CN117821475B - Application of Chinese mango MsHDZ gene in enhancing stress resistance of plants - Google Patents

Abstract

The invention discloses a method for improving plant stress resistance by improving the expression level of Chinese mango MsHDZ gene and/or MsHDZ protein in plants, wherein the amino acid sequence of the Hua Mang MsHDZ protein is shown as SEQ ID NO. 4. The invention obtains MsHDZ gene with drought resistance and salt and alkali resistance in Chinese mango for the first time, and overexpression of MsHDZ gene in Arabidopsis thaliana can improve salt resistance, alkali resistance and drought resistance of plants, and does not influence normal growth of plants. The invention provides a new gene resource for cultivating a new plant variety with high stress resistance, and has important popularization and application values in plant germplasm cultivation and agricultural production for resisting abiotic stress.

Description

Application of Chinese mango MsHDZ gene in enhancing stress resistance of plants

Technical Field

The invention relates to the field of biotechnology and genetic engineering, in particular to application of a Chinese mango MsHDZ gene in enhancing plant stress resistance.

Background

Abiotic stress is an important factor affecting plant growth and yield reduction, including drought, low temperature, soil salinization, flooding, etc. Abiotic stress causes slow growth, accelerated aging, crop yield reduction and even death of plants, seriously damages the planting and production of crops, and causes serious ecological imbalance and economic benefit loss.

In the face of the problem of insufficient cultivated land area caused by increasingly severe abiotic stress, a person skilled in the art adopts a series of soil improvement measures, and the main core technologies are divided into three types: physical modification, chemical modification, and biological modification. The rapid effect of physical improvement is the greatest advantage, but the required cost is high, the engineering quantity is large, and the method is suitable for small areas; the chemical improvement requires comprehensive knowledge of the physical and chemical properties of the soil, and then the modifier is selectively used, so that the chemical improvement is difficult to operate, new ions are easily introduced, secondary pollution is caused, and the chemical improvement is difficult to use in a large area; biological improvement includes phytoremediation and bio-additive methods, and provides economic value by developing and utilizing salt tolerant plants to improve soil structure and physicochemical properties, etc., to increase soil fertility. Phytoremediation is the most economical, effective and sustainable green restoration means at present, and the cultivation of new germplasm for creating plants with strong stress resistance is a new breakthrough point for solving abiotic stress damage.

Disclosure of Invention

In order to solve the problem of developing plant varieties with strong stress resistance in the prior art, the invention provides application of the Chinese mango MsHDZ gene in enhancing the stress resistance of plants.

The first object of the invention is to provide the application of Chinese mango MsHDZ gene and/or MsHDZ protein in enhancing plant stress resistance.

A second object of the present invention is to provide a biomaterial.

A third object of the present invention is to provide the use of said biological material for the preparation of products for enhancing stress resistance in plants.

A fourth object of the invention is to provide a product.

It is a fifth object of the present invention to provide the use of said product for enhancing stress resistance in plants.

It is a sixth object of the present invention to provide a method for enhancing stress resistance in plants.

In order to achieve the above object, the present invention is realized by the following means:

The Miscanthus (Miscanthus) is a perennial herb fiber Miscanthus energy plant originally produced in China, has the characteristics of perennial growth, high biomass, strong stress resistance, wide adaptability and the like, and becomes one of important raw material sources for producing fiber fuel ethanol and forage grass. The miscanthus can normally grow in high-salinity and drought soil, and has a certain repairing effect on saline-alkali soil and heavy metal polluted soil.

The invention researches transcription factors related to saline-alkali stress and drought stress response of Chinese mango (Miscanthus SINENSIS ANDERSS), and obtains MsHDZ gene in families participating in stress response and functions thereof in stress response by carrying out systematic analysis on the whole genome level.

The invention claims the following:

The application of the Chinese mango MsHDZ gene and/or MsHDZ protein in enhancing the stress resistance of plants improves the expression level of the Chinese mango MsHDZ gene and/or MsHDZ protein in the plants; the amino acid sequence of the Chinese mango MsHDZ protein is shown as SEQ ID NO. 4.

Preferably, the nucleotide sequence of the CDS region of the Chinese mango MsHDZ gene is shown as SEQ ID NO. 3.

Preferably, plant stress resistance is stress resistance of a plant to abiotic stress.

More preferably, the abiotic stress includes any one or more of salt stress, alkali stress and drought stress.

Preferably, the enhancing stress resistance of the plant comprises any one or more of promoting germination of seeds of the plant, promoting growth of roots of the plant, improving permeability of cell membranes of the plant, enhancing photosynthesis of the plant and enhancing oxidation resistance of the plant.

Preferably, the plant is arabidopsis thaliana.

The application of the Chinese mango MsHDZ gene in cultivating high stress resistance plants is also within the protection scope of the invention.

The present invention provides a biomaterial which is any one of the following (1) to (5):

(1) A nucleic acid molecule with a sequence shown as SEQ ID NO.3 or a complete complementary sequence of the sequence shown as SEQ ID NO. 3; (2) an expression cassette comprising the nucleic acid molecule of (1); (3) A recombinant expression vector comprising the nucleic acid molecule of (1); (4) A recombinant microorganism comprising the nucleic acid molecule of (1); (5) a protein with an amino acid sequence shown as SEQ ID NO. 4.

Preferably, the recombinant expression vector in (3) uses a PBI121-MYC vector as a skeleton.

More preferably, the recombinant expression vector in (3) uses PBI121-MYC vector as skeleton, and the nucleic acid molecule in (1) is arranged at EcoRI cleavage site.

Preferably, the recombinant microorganism of (4) is a recombinant strain.

More preferably, the recombinant strain is obtained by transforming the recombinant expression vector into agrobacterium.

Further preferably, the agrobacterium is GV3101.

Further preferably, the method of transformation is a freeze-thawing method.

The application of the biological material in preparing the product for enhancing the stress resistance of plants is also within the protection scope of the invention.

The application of the biological material in enhancing plant stress resistance is also within the protection scope of the invention.

The invention provides a product containing the biological material.

The application of the product in enhancing plant stress resistance is also within the protection scope of the invention.

A method for enhancing stress resistance of plants, which comprises treating plants with the product to increase expression level of Chinese mango MsHDZ gene and/or MsHDZ protein in plants.

Preferably, plant stress resistance is stress resistance of a plant to abiotic stress.

More preferably, the abiotic stress includes any one or more of salt stress, alkali stress and drought stress.

Preferably, the enhancing stress resistance of the plant comprises any one or more of promoting germination of seeds of the plant, promoting growth of roots of the plant, improving permeability of cell membranes of the plant, enhancing photosynthesis of the plant and enhancing oxidation resistance of the plant.

Preferably, the plant is arabidopsis thaliana.

