CN117660522B

CN117660522B - Gene for improving salt tolerance of plants and application thereof

Info

Publication number: CN117660522B
Application number: CN202410140517.4A
Authority: CN
Inventors: 郭敬功; 苗雨晨; 李坤; 贾昆鹏; 李瑾; 杨凤博; 朱志娟; 程珂; 李海鹏; 卢延克; 刘博�; 苏亚; 王欣逸; 唐滔
Original assignee: Sanya Research Institute Of Henan University; Henan University
Current assignee: Sanya Research Institute Of Henan University; Henan University
Priority date: 2024-02-01
Filing date: 2024-02-01
Publication date: 2024-04-16
Anticipated expiration: 2044-02-01
Also published as: CN117660522A

Abstract

The invention discloses a gene for improving plant salt tolerance and application thereof, wherein the gene is named GhTSD gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The invention constructs GhTSD over-expression vector p 35S-GhTSD-GFP, utilizes agrobacterium inflorescence infection method to transform wild type Arabidopsis thaliana (Clo-0, WT) to obtain over-expression plant, and analysis result shows that under high salt stress, compared with wild type, ghTSD7 can improve seed germination rate, enhance salt stress tolerance of Arabidopsis thaliana seedling, and show that Arabidopsis thaliana seedling is more sensitive to high salt stress. Then, ghTSD7 silenced cotton plants are obtained in a gene silencing mode, and the result shows that the gene silencing plants are lower in plant height under high salt stress, and the leaf yellowing and withering phenomenon is serious, so that gene resources are provided for crop salt-tolerant molecular breeding.

Description

Gene for improving salt tolerance of plants and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gene for improving salt tolerance of plants and application thereof, and more particularly relates to GhTSD gene and application thereof in improving salt stress tolerance of plants.

Background

Plants are subjected to various abiotic stresses throughout life, with salt stress being one of the major factors limiting crop yield and quality. In the context of current global climate change, salt stress poses a great challenge to plant growth and agricultural yield. Plants are a key component of the ecosystem, and their sensitivity and adaptability to salt stress directly affect crop growth and yield.

First, the effect of salt stress on plants is apparent. The high salt environment can limit the absorption of moisture by plants, leading to dehydration of cells and leaf scorch and wilting, affecting the growth and development of plants. In addition, salt negatively affects the ion balance and nutrient absorption of plants, thereby reducing photosynthesis and nutrient synthesis capacity of the plants. Excessive sodium ion accumulation can disrupt intracellular and extracellular ion balance, affecting water balance and osmotic regulation, leading to cell dehydration and apoptosis. Second, salt stress also causes oxidative stress, leading to accumulation of reactive oxygen radicals, resulting in damage to cell membranes, proteins and nucleic acids. Salt stress also interferes with energy metabolism, photosynthesis and respiration of plants, limiting their normal growth and development. Thus, plants must modulate their physiological and molecular mechanisms to increase tolerance to salt stress.

Plants develop a variety of adaptive mechanisms to cope with salt stress during long-term evolution. These mechanisms include, but are not limited to: regulating root system structure to reduce salt absorption, improving intracellular salt discharge capacity, and accumulating specific ion and lipid substances to protect cells from stress injury, etc. These self-protection mechanisms help plants to maintain survival and basic growth functions under high salt conditions.

In crops, cotton is one of the main raw materials of the textile industry, and has extremely important economic value. However, cotton growth and yield are severely affected by environmental factors such as salt stress. Salt stress results in limited cotton growth, reduced fiber quality, and even reduced overall yield, which results in significant losses to the textile industry.

In order to cope with the vulnerability of cotton under salt stress, scientists and agricultural specialists are continually striving to study and take corresponding measures. Countermeasures against salt resistance of cotton include, but are not limited to:

1. Selecting and breeding a salt-resistant variety: the cotton variety with more salt resistance is cultivated by genetic improvement or gene editing technology.

