIG. 1C.

The qRT-PCR detection result shows that: in the mutant AGL27, the relative expression amount of the RNA of the AGL27 gene was far lower than that of the WT group (wild type) as a control, and was hardly expressed. Thus, it is considered that the mutant agl27 strain can be determined to be a functional deletion homozygous strain.

EXAMPLE 2 Main root Length statistics of mutants agl27 and WT (wild type)

The 10% Bleach formulation (100L) is as follows:

84 disinfectant (blue moon)	10mL
		ddH ₂ O	90mL

The MS medium formulation (1L) is as follows:

10x macroelement mother liquor	100mL
		100x trace element mother liquor	10mL
100x ferric salt mother liquor	10mL
		Sucrose	10g
ddH ₂ O	Make up to 1L

The 10x macroelement mother liquor formulation (1L) is as follows:

NH ₄ NO ₃	16.5g
		KNO ₃	19g
CaCl ₂ ·2H ₂ O	4.4g
		MgSO ₄ ·7H ₂ O	3.7g
KH ₂ PO ₄	1.7g
		ddH ₂ O	make up to 1L

The formula (1L) of the 100x trace element mother solution is as follows:

the 100x ferric salt mother liquor formula (1L) is as follows:

FeSO ₄ ·7H ₂ O	2.78g
		Na ₂ -EDTA·2H ₂ O	3.73g
ddH ₂ O	make up to 1L

Note that: feSO ₄ ·7H ₂ O and Na ₂ -EDTA·2H ₂ O reagent is dissolved separately, mixed to constant volume, and then placed in 65 ℃ water bath for chelation for 2-4 hours, and stored at 4 ℃ in a dark place.

First, appropriate amounts of mutant agl27 and WT (wild-type) (control) seeds were placed in 2mL EP tubes, respectively, to which 10% Bleach was added for hand washing for 15 minutes. Transferring to an ultra-clean workbench, cleaning with sterile pure water for 5 times, and vernalizing at 4deg.C in dark for 2 days. The vernalized seeds are respectively spread on a plurality of MS culture dishes and are vertically cultured under the dark condition of 22 ℃ and 16h illumination and 8 h.

After 5 days of growth, mutant agl27 and WT (wild type) seedlings with consistent growth vigor and near main root length were selected and transferred to MS medium with different concentrations of 0, 250mM mannitol and repeated multiple times. After 5 days of growth on MS medium, photographs were observed and the average main root length of mutants agl27 and WT (wild type) was counted (FIG. 2).

As a result, FIGS. 2A-B show that there was no significant difference in the length of the main roots between the mutant agl27 group and the WT group under normal culture conditions of MS alone; however, the main root of mutant agl27 was significantly longer than that of the WT group with 250mM mannitol treatment.

The result shows that the AGL27 gene has the function of regulating and controlling the elongation of the main root under the condition of osmotic stress. It is presumed that when the gene is deleted, the plant has enhanced tolerance to abiotic stress, and the deletion mutant develops a longer and developed root system. The change is favorable for plants to absorb more nutrition and moisture and ensure the growth and development of the plants, so the inventor carries out verification test of drought resistance.

Example 3 Arabidopsis soil drought test

Firstly, after the mutants agl27 and WT (wild type) seeds are washed and vernalized in the same place, the seeds are germinated on an MS culture dish, and are vertically cultured for 7 days under the conditions of 22 ℃ and 16h illumination and 8h darkness, and then, seedlings with consistent cotyledons and main roots of different strains are selected to be transplanted to 30 small basins with the diameter of 10cm, each basin is 5, the soil content in the small basins is ensured to be consistent with the soil looseness as much as possible, and the number of the seedlings transplanted per small basin is the same.

The optimal condition for seedling growth is controlled at 22-24 ℃ under 16h illumination and 8h darkness. After the seedlings grow for 3 weeks, the seedlings are fully irrigated, and the excessive water at the bottom of each pot is discarded after the water is uniformly absorbed by each pot. The growth status of mutant agl27 and WT (wild-type) material under normal growth conditions was recorded by photographing.