Compared with the prior art, the invention has the following beneficial effects:

The invention obtains MsHDZ gene with drought resistance and salt and alkali resistance in Chinese mango for the first time, and overexpression of MsHDZ gene in Arabidopsis thaliana can improve salt resistance, alkali resistance and drought resistance of plants, and does not influence normal growth of plants. The invention provides a new gene resource for cultivating a new plant variety with high stress resistance, and has important popularization and application values in plant germplasm cultivation and agricultural production for resisting abiotic stress.

Drawings

FIG. 1 shows the results of identifying the expression level of MsHDZ gene in MsHDZ gene over-expression lines and mutant back-filling lines; results of AMsHDZ23 Gene overexpression lines; b is the result of the mutant anaplerotic strain; WT-wild type arabidopsis thaliana.

FIG. 2 shows the phenotypic identification of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines; a is a 5-week-old rosette leaf shape photograph of each plant; b is a single rosette leaf of a 5-week-old plant for separating each plant; c is a 7 week old growth phenotype photograph of each plant; d is the plant height statistical result of each plant; e is the length statistical result of the pods of each plant; f is the statistical result of the length of the leaf, the width of the leaf and the length of the leaf stalk of each plant.

FIG. 3 shows seed germination of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under salt stress; a is a sowing distribution schematic diagram of each plant and a seed germination photo under the control and 100 mM NaCl treatments; the germination rate statistics of each plant seed under the treatment of the 1/2MS agar culture medium of 0mM, 50 mM, 100 mM and 150 mM NaCl are shown in sequence.

FIG. 4 shows root elongation of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under salt stress; a is a root system photo of each plant under the control and 150mM NaCl treatment respectively; the main root length statistics of each plant were obtained by treatment with 1/2MS agar medium with B values of 0mM, 50mM, 100 mM and 150mM NaCl.

FIG. 5 shows the results of phenotype and physiological index measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under long-term salt stress; a is a photo of each plant after 14 days of irrigation with water and saline water respectively; and B-G are statistical results of chlorophyll content, free proline content, relative conductivity, na ⁺ content, K ⁺ content and Na ⁺/K⁺ of each plant after 14 days of water and saline irrigation respectively.

FIG. 6 shows the results of antioxidant indicator measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under long-term salt stress; a to D are the results of the MDA, SOD, POD and CAT measurements in this order.

FIG. 7 shows the DAB and NBT staining results of WT, msHDZ23 gene overexpression lines, hb7 mutant and mutant complementation lines under long-term salt stress.

FIG. 8 shows seed germination of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under alkali stress; a is a sowing distribution schematic diagram of each plant and a seed germination photo under the control and 7mM NaHCO ₃ treatments; and B-C are the statistical results of germination rate of each plant seed treated by the 1/2MS agar culture medium under the conditions of 0 mM and 7mM NaHCO ₃ in sequence.

FIG. 9 shows root elongation of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under alkali stress; a is a root system photo of each plant under the control and 7 mM NaHCO ₃ treatments respectively; the main root length statistics of each plant were obtained by 1/2MS agar medium treatment with B being 0 mM and 7 mM NaHCO ₃.

FIG. 10 shows the results of phenotype and physiological index measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under long-term alkaline stress; a is a photo of each plant after 14 days of watering with water and alkaline water respectively; and B-D are statistical results of chlorophyll content, relative conductivity and free proline content of each plant after 14 days of water and alkaline water irrigation respectively.

FIG. 11 shows the results of antioxidant index measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under long-term alkali stress; a to D are the results of the MDA, SOD, POD and CAT measurements in this order.

FIG. 12 shows the DAB and NBT staining results of WT, msHDZ23 gene overexpression lines, hb7 mutant and mutant complementation lines under long-term alkali stress.

FIG. 13 shows seed germination of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under drought stress; a is a sowing distribution schematic diagram of each plant and a germination photo of seeds under the treatment of control and 200 mM mannitol; the germination rate statistics of the seeds of each plant under the treatment of the 1/2MS agar culture medium of 0mM, 100 mM and 200 mM mannitol are sequentially shown as the B-D.

FIG. 14 shows root elongation of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under drought stress; a is a root system photo of each plant under the treatment of control and 200mM mannitol respectively; the main root length statistics of each plant were obtained by treatment with 1/2MS agar medium containing 0mM, 50 mM, 100 mM, 150 mM and 200mM mannitol.

FIG. 15 is a graph showing the determination of phenotype, survival rate and relative water content of WT, msHDZ23 gene overexpression lines, hb7 mutant and mutant complementation lines under long-term drought stress; a is a photo of each plant after 14 days of watering and drought and 3 days of rehydration respectively; b is the survival rate result of each plant before and after rehydration; c is the relative water content results of each plant before and after rehydration.

FIG. 16 shows the results of physiological index measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under long-term drought stress; and A-C are statistical results of chlorophyll content, relative conductivity and free proline content of each plant after water irrigation and drought treatment respectively.

FIG. 17 shows the results of antioxidant indicator measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant complementation lines under long-term drought stress; a to D are the results of the MDA, POD, SOD and CAT measurements in this order.

FIG. 18 shows the DAB and NBT staining results of WT, msHDZ23 gene overexpression lines, hb7 mutant and mutant complementation lines under long-term drought stress.

FIG. 19 shows the results of relative water loss measurements of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant anaplerotic lines under long-term drought stress.

FIG. 20 shows stomatal morphology and opening and closing of WT, msHDZ23 gene overexpression lines, hb7 mutant lines and mutant anaplerotic lines under long-term drought stress; a is a pore photo; b is the result of measuring the degree of opening and closing of the air holes.

Detailed Description

The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1MsHDZ construction of Gene overexpression Strain and mutant anaplerotic Strain

1. Experimental method

1. Construction of MsHDZ Gene overexpression lines

(1) 35S MsHDZ construction of a MYC over-expression vector

The CDS sequence of MsHDZ Gene (Gene loci: misin G257500) located in Chinese mango (Miscanthus sinensis) was retrieved from genome annotation information (http:// www.phytozome.net /) obtained at Phytozome, and the upstream amplification primer MsHDZ-CDS-F was designed: 5'-ATGGACGGCGAGGACGACGT-3' (SEQ ID NO. 1) and downstream amplification primer MsHDZ-CDS-R: 5'-TCACTCACTGAGGGACTCGAATTC-3' (SEQ ID NO. 2), PCR amplification was performed using 2X Phanta Max Master Mix high-fidelity enzyme using cDNA obtained by reverse transcription of RNA from wild type Chinese mango leaves as a template.

The PCR reaction system is ：MsHDZ23-CDS-F（SEQ ID NO.1）（10 μM）,1 μL;MsHDZ23-CDS-R（SEQ ID NO.2）（10 μM）,1 μL;2×Phanta Max Master Mix,25 μL; templates, 1 mu L; ddH ₂ O was made up to 50. Mu.L. The PCR reaction procedure was: 95 ℃,5 min ℃;95 ℃,15 sec,58 ℃,15 sec,72 ℃,1 min,72 ℃,5 min,35 cycles.