2. An improved irrigation system: the irrigation mode is optimized, the accumulation of salt in soil is reduced, and the salt content of cotton fields is reduced.

3. The biotechnology means: the tolerance of cotton to salt stress is improved by means of genetic engineering, growth regulator and the like.

The ability of cotton to combat salt is of great importance to agriculture and industry. Under the conditions of global climate change and natural disaster frequency, improving the adaptability of cotton to salt stress is a key factor for ensuring the yield and quality of the cotton. By adopting salt-resistant measures for cotton, the negative influence of salt stress on cotton production can be reduced, and the stable supply of textile industry is promoted, so that the economic development is promoted and the life quality of people is improved.

In conclusion, the influence of plants on salt stress and the adaptability mechanism thereof are important directions of current researches, and especially, the improvement of the salt resistance of key crops such as cotton is of great significance to agricultural production and industrial development. The GhTSD gene is found in the research process, and early research shows that the GhTSD gene plays an important role in drought resistance, and as the research is in progress, the salt tolerance mechanism of the GhTSD gene is in progress, and as a result, the GhTSD gene plays an important role in improving the salt stress tolerance of plants.

Disclosure of Invention

The invention aims to provide a gene for improving salt tolerance of plants and application thereof, wherein the gene is named GhTSD gene, and the main aim is to provide functions of the gene in improving salt stress tolerance of plants.

In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:

The GhTSD gene adopted by the invention has the gene Sequence number (Sequence ID) of XM_016859597.2 in NCBI, the nucleotide Sequence is shown as SEQ ID NO.1, the length of the messenger RNA (mRNA) Sequence of GhTSD gene is 1461bp, and the length of the coding Sequence of GhTSD7 gene is 1152 bp, which comprises 383 amino acids.

The invention also constructs a series of plant expression vectors, expression vectors containing the genes, transgenic plant lines and host cells containing the vectors, and the functions of improving the salt stress resistance of plants also fall into the protection scope of the invention. The functions of the genes protected by the invention not only comprise GhTSD genes, but also comprise the functions of homologous genes with higher homology (up to 96.7% of homology) with GhTSD genes in the aspect of salt stress resistance.

The biological function of GhTSD gene in plant salt stress tolerance disclosed by the invention is specifically expressed in: under salt stress, seed germination rate of GhTSD7 over-expression strain is higher than that of wild type, yellowing degree of leaf is lower than that of wild type, damage degree of leaf is lower than that of wild type, and plant height of GhTSD gene silencing strain is lower than that of wild type, yellowing and withering phenomenon of leaf is higher than that of wild type, and damage degree of leaf is higher than that of wild type.

According to the function thereof, a salt stress resistant plant can be obtained by means of a transgene, in particular, a transgenic plant can be obtained by introducing GhTSD gene into a target plant, and the salt stress resistant capacity of the plant is higher than that of the target plant.

Specifically, ghTSD gene can be introduced into the plant of interest specifically by the recombinant expression vector. In the method, the recombinant expression vector may be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and the transformed plant tissues are cultivated into plants.

In order to improve the excellent properties of plants, the present invention also provides a novel plant breeding method for obtaining plants having salt stress tolerance higher or/and lower than that of the target plants by regulating the expression of GhTSD gene in the target plants.

The expression of GhTSD gene in the plant of interest is regulated in such a way that the GhTSD gene is overexpressed, silenced or directionally mutated.

More specifically, the method may be one comprising (1) or (2) or (3):

(1) Obtaining a plant with salt stress tolerance stronger than that of the target plant by increasing the activity of GhTSD protein in the target plant;

(2) By promoting the expression of GhTSD gene in the target plant, obtaining a plant with stronger salt stress tolerance than the target plant;

(3) By inhibiting the expression of GhTSD gene in the plant of interest, a plant with lower salt stress tolerance than the plant of interest is obtained.