Drought treatment was then performed and changes in aerial leaves of mutant agl27 and WT (wild-type) material were observed daily. And during drought treatment, watering is not performed at all. Drought treatment was stopped and the record was photographed 18 days after drought treatment, watering was continued sufficiently to allow the material to rehydrate and the record was photographed and the survival rates of mutant agl27 and WT (wild type) strains were counted (fig. 3A-B).

The inventors observed that after 18 days of drought treatment, significant manifestations (varying degrees of wilting or plant death) occurred between the aerial leaves of the mutant agl27 and WT (wild-type) lines. The statistics of FIG. 3B show that in each group of # plants, the survival rate of the mutant agl27 after rehydration was much higher than that of the WT group, the survival rate of the mutant agl27 was 78.05%, significantly higher than that of the WT group by 30.67%, and the difference was significant.

The result further verifies that the agl27 function-deleted mutant has obviously enhanced drought resistance.

EXAMPLE 4 Arabidopsis thaliana stomatal density statistics

The epidermis was removed from 3-week-old leaves of the mutants agl27 and WT (wild type) grown in soil, respectively, and the epidermis was removed with forceps, pelleted, and observed under a microscope (magnification 700), photographed, and counted for stomata, as shown in FIG. 4A, B. When the materials are obtained, the same parts of the lower epidermis of different materials are selected as much as possible, and the number of the samples of the epidermis taken by each group is not less than 30 parts.

The results showed that, as shown in FIG. 4A, the deletion mutant agl27 strain was observed to have a smaller number of stomata than the WT group; further statistics confirm that the pore density of mutant agl27 was significantly lower than the control (FIG. 4B).

Discussion: mutants lacking agl27 function have reduced stomatal density, which reduces leaf moisture loss, thereby ensuring that plants have stronger stress resistance and can maintain normal growth under the condition of water deficiency. This result further verifies the results of example 3 that plants have significantly increased drought resistance when AGL27 gene is not expressed normally.

EXAMPLE 5 Arabidopsis thaliana stomatal pore size statistics

Sensitivity of reaction experiment

Specifically, firstly, the leaves of different strains are respectively soaked in an air hole opening buffer solution for 4-8 hours, so that the air holes are fully opened, then, the leaves are transferred into an air hole closing buffer solution added with 0 and 10uM ABA respectively, the treatment is continued for 2 hours, and the pictures are taken under a microscope (the magnification is 700 times) and the size of the air hole diameters is counted.

The pore opening buffer formulation is as follows (1L):

KCl	3.726g
		K ⁺ -MES	1.952g
CaCl ₂	0.0012g
		ddH ₂ O	make up to 1L

That is, the pore opening buffer comprises 50mM KCl, 10mM K ⁺ MES and 10. Mu.M CaCl ₂ The pH was adjusted to 6.15.

The pore closing buffer formulation (1L) is as follows:

KCl	0.746g
		K ⁺ -MES	0.976g
CaCl ₂	0.006g
		water and its preparation method	Make up to 1L

That is, the pore-closing buffer comprises 10mM KCl, 5mM K ⁺ MES and 50. Mu.M CaCl ₂ The pH was adjusted to 5.6.

The sensitivity of stomata to abscisic acid (ABA, purchased from Sigma) was tested by taking several leaves of mutants agl27 and WT (wild type) grown in soil for 3 weeks, respectively, and the results are shown in fig. 5A, B.

The results show that there is no significant difference in pore size between mutant agl27 and WT (wild type) when ABA treatment is not used (0 uM group); when treated with 10uM ABA, the pore size of mutant agl27 (average size 1.68 μm) was significantly smaller than the WT group (average size 2.82 μm) (FIG. 5B).

The results illustrate that: after the gene AGL27 is deleted, the arabidopsis plant is more sensitive to the induction of ABA, has smaller pore diameter after ABA treatment, can be presumed that the plant can store more water and prevent excessive water evaporation, thereby achieving the aim of better drought resistance. It was suggested that the mechanism of action of the AGL27 gene may depend on ABA signaling pathway.