The PCR product is detected by agarose gel electrophoresis, and is recovered by StedyPure DNA gel recovery kit, so as to finally obtain CDS fragment (SEQ ID NO. 3) of MsHDZ gene 732 bp, and the amino acid sequence of MsHDZ protein encoded by the CDS fragment is shown as SEQ ID NO. 4.

According to a single enzyme digestion and homologous recombination method, the CDS fragment (SEQ ID NO. 3) of MsHDZ gene is inserted into EcoRI site of PBI121-MYC vector (namely PBI121 vector with MYC tag), and the vector with correct sequence is identified by sequencing, namely the over-expression vector of MsHDZ gene is marked as 35S: msHDZ23-MYC.

(2) Construction of Agrobacterium

35S:: msHDZ-MYC was introduced into GV3101 Agrobacterium (Agrobacterium tumefaciens, available from Shanghai Biotechnology Co., ltd.) by freeze thawing, and the resulting Agrobacterium was designated 35S:: msHDZ-MYC-GV 3101.

(3) Transformation by immersion

Wild type Arabidopsis thaliana (Arabidopsis thaliana, marked as WT) which is strong in growth and takes a flowering vigorous period is selected, 35S is obtained by converting an inflorescence of Arabidopsis thaliana into MsHDZ-MYC-GV 3101 by a flower dipping method, and 10:00-11:00 in the morning is generally selected, and flowering is the most vigorous at this time, and the infection is suitable. The infection steps are as follows:

a. Adding 1 mL LB liquid containing california rifampicin antibiotic into a 2 mL sterilization centrifuge tube on an ultra-clean workbench, scraping 35S with toothpick, shaking MsHDZ-MYC-GV 3101 in the centrifuge tube, placing in a shaking table at 28 ℃ and activating overnight;

b. 35S which is activated in the last step is MsHDZ-MYC-GV 3101, the LB liquid containing the kanaforifampicin antibiotic is transferred into 200 mL on an ultra-clean workbench, and the mixture is cultured overnight at 28 ℃ by shaking table, and the OD600 is 0.8-1.0, so that the infection is easier. Watering the seedling to be infected in advance to ensure sufficient water;

c. 35S of overnight culture, msHDZ-MYC-GV 3101, was taken out, and the cells were collected by sub-packaging into 50 mL centrifuge tubes, 5 rpm, and centrifugation at 28℃for 15 min. Discarding the supernatant and reserving thalli;

d. The cells were resuspended in 200 mL conversion Buffer.

E. pouring the suspended bacterial liquid into a 50mL centrifuge tube, immersing the inflorescence of the arabidopsis into the centrifuge tube for 15-20 sec, and then placing the arabidopsis in a seedling raising basin;

f. covering the seedling pot with a cover, keeping humidity, and culturing in the dark overnight for 1 d;

g. the following day, the overnight dark-cultured Arabidopsis thaliana was taken out and erected, and cultured under normal light conditions (typically, once every other week for three times before seed collection).

(4) Transgenic positive plant selection

After the converted arabidopsis thaliana is ripe, collecting seeds, and sterilizing by a sodium hypochlorite-alcohol sterilization method, wherein the method comprises the following specific steps of: placing the collected dry seeds into a2 mL centrifuge tube (not more than 0.3 mL), then operating in an ultra-clean workbench, adding 1 mL of 75% alcohol to disinfect about 2 min, and mixing uniformly upside down; sucking out 75% alcohol with a pipette, repeating for one time, then adding 10% (v/v) NaClO solution for sterilization of about 5 min, and sucking out the solution; adding 1 mL sterilized water, mixing, sucking out, and repeating.

The sterilized seeds are uniformly sown in a 1/2MS culture medium, positive plants obtained through hygromycin screening are T1 generation MsHDZ gene over-expression lines, which are marked as 35S: msHDZ 23:MYC lines, and then repeated screening is carried out for 2 generations, so that T3 generation 35S: msHDZ 23:MYC lines are obtained, and the seeds of the T3 generation are collected.

2. Construction of mutant anaplerotic lines

(1) Identification of homozygous mutants

Arabidopsis hb7 mutant (i.e., CS875449, a pool of Arabidopsis thaliana biological resource center mutants purchased from the United states) belongs to a T-DNA insertion mutant that inserts a T-DNA sequence in the nucleotide sequence so that the gene is not expressed.

Genomic DNA obtained by extracting the hb7 mutant by using an Edward buffer method is used as a template, and a three-primer PCR method is used for identifying the homozygous mutant.

The homozygous mutant of the hb7 mutant was maintained by agarose gel electrophoresis of the resulting PCR product from the three-primer system of genomic DNA of the hb7 mutant using wild type Arabidopsis thaliana (WT) as a control.

(2) Transformation and screening

According to the procedure of "transformation by dipping method" and "screening transgenic positive plants" in example 1, msHDZ-MYC-GV 3101 was used to infect the homozygous mutant of the hb7 mutant, and the positive plants obtained by screening were identified as mutant anaplerotic lines, which were designated MsHDZ/hb 7. And repeatedly screening for 2 generations to obtain the T3 generation MsHDZ/hb 7 strain.

3. Identification of the expression level of MsHDZ Gene

Extracting RNA of 35S of T3 generation, msHDZ S of MYC strain, msHDZ/hb 7 strain of T3 generation and wild Arabidopsis (WT) respectively, and carrying out reverse transcription to obtain cDNA. PCR amplification and agarose gel electrophoresis were performed using cDNA of each plant as a template, the upstream primer for detecting MsHDZ gene was MsHDZ-F: 5'-AGCGAGGAGCAGATCAAGTC-3' (SEQ ID NO. 5), the downstream primer was MsHDZ-R: 5'-CGTGCTTCTCCTTCTTGAGG-3' (SEQ ID NO. 6), the upstream primer for detecting internal reference Actin2 gene was Actin2-F:5'-GGTAACATTGTGCTCAGTGGTGG-3' (SEQ ID NO. 7), and the downstream primer was Actin2-R:5'-AACGACCTTAATCTTCATGCTGC-3' (SEQ ID NO. 8).

The PCR reaction system is as follows: an upstream primer (10. Mu.M), 1. Mu.L; downstream primer (10. Mu.M), 1. Mu.L; 2×Taq Mix, 12.5. Mu.L; template, 0.5 μl; ddH ₂ O was made up to 25. Mu.L. The PCR reaction procedure was: 95 ℃,5 min ℃;95 ℃,15 sec,58 ℃,15 sec,72 ℃,1 min,72 ℃,5 min,23 cycles.