The "promoting expression of GhTSD gene in the plant of interest" may be achieved as follows (1) or (2) or (3):

(1) Introducing GhTSD gene into target plant;

(2) Introducing strong promoters and/or enhancers;

(3) Other methods are common in the art.

Wherein the target plant is cotton or Arabidopsis thaliana.

Genes of interest, also known as target genes, are used in genetic engineering design and manipulation to recombine genes, alter receptor cell traits and obtain desired expression products. May be of the organism itself or from a different organism.

The "method of modulating expression of GhTSD gene in a plant" is over-expression, silencing, or directed mutation of GhTSD gene.

Regulating the level of gene expression includes regulating the GhTSD expression using DNA homologous recombination techniques, virus-mediated gene silencing techniques, and agrobacterium-mediated transformation systems to obtain transgenic plant lines. In the present invention, the plant suitable for the present invention is not particularly limited as long as it is suitable for performing a gene transformation operation such as various crops, flower plants, forestry plants, or the like. The plant may be, for example (without limitation): dicotyledonous, monocotyledonous or gymnosperm plants.

As a preferred mode, the "plant" includes, but is not limited to: cotton, arabidopsis, and especially upland cotton (Gossypium hirsutum), all genes having this gene or being homologous thereto are suitable.

As used herein, the term "plant" includes whole plants, parent and progeny plants thereof, and various parts of plants, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, in which the gene or nucleic acid of interest is found. Reference herein to "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the foregoing comprises the gene/nucleic acid of interest.

The present invention includes any plant cell, or any plant obtained or obtainable by a method therein, as well as all plant parts and propagules thereof. The present patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the sub-representations exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of this patent have the same characteristics.

The invention also extends to harvestable parts of a plant as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. And further to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starches or proteins.

The invention has the advantages that:

(1) The invention adopts a method of comparing transcriptomics to innovatively clone protein GhTSD7 responding to adversity stress in upland cotton (Gossypium hirsutum). The p 35S-GhTSD-GFP of GhTSD7 is constructed, the wild Arabidopsis thaliana (Clo-0, WT) is transformed by using an agrobacterium inflorescence infection method, an over-expression plant is obtained, and an analysis result shows that under high salt stress, compared with the wild Arabidopsis thaliana, ghTSD7 can improve seed germination rate and strengthen salt stress tolerance of Arabidopsis thaliana seedlings. Further constructing GhTSD gene silencing vector TRV2-GhTSD7, injecting agrobacterium containing TRV2-GhTSD7 into cotton leaves by using a method of infecting the cotton leaves with agrobacterium, obtaining GhTSD7 silenced cotton plants, and the result shows that the gene silencing plants are dwarfed under high salt stress and serious in wilting and yellowing phenomena, are more sensitive to high salt drought treatment, and provide gene resources for crop salt-tolerant molecular breeding.

(2) The plant with salt tolerance and drought tolerance can be obtained by a transgenic mode, in particular, the transgenic plant can be obtained by introducing GhTSD genes into a target plant, the salt tolerance and drought tolerance of the plant is higher than that of the target plant, and a new way is provided for plant salt tolerance and drought tolerance breeding.

Drawings

FIG. 1 is an analysis of the levels of GhERF and GhTSD7 gene expression under salt stress conditions (FIG. 1A);

FIG. 2 is a diagram showing the results of GhTSD transgenic Arabidopsis expression level analysis and fluorescence detection; wherein, FIG. 2A is a GhTSD7 transgenic Arabidopsis expression level analysis; FIG. 2B is a GhTSD transgenic Arabidopsis fluorescence detection result;

FIG. 3 is an analysis of germination rate of Arabidopsis thaliana seeds overexpressing GhTSD under high salt stress; wherein, FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D are analysis of germination rate of Arabidopsis thaliana seeds overexpressed GhTSD under different salt concentration treatments;