In summary, the group of mutants AGL27 deleted for gene AGL27 had significantly longer length of main root than WT group under mannitol treatment conditions; under the soil drought experimental treatment condition, the survival rate of the mutant agl27 is 78.05 percent, which is obviously higher than that of the WT group (30.67 percent); mutant agl27 had relatively less stomatal density (121.68/mm) ² ) And has smaller pore size under abscisic acid (ABA) treatment conditions. Therefore, the gene AGL27 can be used for negatively regulating plant stress resistance, and when the gene is deleted, the drought resistance of the plant is obviously improved.

The discovery is favorable for further understanding the molecular mechanism of the plant stress-resistance strategy, and provides a new candidate gene for crop improvement and cultivation of new varieties of stress-resistance crops.

Industrial applicability

The invention provides a gene AGL27 for enhancing plant stress resistance, especially drought resistance, and a method for improving plant drought resistance, cultivating drought-resistant transgenic plants and breeding new varieties of drought-resistant crops by using the gene AGL27. The new variety of drought-resistant crops is expected to be provided, so that the contradiction between modern agriculture and the population which is rapidly increased can be relieved, and a foundation is laid for sustainable agricultural production.

It should be understood that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. In the embodiments of the present invention, although the present invention has been described in detail, it should be understood by those skilled in the art that the words which have been used are words of description and illustration, rather than words of limitation, and that various changes in the details of the embodiments may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method of increasing stress resistance in a plant comprising: the function of stress resistance related protein in plants or the coding gene thereof is deleted or reduced,

wherein the stress resistance related protein is a protein consisting of an amino acid sequence shown as SEQ ID NO.1, the stress resistance is drought resistance, and the plant is Arabidopsis thaliana.

2. The method according to claim 1, wherein the coding gene is a DNA molecule consisting of the DNA sequence shown in SEQ ID NO. 2.

3. The method according to claim 1 or 2, wherein,

the loss of function or the reduction of function is achieved by gene editing, thereby obtaining a loss-of-function mutant.

4. The method of claim 3, wherein the gene editing is performed by means of T-DNA insertion.

5. The method according to claim 4, wherein the flanking sequence of the T-DNA insertion site is the DNA sequence shown in SEQ ID NO. 3.

6. A method of growing plants with improved drought resistance comprising:

selecting a coding gene sequence of an amino acid sequence shown as SEQ ID NO.1 in the plant, and constructing the coding gene sequence into a gene knockout vector;

identifying the obtained transgenic plant, and determining the functional deficiency or reduction of the coding gene, thereby obtaining a functional deficiency plant line, wherein the plant is arabidopsis thaliana.

7. The cultivation method according to claim 6, comprising screening the loss-of-function strain based on drought resistance or osmotic stress resistance.

8. The method of claim 7, wherein the screening comprises:

plant plants obtained by the method of claim 6 or 7, which are selected to have increased drought resistance as plants having a higher rehydration survival rate after soil drought treatment at a specific time, compared to corresponding unedited control plants.

9. The method of claim 7, wherein the screening comprises:

selecting as plants with increased drought resistance, a plant line that after a soil drought treatment at a specific time corresponds to at least one of the following drought-related phenotypes, as compared to corresponding unedited control plants, plant plants obtained using the method of claim 6 or 7:

the phenotype of the root system is more developed,

the stomatal phenotype is more drought tolerant.

10. The cultivation method as claimed in claim 9, wherein the root phenotype is developed more the length of the main root is longer, and the stomatal phenotype is more drought tolerant as the stomatal density is smaller and the stomatal size is smaller.

11. A protein consisting of an amino acid sequence shown in SEQ ID NO. 1; or the application of a DNA molecule consisting of a DNA sequence shown as SEQ ID NO. 2 in improving the stress resistance of plants, wherein the stress resistance is drought resistance, and the plants are arabidopsis thaliana.