2. Experimental results

As shown in A-B of FIG. 1, from the resulting overexpressed strain 35S:: msHDZ:: MYC strains, 2 plants with high MsHDZ gene expression (i.e., #3 and # 5) were retained, and from the mutant complement strain MsHDZ/hb 7 strains, 2 plants with high MsHDZ gene expression (i.e., #10 and # 3) were retained, and subsequent experiments were performed.

Example 2 phenotype identification of MsHDZ Gene overexpression lines and mutant-complementation lines

1. Experimental method

1. Plant cultivation

Wild-type Arabidopsis thaliana (Arabidopsis thaliana) (i.e., WT), #335S obtained in example 1:: msHDZ:: MYC strain (i.e., msHDZ 23-OE-3), # 535S::: msHDZ:: MYC strain (i.e., msHDZ-OE-5), hb7 mutant strain (i.e., CS 875449), #3MsHDZ23/hb7 strain (i.e., msHDZ/hb 7-3) and #10MsHDZ23/hb7 strain (i.e., msHDZ23/hb 7-10) were cultivated in a greenhouse with light intensities of 120-150. Mu. Mol/(m ². S), 16h illumination, 8h dark period, growth temperature 25℃for 4 weeks.

2. Phenotypic analysis

After the cultivation is finished, each plant is photographed and recorded, and the growth index (namely, the plant height, the length of the pod, the length of the leaf, the width of the leaf and the length of the leaf stalk) is measured.

2. Experimental results

As shown in FIGS. 2A-F, there was no significant difference in growth morphology and growth index between WT and hb7 mutant, whereas each of the growth indexes MsHDZ-OE-3 and MsHDZ-OE-5 and MsHDZ23/hb7-3 and MsHDZ/hb 7-10 was significantly lower than that of WT, and the growth index of 2 overexpressed lines was significantly lower than that of 2 mutant anaplerotic lines. It was shown that overexpression MsHDZ stunted Arabidopsis thaliana.

Example 3 overexpression of MsHDZ Gene to enhance tolerance of Arabidopsis plants to NaCl stress

1. Experimental method

1. Seed sterilization

Seeds of wild type Arabidopsis thaliana (Arabidopsis thaliana) (i.e., WT), msHDZ23-OE-3, msHDZ23-OE-5, hb7 mutant (i.e., CS 875449), msHDZ23/hb7-3, msHDZ23/hb7-10 were sterilized according to the "sodium hypochlorite-alcohol sterilization" method of example 1.

2. Germination percentage measurement

Seeds of sterilized WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ/hb 7-3 and MsHDZ/hb 7-10 were sown in 1/2MS agar medium containing concentrations of 0mM (i.e., control group, CK or Control), 50mM, 100mM and 150 mM NaCl, respectively, and cultured for 5 days, and the germination rates of the seeds of each plant were counted daily according to the growth conditions of cotyledons, and the calculation formula was: germination rate = number of germinated seeds/total number of seeds x 100%.

3. Root elongation test

Seeds of sterilized WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ/hb 7-3 and MsHDZ/hb 7-10 were sown in 1/2MS agar medium, respectively, and sprouted for 5 days, and then transferred to 1/2MS agar medium containing concentrations of 0mM (i.e., control group, denoted as CK), 50 mM, 100 mM and 150 mM NaCl, respectively, to vertically culture seedlings, and after further culture for 14 days, the main root length, lateral root length and lateral root number of each plant were measured.

4. Long-term salt stress test

(1) Salt stress treatment

Seeds of sterilized WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ23/hb7-3 and MsHDZ/hb 7-10 were sown in 1/2MS agar medium, routinely cultured to 3 weeks of age, dehydrated and drought treated for one week, i.e., the soil was not watered for one week at the beginning of no moisture.

Then 150mM NaCl solution (i.e. saline) or water is used for irrigation every day, continuous irrigation is carried out for 14 days, and the leaves of each plant (the leaves of the same part of each plant are respectively cut off, and 3 plants are taken for each plant line) are collected for measuring each index.

(2) Determination of chlorophyll content (Chlorophyll content)

Weighing 0.5 g of leaves, placing the leaves into a 50 mL centrifuge tube, adding 25 mL of 95% ethanol for sealing, extracting at room temperature under a dark condition for 24-36 h, diluting the extracting solution, performing colorimetric detection under wavelengths 665 nm, 649 nm and 470 nm respectively by using a Cary6003040426 ultraviolet-visible spectrophotometer, and calculating the content of chlorophyll in the leaves according to the result, wherein the calculation formula is as follows:

Ca (chlorophyll a) = 13.95D665-6.88D649;

cb (chlorophyll b) = 24.96D649-7.32D665;

Cx.c. (carotenoid) = (1000D 470-2.05 Ca-114.8 Cb)/245;

chlorophyll content in leaf (mg/g) =pigment concentration (ca+cb+cx.c) (mg/L) x total volume of extract (mL) x dilution factor/sample mass (g).

(3) Determination of free proline content (Free proline content)

Accurately weighing 25 mg proline, dissolving with distilled water, and fixing the volume to 250 mL to obtain mother solution with the concentration of 100 mug/mL. And then taking 10mL mother liquor, and diluting to 100 mL by distilled water to obtain the proline standard solution with the concentration of 10 mug/mL, and preparing a standard curve.

Placing 0.2-0.5 g blade into a large test tube, adding 5mL 3% sulfosalicylic acid solution, heating with glass ball boiling water for 10: 10 min, taking out, cooling to room temperature, and collecting supernatant to obtain extractive solution. After mixing 2mL extract 2mL glacial acetic acid and 3 mL of 2.5% acidic ninhydrin color solution, boiling water was heated to 40 and min, and after cooling, 5mL toluene was added to each tube and the mixture was thoroughly shaken. Standing, layering, sucking the toluene layer in each tube, and measuring the absorbance at the wavelength 520 nm. The absorbance value of the toluene layer is brought into a linear regression equation of a standard curve, the concentration (mug) of the proline in the extracting solution is calculated, and then the result is substituted into the following formula to calculate the content of the free proline in the sample:

Proline (μg/g FW) = (c×v/a)/W;

Wherein, the concentration of proline in the C-extracting solution (mug), the total volume of the V-extracting solution (mL), the volume of toluene layer (mL) sucked during a-measurement and the weight of the W-blade sample (g).