FIG. 4 is an analysis of the growth status of Arabidopsis seedlings overexpressing GhTSD under high salt stress on MS medium with different concentrations of NaCl (FIGS. 4A-4D); FIG. 4E is root elongation statistics of over-expressed GhTSD Arabidopsis seedlings on MS medium at different concentrations of salt treatment; FIG. 4F is a plot of chlorophyll content statistics of over-expressed GhTSD A.thaliana seedlings on MS medium under different salt concentrations; FIG. 4G is a graph showing anthocyanin content statistics of Arabidopsis seedlings overexpressing GhTSD A.thaliana seedlings in MS medium under different salt treatments;

FIG. 5 is a plant growth of Arabidopsis thaliana over-expressed GhTSD under saline watering; wherein, FIG. 5A is a growth phenotype diagram; FIG. 5B is an ion leakage rate statistic for over-expressed GhTSD Arabidopsis plants under saline watering; FIG. 5C is fresh weight statistics of Arabidopsis plants overexpressing GhTSD under saline watering; FIG. 5D is chlorophyll content statistics of Arabidopsis plants overexpressing GhTSD under saline watering;

FIG. 6 is an analysis of GhTSD expression levels in GhTSD gene-silenced plants; wherein, fig. 6A is a normal whitening plot for a control CLA; FIG. 6B is an analysis of GhTSD7 expression levels in GhTSD gene-silenced plants;

FIG. 7 is a phenotypic and physiological index analysis of cotton GhTSD under salt stress 7-silenced plants (TRV:: ghTSD) and normal plants (TRV:: 00); wherein FIG. 7A, FIG. 7B is a comparison of cotton GhTSD 7-silenced plants (TRV:: ghTSD 7) and normal plants (TRV:: 00) phenotypes under salt stress; FIG. 7C is an ion leakage rate statistic for cotton GhTSD 7-silenced plants (TRV:: ghTSD) and normal plants (TRV:: 00) under salt stress; FIG. 7D is a graph showing proline content statistics for cotton GhTSD 7-silenced plants (TRV:: ghTSD) and normal plants (TRV:: 00) under drought stress;

FIG. 8 is a DAB staining and physiological index analysis of cotton GhTSD 7-silenced plants (TRV:: ghTSD) and normal plants (TRV: 00) under salt stress; FIG. 8A is a plot of DAB staining of cotton GhTSD silent plants (TRV:: ghTSD 7) and normal plants (TRV:: 00) under salt stress; FIG. 8B is a detailed numerical statistic of DAB staining of cotton GhTSD 7-silenced plants (TRV:: ghTSD) and normal plants (TRV:: 00) under salt stress; FIG. 8C; FIG. 8D; FIG. 8E; FIG. 8F; FIG. 8G; FIG. 8H shows hydrogen peroxide content of cotton GhTSD silent plants (TRV:: ghTSD 7) and normal plants (TRV:: 00) after 3 days of salt stress, respectively; super-oxyanion content; malondialdehyde content; catalase activity; superoxide dismutase activity; proline content;

in the above figures, CK is the case of growth under normal conditions (control).

Detailed Description

The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.

Biological material

Cotton TM-1 seeds were stored for laboratory; the arabidopsis Col-0 seeds are preserved in a laboratory;

The overexpression vector pSuper-1300-GFP is stored in a laboratory; ghCLA is a laboratory preservation;

Coli DH 5. Alpha. And Agrobacterium GV3101 were kept in the laboratory;

Primer synthesis and sequencing were performed by zheng state qing department of biology.

Experimental reagent

RNA extraction kits, reverse transcription kits, and fluorescent quantification kits were purchased from nuuzan biotechnology limited;

common reagents such as NaCl are purchased from Soy Corp;

Hygromycin is purchased from soribao biosystems;

MS media was purchased from beijing cool pacing technologies limited;

Various endonucleases were purchased from monate biotechnology limited;

one-step cloning enzyme was purchased from nuuzan biotechnology limited;

plasmid miniprep and gel recovery kits were purchased from beijing tiangen biotechnology limited.