(4) Determination of relative conductivity (Relative electrolyte leakage, REL)

The leaf was rinsed 2 times with ultrapure water, and the water on the leaf surface was sucked off with filter paper. Taking leaf discs by using a puncher, taking 15 leaves from each plant of leaves, and putting the leaves into a clean 15mL test tube. 10mL ultra pure water was added to each test tube, and the test tube was placed in a vacuum dryer, and 10min was evacuated by a vacuum pump to eliminate air in the cell gap, resulting in easy exudation of electrolytes in leaf tissues. The tube was shaken in a shaker for 1 h, and after sufficient shaking, the initial conductivity (K ₁) was determined using a Lei Ci DDSJ-308A conductivity meter. After the measurement, the test tube was sealed and placed in a boiling water bath to boil for 15min a to kill the whole electrolyte in the cells of the plant tissue, and after cooling to room temperature, the conductivity value in the test tube was measured (K ₂). Meanwhile, another test tube was used for 10mL ultra pure water as a control, and a blank conductance value K ₀ was measured. According to the formula: relative conductivity (%) = (K ₁-K₀）/（K₂-K₀) ×100% was calculated.

(5) Determination of Na ⁺ and K ⁺ content

Pulverizing the air-dried leaf blade with a plant pulverizer, sieving with a 1mm sieve, accurately weighing 1g sample, placing in a digestion tube, adding mixed solution of 10 mL HNO ₃ (high-grade pure) and 2mL HCIO ₄ (high-grade pure) mixed acid, covering, and standing overnight. The next day is digested by covering the temperature control digestion instrument, and the temperature control digestion instrument is gradually heated to a micro-boiling state (160 ℃ -170 ℃), a large amount of brown NO ₂ gas is emitted at the moment, after about 1 hour, the cover on the digestion tube is taken off, and the heating is continued until the brown gas disappears and white smoke is emitted. When the sample solution was a pale yellow (or colorless) transparent liquid, the sample had digested completely, and heating was continued to evaporate most of HNO ₃ and HCIO ₄, leaving about 1 mL solution. After cooling, adding about 10 mL deionized water, heating to remove acid, slightly boiling for about 5 minutes, taking down, transferring the sample solution into a 25 mL volumetric flask while the sample solution is still hot, rinsing the digestion tube with a small amount of deionized water for multiple times, pouring into the volumetric flask, cooling, and then fixing the volume to the scale with deionized water, and uniformly mixing to obtain the sample solution to be measured. A sample blank solution was prepared simultaneously. The Na ⁺ and K ⁺ content of each sample was determined by inductively coupled plasma emission spectroscopy (ICP-OES) Optima 8000 and the ratio of Na ⁺ content to K ⁺ content (i.e. Na ⁺/K⁺) was calculated.

(6) Antioxidant index determination

MDA is a membrane lipid oxidation index (the lower the MDA value, the stronger the oxidation resistance), and SOD, POD and CAT are antioxidant enzymes. Leaves of each plant were examined with Malondialdehyde (MDA) content detection kit (purchased from Solarbio company), superoxide dismutase (SOD) activity detection kit (purchased from Solarbio company), peroxidase (POD) activity detection kit (purchased from Solarbio company) and Catalase (CAT) activity detection kit (purchased from Solarbio company).

(7) Reactive Oxygen Species (ROS) assay

Stress usually results in excessive accumulation of ROS, and eventually oxidative stress, and in this example, 3' -Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining methods are used to detect in-situ accumulation of hydrogen peroxide (H ₂O₂) and superoxide (O ^2-) in leaves, which comprises the following steps: soaking the leaves in DAB solution (1 mg/mL, pH=3.8) or NBT solution (0.1 mg/mL), and dyeing at 28 ℃ in a dark place for 5-8 h; after staining, leaves were soaked in 95% ethanol for 2 h until chlorophyll was removed, and images were taken with a microscope.

2. Experimental results

1. Salt stress germination percentage measurement results

As shown in FIG. 3A-E, the germination rates of the individual lines were significantly different at 50 mM-150 mM NaCl compared to the control group, and especially at 100 mM and 150mM NaCl, the germination rates of the MsHDZ gene over-expressed lines (MsHDZ 23-OE-3 and MsHDZ 23-OE-5) were significantly higher than those of the WT, hb7 mutant and mutant complementation lines (MsHDZ 23/hb7-3 and MsHDZ23/hb 7-10), wherein the germination rates of the mutant complementation lines (MsHDZ/hb 7-3 and MsHDZ23/hb 7-10) were significantly higher than those of the hb7 mutant. The over-expression MsHDZ gene is shown to reduce the influence of salt stress on seed germination.

2. Root elongation test results under salt stress

As shown in A-B in FIG. 4, the root elongation of each strain was significantly different under 50 mM-150 mM NaCl treatment compared to the control group, and the main root length, the lateral root length and the lateral root number of the MsHDZ gene-overexpressing strain (MsHDZ-OE-3 and MsHDZ-OE-5) were significantly greater than those of WT, the hb7 mutant strain and the mutant complementation strain (MsHDZ 23/hb7-3 and MsHDZ/hb 7-10), and the main root length, the lateral root length and the lateral root number of the mutant complementation strain (MsHDZ/hb 7-3 and MsHDZ/hb 7-10) were significantly greater than those of the hb7 mutant strain. The overexpression MsHDZ gene was shown to reduce the effect of salt stress on root growth.

3. Long-term salt stress test results

(1) Results of chlorophyll content, free proline content, relative conductivity, na ⁺ and K ⁺ content

As shown in A in FIG. 5, compared with water irrigation, the rosette leaves of both WT and hb7 mutant strains show obvious wilting, yellowing and the like under long-term saline irrigation treatment, while the growth condition of MsHDZ gene over-expression strain is obviously better than that of WT, the leaves still maintain the state of green leaves, and the growth condition of mutant anaplerotic strain is also obviously better than that of hb7 mutant strain.

As shown in B-G in FIG. 5, under the water irrigation treatment, the physiological indexes of each strain have no obvious difference; compared with wild arabidopsis, under long-term saline irrigation treatment, the chlorophyll content of MsHDZ gene over-expression strain is increased by 25% -43%, the free proline content is increased by 27% -68%, the relative conductivity is reduced by 5% -9%, the Na ⁺ content is obviously increased, the K ⁺ content is obviously reduced, and the Na ⁺/K⁺ content is obviously increased; compared with wild type arabidopsis, the chlorophyll content of the hb7 mutant is reduced by 9%, the free proline content is reduced by 31%, the relative conductivity is increased by 4%, the Na ⁺ content is obviously reduced, the K ⁺ content is obviously increased, and the Na ⁺/K⁺ content is obviously reduced. The over-expression MsHDZ gene is helpful for plant cells to contain more proline solution, provides enough free water, maintains normal life activities of plants, enhances the resistance of the plants to salt stress, reduces the damage degree of cell membranes caused by salt stress, and reduces the loss of membrane selectivity.

The above results indicate that MsHDZ, by improving plant cell membrane permeability, providing more proline solution to cells, enhancing plant photosynthesis and maintaining Na ⁺/K⁺ balance, increases plant resistance to salt stress to increase salt tolerance.