Experimental equipment

PCR apparatus was purchased from Bio-rad company;

the refrigerated centrifuge is purchased from Eppendorf corporation;

Quantitative PCR instrument was purchased from Bio-rad company;

confocal laser microscopy was purchased from zeiss corporation;

the autoclave MLS-3750 was purchased from Sanyang, japan;

nucleic acid detector nanodrop 2000C was purchased from Thermo Scientific company;

normal temperature centrifuge and microplate reader SpectraMax iD5 were purchased from Thermo Scientific.

Example 1 cloning of GhTSD Gene and analysis of coding sequence thereof

RNA of upland cotton TM-1 growing for 15 days is extracted, cDNA obtained by reverse transcription reaction is used as a template, a gene sequence obtained from NCBI database is subjected to PCR reaction by a Primer premier5.0 design specific Primer, and a coding sequence of GhTSD gene is cloned.

The coding sequence of the GhTSD7 gene, comprising 1152 bp bases, is available in CottonMD. The encoded protein comprises 383 amino acids.

To analyze whether GhTSD7 is involved in the cotton response to high salt stress, first, the expression pattern of GhTSD under high salt stress conditions was analyzed. Wild-type cotton plants TM-1 grown in 400 mM NaCl solution for 18 days were soaked, leaves were sampled at 0 h,2 h,4 h,8 h,12 h and 24 and h, respectively, and the expression level of GhTSD was detected by real-time fluorescent quantitative PCR (qRT-PCR).

As a result, it was found that the expression level of the marker gene GhERF for high salt stress-induced expression was increased to about 5-fold in the salt stress treatment of 8h and to about 8-fold in the salt stress treatment of 12 h, indicating that cotton seedlings were indeed subjected to high salt stress treatment; the expression level of GhTSD was increased at 2 h, 4h, 8h, 12 h, and at 24: 24 h to 5-fold higher than that at the same time (FIG. 1A). This result suggests that GhTSD7 may be involved in regulating the response process of cotton to high salt stress. The following is an experiment on this gene expansion around this aspect.

EXAMPLE 2 construction of the overexpression vector p 35S-GhTSD-GFP

To analyze GhTSD' S function, the inventors constructed the GhTSD7 overexpression vector p 35S-GhTSD 7-GFP, obtaining an overexpression Arabidopsis plant. The specific procedure is briefly described as follows.

First, primers were designed with restriction enzyme pst1 and kpn1 cleavage sites, the sequences were as follows:

1300-GhTSD7-F:5' - GGGGCCCGGGCTGCAGATGTCGTGTTCTTCG-3'

1300-GhTSD7-R:5'- GTATTTAAATGGTACCCTCACAAGCCAGTTT-3'

secondly, performing PCR amplification by using the cDNA sample prepared in the example 1 as a template, and purifying and recovering an amplification product;

Thirdly, carrying out double enzyme digestion on the 1300-GFP vector by using pst1 and kpn1, and purifying enzyme digestion products;

Fourthly, carrying out homologous recombination connection on the PCR amplification product and the vector after enzyme digestion to construct a 1300-GhTSD 7 over-expression vector;

Fifthly, converting the connection product into escherichia coli DH5 alpha by adopting a heat shock conversion method, carrying out K ⁺ (kanamycin, 50 mug/mL) resistance screening, selecting positive colonies for PCR detection, amplifying and sequencing correct colonies identified by the PCR detection, and extracting plasmids from bacterial liquid with correct sequencing for later use;

sixthly, the extracted plasmid is transformed into an agrobacterium competent cell GV3101, and the plasmid is preserved at the temperature of minus 80 ℃ for standby;

Seventh, wild type Arabidopsis thaliana (Clo-0, WT) is transformed by using an Agrobacterium inflorescence infection method, the harvested seeds are screened on MS culture medium containing hygromycin, the seeds are harvested for single plants of potential transgenic plants, and the seeds are screened on the culture medium containing hygromycin again until T ₃ generation screening is carried out to obtain potential homozygous transgenic plants.

The expression level of GhTSD in potentially transgenic plants was analyzed using qRT-PCR. As a result, it was found that the expression level of GhTSD7 was up-regulated about 400-1200 times in GhTSD potential transgenic plants in 3 different lines (FIG. 2A). The result of observing GFP fluorescence of the root of the over-expressed arabidopsis thaliana by a laser confocal microscope shows that GFP fluorescence can be detected in the root of the GhTSD transgenic plants which are screened (figure 2B). These results indicate that the GhTSD transgenic Arabidopsis constructed is a GhTSD-GFP overexpressing plant.

Example 3 overexpression of Arabidopsis thaliana verifies the function of GhTSD gene in Arabidopsis thaliana salt stress tolerance

High salt stress can inhibit seed germination, in order to further analyze whether GhTSD7 participates in regulating the process of inhibiting seed germination by high salt stress, we seed WT, over-expressed GhTSD7 Arabidopsis seeds on MS culture medium containing different concentrations of NaCl (0 mM, 50 mM, 100 mM and 150 mM), treat 3 d at low temperature of 4 ℃, then place in a greenhouse (21 ℃) with 12h illumination/12 h darkness, and count seed germination at intervals of 12h under a split microscope.

The results showed that the germination rate of the overexpressed seeds grown on MS medium was substantially identical to that of WT, representing that the seed germination rate of overexpressed GhTSD7 and WT was nearly 100% at 36 h (fig. 3A); the NaCl treatment with different concentrations obviously inhibits the germination of seeds, and the effect of inhibiting the germination of the seeds is more obvious along with the increase of the NaCl concentration in the MS culture medium. Seed grown for 48h on MS medium with 50 mM had a germination rate of over-expressed GhTSD7 of over 95% and seed germination rate of WT of less than 95% (FIG. 3B). Growing 48h seed on an MS medium containing 100 mM, over-expressing GhTSD7 seed germination rate of about more than 30% and WT seed germination rate of about less than 20%, over-expressing GhTSD7 seed germination rate of about 100%, about 90% and WT seed germination rate of less than 90% after 84h growth (fig. 3C); 84h seed was grown on MS medium containing 150 mM with approximately 60% germination of over-expressed GhTSD7 and approximately 30% germination of WT, and 108 hours after growth approximately 100% germination of over-expressed GhTSD and approximately 70% germination of WT (FIG. 3D).

These results indicate that overexpression GhTSD of 7 in arabidopsis can promote germination of seeds under high salt stress.

Meanwhile, in order to further analyze whether the transgenic plants of the over-expression GhTSD7 participate in the process of regulating and controlling the growth of the high-salt stress inhibition seedlings, we put WT and the seedlings of the over-expression GhTSD7 Arabidopsis thaliana (which vertically grow on MS medium for 5 days) on MS medium containing NaCl with different concentrations (0 mM, 100 mM, 150mM and 200 mM) respectively, then put the seedlings in a greenhouse (21 ℃) with 12h illumination/12 h darkness, and observe the growth condition of the seedlings. After seedlings were grown in different concentrations of medium for 10 days, we can clearly observe that the growth of WT on MS medium of 0mM NaCl and that of the overexpressed GhTSD7 arabidopsis seedlings were substantially identical (fig. 4A), whereas the root length of WT on MS medium of 100 mM and 150mM NaCl was significantly lower than that of the overexpressed GhTSD arabidopsis root length (fig. 4b,4 c), which was counted in specific values using imageJ and Prism9, found that the root length of WT on MS medium of 100 mM and 150mM NaCl was significantly lower than that of the overexpressed GhTSD arabidopsis root length (fig. 4E); and the WT leaves appeared significantly whitened on the MS medium of 200 mM NaCl compared to those overexpressing GhTSD7 (fig. 4D). We also performed statistics of specific data for chlorophyll and anthocyanin (fig. 4f,4 g).