(2) Oxidation index and reactive oxygen species results

As shown in a-D in fig. 6, under the long-term saline irrigation treatment, the MDA content of MsHDZ gene over-expression strain is significantly reduced by 53% -56% compared with wild type arabidopsis, the SOD content is significantly increased by about 80%, the POD content is significantly increased by 38% -66%, and the CAT content is significantly increased by about 300%; compared with WT, the hb7 mutant has 24% raised MDA content, 62% lowered SOD content, 64% lowered POD content and 56% lowered CAT content. It is shown that overexpression MsHDZ gene enhances oxidation resistance of Arabidopsis under salt stress.

As shown in fig. 7, under the water irrigation treatment, there was no obvious difference between DAB staining and NBT staining of each strain; under prolonged saline water treatment, both DAB and NBT staining of MsHDZ gene over-expression lines (MsHDZ-OE-3 and MsHDZ-OE-5) were significantly lighter than that of WT, and both DAB and NBT staining of hb7 mutant were significantly darker than that of WT. It was demonstrated that overexpression of MsHDZ gene reduced ROS in situ accumulation in arabidopsis under salt stress.

The result shows that MsHDZ < 23 > increases the enzyme activity of antioxidant enzyme in the arabidopsis plant body, improves the capability of scavenging active oxygen, and further improves the salt tolerance of the plant.

Example 4 overexpression of MsHDZ Gene to enhance tolerance of Arabidopsis plants to alkaline stress

1. Experimental method

1. Seed sterilization

The procedure was the same as in "seed sterilization" of example 3.

2. Germination percentage and root elongation measurement

The procedure was essentially the same as in "germination rate measurement" and "root elongation test" of example 3, except that: 1/2MS agar medium containing NaHCO ₃ at concentrations of 0mM (i.e., control, CK or Control) and 7 mM (pH 8.2), respectively, was used.

3. Root elongation test

The procedure was essentially the same as in "root elongation test" of example 3, except that: after 5 days of germination culture of WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ/hb 7-3 and MsHDZ/hb 7-10, seedlings were cultured vertically on 1/2MS agar medium containing concentrations of 0mM (i.e., control group, CK or Control) and 7 mM NaHCO ₃ (pH 8.2), respectively.

4. Long-term alkali stress test

(1) Alkali stress treatment

The procedure was essentially the same as in "salt stress treatment" of example 3, except that: and (3) carrying out drought treatment for one week, then irrigating with 150 mM NaHCO ₃ solutions (namely alkaline water) or water every day, continuously irrigating for 14 days, collecting the leaves of each plant (respectively cutting the leaves of the same part of each plant, and taking 3 plants for each plant line), and measuring each index.

(2) Determination of chlorophyll content

The procedure was the same as in example 3, "determination of chlorophyll content (Chlorophyll content)".

(3) Free proline content determination

The procedure is the same as in example 3, "free proline content (Free proline content) assay".

(4) Determination of relative conductivity

The procedure is the same as in example 3 "determination of relative conductivity (Relative electrolyte leakage, REL)".

(5) Antioxidant index determination

The procedure was the same as in "antioxidative index assay" of example 3.

(6) Reactive Oxygen Species (ROS) assay

The procedure was the same as in "antioxidative index assay" of example 3.

2. Experimental results

1. Determination of germination percentage under alkali stress

As shown in A-C in FIG. 8, the germination rates of the respective strains under the treatment of 7 mM NaHCO ₃ were significantly different from that of the control group, the germination rates of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly higher than those of WT, hb7 mutant and MsHDZ/hb 7-3 and MsHDZ/hb 7-10, the germination rates of hb7 mutant were severely affected, and the germination rates of MsHDZ23/hb7-3 and MsHDZ/hb 7-10 were significantly higher than those of hb7 mutant. The over-expression MsHDZ gene is shown to reduce the influence of alkali stress on seed germination.

2. Root elongation test results under alkali stress

As shown in FIGS. 9A-B, the root elongation of each strain was significantly different under 7 mM NaHCO ₃ treatment compared to the control, and the length of the main roots, the length of the side roots and the number of side roots of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly greater than those of WT and hb7 mutants, and MsHDZ/hb 7-3 and MsHDZ23/hb7-10 were significantly greater than those of hb7 mutants. The overexpression MsHDZ gene is shown to reduce the influence of alkali stress on root growth.

3. Long-term alkali stress test results

(1) Results of chlorophyll content, relative conductivity and free proline content

As shown in FIG. 10A, compared with water irrigation, the rosette leaves of both WT and hb7 mutants showed significant wilting, yellowing, etc. under the long-term alkaline water irrigation treatment, while the growth conditions of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly better than those of WT and hb7 mutants; msHDZ23/hb7-3 and MsHDZ/hb 7-10 also showed significantly better growth status than the hb7 mutant.

As shown in B-D in FIG. 10, under the long-term alkaline water irrigation treatment, compared with WT, the chlorophyll content of MsHDZ-OE-3 and MsHDZ-OE-5 is increased by 31-38%, the relative conductivity is reduced by 19-22%, and the free proline content is increased by 49-53%; compared with WT, the chlorophyll content of the hb7 mutant is reduced by 16%, the free proline content is reduced by 60%, and the relative conductivity is increased by 26%. The over-expression MsHDZ gene is helpful for plant cells to contain more proline solution, provides enough free water, maintains normal life activities of plants, enhances the resistance of the plants to alkali stress, reduces the damage degree of cell membranes caused by the alkali stress, and reduces the loss of membrane selectivity.

The result shows that MsHDZ gene can provide more proline solution to cells by improving the permeability of plant cell membrane, enhance the photosynthesis of plants to improve the resistance of plants to alkali stress so as to improve alkali resistance.

(2) Oxidation index and reactive oxygen species results

As shown by A-D in FIG. 11, under the long-term alkaline water irrigation treatment, compared with the WT, the MDA content of MsHDZ-OE-3 and MsHDZ-OE-5 is obviously reduced by 39% -42%, the SOD content is obviously increased by about 90%, the POD content is obviously increased by about 38%, and the CAT content is obviously increased by about 56% -160%; compared with WT, the hb7 mutant has MDA content increased by 12%, SOD content decreased by 41%, POD content decreased by 24% and CAT content decreased by 73%.

As shown in FIG. 12, under prolonged alkaline water treatment, both DAB and NBT stains of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly lighter than that of WT, both DAB and NBT stains of hb7 mutant were significantly darker than that of WT, and both DAB and NBT stains of MsHDZ23/hb7-3 and MsHDZ23/hb7-10 were also significantly lighter than that of hb7 mutant. It was demonstrated that overexpression of MsHDZ gene reduced ROS in situ accumulation under alkali stress.

The result shows that MsHDZ < 23 > increases the enzyme activity of the antioxidant enzyme in the arabidopsis plant body, and improves the capability of removing active oxygen, thereby improving the alkali resistance of the plant.