These results indicate that overexpression GhTSD of 7 in arabidopsis can promote seedling growth under high salt stress.

Furthermore, we treated arabidopsis WT and overexpressed GhTSD7 transgenic plants with aqueous solution containing 150 mM NaCl, and found that after 25 days of high salt stress, in the control group (water treatment without NaCl), the overexpressed GhTSD transgenic plants were not significantly different from WT, whereas the high salt stress significantly inhibited the growth of the overexpressed GhTSD transgenic plants and WT, showing smaller rosette leaves, and that the leaves of WT showed significant yellowing, compared to WT, the overexpressed GhTSD transgenic plants did not show significantly yellowing leaves, showing a stronger high salt tolerance (fig. 5A).

To analyze the extent of damage to leaves of WT and over-expressed GhTSD transgenic plants under high salt treatment, we examined the ion leakage rate of the leaves. As a result, it was found that under high salt treatment conditions, both WT and over-expressed GhTSD7 transgenic plant leaves showed an increase in ion leakage rate, while over-expressed GhTSD transgenic plant leaves showed a lower ion leakage rate compared to WT (FIG. 5B). We also performed statistics of chlorophyll and fresh weight, which found that WT chlorophyll was significantly lower than that of the over-expressed GhTSD transgenic plant and fresh weight was also lower than that of the over-expressed GhTSD transgenic plant under high salt treatment conditions. The above results all indicate a greater degree of WT damage (fig. 5c,5 d).

These results indicate that overexpression GhTSD of 7 in arabidopsis can promote plant growth under high salt stress.

Example 4 silencing GhTSD Gene its function in cotton salt stress tolerance was verified

In order to deeply study the function of GhTSD in response to high salt stress of upland cotton, the inventor constructs GhTSD7 gene silencing vector TRV2-GhTSD7 by using a virus-induced GENE SILENCING (VIGS) system aiming at the coding region of GhTSD7, and obtains GhTSD7 silenced cotton plants (GhTSD 7 has higher homology, and the constructed TRV2-GhTSD vector can silence GhTSD7 genes and homologous genes thereof). The specific procedure is briefly described as follows.

First, a primer with restriction enzyme Bamh I and KpnI cleavage sites was designed, and the sequence was as follows:

TRV2-GhTSD7-F：GCCTCCATGGGGATCCAGTCCCAGCCGGCAATT

TRV2-GhTSD7-R：CGCGTGAGCTCGGTACCTGGGTCTACAGTAGCAGACAG

thirdly, carrying out double digestion on the TRV2 vector by adopting Bamh I and KpnI, and purifying the digested product;

fourthly, carrying out homologous recombination connection on the PCR amplification product and the digested vector to construct a TRV2-GhTSD7 expression vector;

Seventh, the agrobacteria containing TRV2-GhTSD, TRV2, TRV-CLA and TRV1 are injected into cotton leaves by using a method of infecting cotton leaves with the agrobacteria to obtain GhTSD silenced plants.

The results showed that 7 days after Agrobacterium infection, the transformed TRV:: ghCLA (TRV:: 00) plants as a positive control group exhibited a leaf albino phenotype (FIG. 6A), indicating that the gene silencing system was functioning. The expression level of GhTSD7 in the different plants with GhTSD7 silencing created by the VIGS system was tested by qRT-PCR experiments, and the results showed that the expression level of GhTSD7 in the 3 cotton seedlings tested was 1/5 of the control group, whereas the expression level of GhTSD7 in the 3 plants was only less than 1/10 of the control group (fig. 6B), which indicated that GhTSD7 silencing plants were created successfully.

Based on the analysis of the results of example 3 above, genetically silenced plants TRV:: ghTSD and TRV: 00 were treated with an aqueous solution containing 400 mM NaCl, after 12 days of treatment TRV::00 plant leaves remained green, while TRV:: ghTSD7 plant leaves wilted severely and plants were dwarf (FIGS. 7A, 7B). This result indicates GhTSD positive control of cotton tolerance to high salt stress.