Example 5 overexpression MsHDZ Gene to enhance drought resistance in Arabidopsis plants

1. Experimental method

1. Seed sterilization

The procedure was the same as in "seed sterilization" of example 3.

2. Germination percentage measurement and root elongation test

The procedure was essentially the same as in "germination rate measurement" and "root elongation test" of example 3, except that: 1/2MS agar medium containing mannitol at concentrations of 0mM (i.e., control, CK or Control), 50 mM, 100 mM, 150 mM, and 200 mM, respectively, was used.

4. Long-term drought stress test

(1) Drought stress treatment

Seeds of sterilized WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ/hb 7-3 and MsHDZ/hb 7-10 were sown in 1/2MS agar medium, respectively, cultured conventionally to 3 weeks old, and rehydrated for 3 days (Re-watering) after 2 weeks of stopping watering (i.e., drought, drought or Dehydration), or irrigated continuously with water (i.e., control or CK). Before and after rehydration, the leaves of each plant (the leaves of the same part of each plant are cut off respectively, 3 plants are taken for each plant line) are collected for measuring various indexes, the survival condition of the plant is counted, and the survival rate (%) = survival number/total number multiplied by 100% is calculated.

(2) Determination of relative Water content

The fresh weight (Wf) of the leaf is weighed before rehydration. Immersing the leaves in ultra-pure water for 10 h, weighing until the saturated weight of the sample is similar, and obtaining the saturated fresh weight (Wt) of the leaves. The leaf blade weighed out with saturated fresh weight is wrapped with tinfoil paper and put into an oven at 85 ℃ to be baked to constant weight, and then dry weight (Wd) is weighed. Substituting the data into a calculation formula: relative Water Content (RWC) = (Wf-Wd)/(Wt-Wd) ×100%, to obtain the relative water content of the blade.

(3) Determination of chlorophyll content

The procedure was the same as in example 3, "determination of chlorophyll content (Chlorophyll content)".

(4) Free proline content determination

The procedure is the same as in example 3, "free proline content (Free proline content) assay".

(5) Determination of relative conductivity

The procedure is the same as in example 3 "determination of relative conductivity (Relative electrolyte leakage, REL)".

(6) Antioxidant index determination

The procedure was the same as in "antioxidative index assay" of example 3.

(7) Determination of relative Water loss

10 Leaves were cut from WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ/hb 7-3 and MsHDZ/hb 7-10 seedlings grown under continuous irrigation with water, respectively, and weighed on a balance to obtain W ₁, after 30-min, and weighed to obtain W ₂, the weighing was repeated until the weight remained unchanged, and the results were recorded according to the formula: the percent loss = (W ₁-W₂）/W₁ x 100% (W1: weight before loss of water from the detached blade; W2: weight per half hour of loss of water from the detached blade) was calculated and a line graph was drawn.

(8) Air hole observation and opening and closing degree measurement

Rosette leaves were taken from WT, msHDZ23-OE-3, msHDZ-OE-5, CS875449, msHDZ/hb 7-3 and MsHDZ/hb 7-10 seedlings grown under continuous irrigation with water, respectively, and air holes were observed by the "nail polish stamping method". Three replicates were performed for each strain with 3 leaves counted.

(9) Reactive Oxygen Species (ROS) assay

The procedure was the same as in "antioxidative index assay" of example 3.

2. Experimental results

1. Determination of germination percentage of drought stress

As shown in A-D of FIG. 13, the germination rates of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly higher than that of WT under different concentrations of mannitol, the germination rates of hb7 mutants were significantly lower than that of the hb7 mutants, msHDZ/hb 7-3 and MsHDZ/hb 7-10 were significantly higher than that of the hb7 mutants. The result shows that the over-expression MsHDZ gene reduces the influence of drought stress on seed germination.

2. Root elongation test results for drought stress

As shown in FIGS. 14A-B, the length of the main roots, the length of the side roots and the number of the side roots of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly greater than those of the WT and hb7 mutants, and the length of the main roots, the length of the side roots and the number of the side roots of MsHDZ/hb 7-3 and MsHDZ/hb 7-10 were significantly greater than those of the hb7 mutants in the mannitol treatment at different concentrations. The over-expression MsHDZ gene is shown to reduce the influence of drought stress on root growth.

3. Long-term drought stress test results

(1) Survival rate after drought and rehydration and relative water content results

As shown in a of fig. 15, rosette leaves of both WT and hb7 mutant showed wilting and withering after long-term drought stress and failed to resume normal growth after rehydration; however, msHDZ-OE-3 and MsHDZ-OE-5 grew slowly but grew significantly better than the WT and hb7 mutants and recovered from normal growth after rehydration; msHDZ23/hb7-3 and MsHDZ/hb 7-10 were superior to hb7 mutants in terms of growth state.

As shown in B and C in fig. 15, after long-term drought stress, the survival rate of WT was only 33% and the survival rate of hb7 mutant was as low as 8% and no significant recovery of growth was seen after rehydration, even with a lethal phenomenon (survival rate of 0%), the relative water content of the leaves of WT and hb7 mutant was very low; whereas MsHDZ-OE-3 and MsHDZ-OE-5 have significantly higher survival rates before rehydration than the WT and hb7 mutants and continue to survive after rehydration, the relative water content of the leaves before rehydration is 40% and 77%, respectively, significantly higher than that of the WT and hb7 mutants, and the relative water content of the leaves after rehydration is increased; msHDZ23/hb7-3 and MsHDZ/hb 7-10 also had significantly higher survival rates before rehydration than the hb7 mutant.

The above results indicate that expression of the MsHDZ gene enhances drought stress tolerance in arabidopsis.

(2) Results of chlorophyll content, relative conductivity and free proline content

As shown in A-C in FIG. 16, chlorophyll content of MsHDZ-OE-3 and MsHDZ-OE-5 is increased by 13% -26% and relative conductivity is reduced by 17% -45% and free proline content is increased by 64% -71% under long-term drought treatment compared with WT; compared with WT, the chlorophyll content of the hb7 mutant is reduced by 20%, the free proline content is reduced by 18%, and the relative conductivity is increased by 9%; the over-expression MsHDZ gene is helpful for plant cells to contain more proline solution, provides enough free water, maintains normal life activities of plants, enhances the resistance of the plants to drought stress, reduces the damage degree of cell membranes caused by the drought stress, and reduces the loss of membrane selectivity.

The result shows that MsHDZ gene can provide more proline solution to cells by improving the permeability of plant cell membrane, enhance the photosynthesis of plants to improve the resistance of plants to alkali stress so as to improve drought tolerance.

(3) Oxidation index and reactive oxygen species results

As shown in A-D in FIG. 17, under long-term drought treatment, both the MDA, SOD, POD and CAT contents of MsHDZ-OE-3 and MsHDZ-OE-5 before rehydration are increased, and both the MDA, SOD, POD and CAT contents after rehydration are brought to normal growth levels, as compared with normal water flooding treatment; under long-term drought treatment, MDA content of MsHDZ23 over-expression line is significantly reduced by 37% -39% compared with WT, while mutant is raised by 39%. After rehydration, compared with drought treatment, the MDA content of the WT is reduced by 26%, the overexpressed strain is reduced by 6% -17%, and the mutant is reduced by 28%, and the MDA content of the overexpressed strain is still maintained to be lower than that of other strains after rehydration, which possibly shows that the plant still starts a defense mechanism after rehydration and is relieved by stress hazard; the SOD content of the over-expression system is obviously increased by 24% -34% compared with that of the WT, the mutant is reduced by 75%, and after rehydration, the SOD content of the WT is reduced by 66% compared with that of drought treatment, the over-expression system is reduced by 47% -61%, and the mutant is reduced by 13%; the POD content of the over-expression line is significantly increased compared with that of the WT, and the mutant is reduced by 24%. After rehydration, compared with drought treatment, the POD content of the WT is reduced by 63%, the over-expression strain is reduced by 70% -78%, and the mutant is reduced by 66%; the CAT content of the over-expression line is obviously increased by more than 53 percent compared with that of the WT, the CAT content of the WT is reduced by 64 percent compared with drought treatment after rehydration, the CAT content of the over-expression line is reduced by 20 to 30 percent, and the CAT content of the mutant is reduced by 51 percent.

As shown in FIG. 18, under prolonged drought treatment, both DAB and NBT staining of MsHDZ-OE-3 and MsHDZ-OE-5 were significantly lighter than that of WT, both DAB and NBT staining of hb7 mutant were significantly darker than that of WT, and both DAB and NBT staining of MsHDZ23/hb7-3 and MsHDZ23/hb7-10 were also significantly lighter than that of hb7 mutant. It was demonstrated that overexpression of MsHDZ gene reduced ROS in situ accumulation under drought stress.

The result shows that MsHDZ < 23 > increases the enzyme activity of antioxidant enzyme in the arabidopsis plant body, improves the capability of scavenging active oxygen, and further improves the drought tolerance of the plant.

(4) Results of relative Water loss and air holes

As shown in FIG. 19, after the leaves are 6 h isolated, the water loss rates of MsHDZ-OE-3 and MsHDZ-OE-5 reach 74% and 59% respectively, and then the water loss rates of WT and MsHDZ/hb 7-3 and MsHDZ/hb 7-10 are maintained at 81% -88%, the water loss rate of the hb7 mutant is maintained at about 95%, the water loss rate of the strain overexpressing MsHDZ23 gene is significantly lower than that of WT and hb7 mutant, and the water loss rate of the variant anaplerotic strain is significantly higher than that of hb7 mutant and similar to that of WT, proving that anaplerotic MsHDZ23 gene compensates drought sensitivity of hb7 mutant.

As shown in A-B in FIG. 20, under normal water irrigation treatment, the stomata of each strain of leaves are in an open state, wherein the stomata aperture of the hb7 mutant strain is the largest; under drought stress conditions, except that the hb7 mutant strain is still in an open state, the stomata of other strains are all in a closed state, the stomata pore diameter of the MsHDZ gene over-expression strain is obviously smaller than that of the WT, and the stomata pore diameter of the variant anaplerotic strain is basically consistent with that of the WT. The MsHDZ gene is shown to be capable of improving the sensitivity of stomata opening and closing to drought stress response, controlling the movement change of stomata aperture and reducing the transpiration rate, thereby improving the drought tolerance of plants.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (12)

1. The application of the Chinese awn MsHDZ gene and/or MsHDZ protein in enhancing the stress resistance of plants is characterized in that the expression level of the Chinese awn MsHDZ gene and/or MsHDZ protein in the plants is improved;

The amino acid sequence of the Chinese mango MsHDZ protein is shown as SEQ ID NO. 4;

The plant stress resistance is stress resistance of the plant to abiotic stress; the abiotic stress comprises any one or more of salt stress, alkali stress and drought stress.

2. The use according to claim 1, wherein the enhancing plant stress resistance comprises any one or more of promoting germination of plant seeds, promoting growth of plant roots, improving cell membrane permeability of plants, enhancing photosynthesis of plants, and enhancing oxidation resistance of plants.

3. The use according to any one of claims 1 to 2, wherein the plant is arabidopsis thaliana.

4. Use of a biomaterial in the preparation of a product for enhancing stress resistance in a plant, wherein the biomaterial is any one of the following (1) to (5):

(1) A nucleic acid molecule with a sequence shown as SEQ ID NO.3 or a complete complementary sequence of the sequence shown as SEQ ID NO. 3;

(2) An expression cassette comprising the nucleic acid molecule of (1);

(3) A recombinant expression vector comprising the nucleic acid molecule of (1);

(4) A recombinant microorganism comprising the nucleic acid molecule of (1);

(5) A protein with an amino acid sequence shown as SEQ ID NO. 4;

The plant stress resistance is stress resistance of the plant to abiotic stress; the abiotic stress comprises any one or more of salt stress, alkali stress and drought stress.

5. The use according to claim 4, wherein the enhancing stress resistance of plants comprises any one or more of promoting germination of plant seeds, promoting growth of plant roots, improving cell membrane permeability of plants, enhancing photosynthesis of plants, and enhancing oxidation resistance of plants.

6. The use according to any one of claims 4 to 5, wherein the plant is arabidopsis thaliana.

7. Use of a product for enhancing stress resistance in plants, characterized in that the product comprises a biomaterial according to claim 4; the plant stress resistance is stress resistance of the plant to abiotic stress; the abiotic stress comprises any one or more of salt stress, alkali stress and drought stress.

8. The use according to claim 7, wherein the enhancing stress resistance of plants comprises any one or more of promoting germination of plant seeds, promoting growth of plant roots, improving cell membrane permeability of plants, enhancing photosynthesis of plants, and enhancing oxidation resistance of plants.

9. The use according to any one of claims 7 to 8, wherein the plant is arabidopsis thaliana.

10. A method for enhancing stress resistance of a plant, characterized in that the plant is treated with the product of claim 7 to increase the expression level of the chinese mango MsHDZ gene and/or MsHDZ protein in the plant; the plant stress resistance is stress resistance of the plant to abiotic stress; the abiotic stress comprises any one or more of salt stress, alkali stress and drought stress.

11. The method of claim 10, wherein the enhancing stress resistance of the plant comprises any one or more of promoting germination of plant seeds, promoting growth of plant roots, improving cell membrane permeability of the plant, enhancing photosynthesis of the plant, and enhancing oxidation resistance of the plant.

12. The method according to any one of claims 10 to 11, wherein the plant is arabidopsis thaliana.