To analyze the damage level of the leaves of the plants with TRV:: ghTSD and TRV::00 in the high salt treatment, we examined the ion leakage rate of the leaves. As a result, it was found that under the high salt treatment conditions, both TRV:: ghTSD and TRV: 00 increased the ion leakage rate in the plant leaves, whereas the ion leakage rate in the TRV:: ghTSD7 leaves was higher than that in TRV: 00 (FIG. 7C). This result shows that the TRV GhTSD plants are damaged to a greater extent.

Furthermore, to further analyze the level of plant stress resistance under high salt stress, we examined the content of Proline (PRO) in leaves, and the results showed that high salt stress resulted in more PRO accumulated in leaves, whereas more proline accumulated in leaves of TRV:: ghTSD compared to TRV: 00 (fig. 7D). The content of free proline in the plant body reflects the stress resistance of the plant to a certain extent, and under the stress conditions of drought, salting and the like, a large amount of proline in the plant body is accumulated, and the accumulated proline not only serves as an inner penetration regulating substance of plant cytoplasm, but also plays an important role in stabilizing a biological macromolecular structure, reducing cell acidity, relieving ammonia toxicity, regulating cell redox potential as an energy reservoir and the like. Excessive accumulation of proline indicates that GhTSD gene silencing results in a higher degree of leaf damage under high salt stress conditions.

Finally we analyzed the levels of active oxygen in the leaves of plants at salt stress of GhTSD and at TRV of 00, and after 14 days salt treatment of the second true leaves at salt of GhTSD and at TRV of 00 were subjected to DAB (3, 3-diaminobenzidine tetrahydrochloride) staining, which revealed that the tan deposit was significantly more in the leaves after salt treatment than in the leaves of the control group (H ₂ O watering), and that the tan sites appeared in the leaves at TRV of GhTSD to a deeper extent than in the leaves at TRV of 00 (FIG. 8A), indicating that more H ₂O₂ was accumulated in the leaves at high salt stress of TRV of GhTSD. Quantitative results of staining levels of the leaves, taken with Image J at the same sites of the different leaves, also showed that high salt stress caused accumulation of excess H ₂O₂ in the GhTSD leaves (FIG. 8B).

To analyze the levels of H ₂O₂ and superoxide anions in leaves of plants with TRV:: ghTSD and TRV::00 under high salt stress, we examined the levels of H ₂O₂ and superoxide anions under high salt stress 3 days after salt treatment. As a result, it was found that under the high salt treatment conditions, the contents of H ₂O₂ and superoxide anions in the leaves were increased, whereas the contents of H ₂O₂ and superoxide anions in the leaves of TRV: ghTSD7 were higher than those in the leaves of TRV:00 (FIGS. 8C, 8D), which is consistent with the results of DAB staining.

To further analyze the levels of intracellular reactive oxygen species under high salt stress, we also examined the levels of Malondialdehyde (MDA) in leaves three days after salt treatment, and the results showed that high salt stress resulted in more MDA accumulated in leaves, whereas TRV:: ghTSD accumulated more MDA in leaves than TRV: 00 (FIG. 8E). MDA is one of common indexes for measuring the degree of oxidative stress, can reflect the degree of peroxidation of plant membrane lipid, and is excessively accumulated, so that TRV:: ghTSD gene silencing leads to higher degree of peroxidation of leaf membrane lipid under high-salt stress conditions. In addition, to analyze the effect of other enzymes scavenging active oxygen on intracellular active oxygen levels, we also examined the activities of Catalase (CAT) and superoxide dismutase (SOD) in leaves after three days of salt treatment, respectively. The results show that high salt stress reduced CAT and SOD activity compared to the control, and that GhTSD gene silencing resulted in lower in leaf CAT and SOD activity than TRV::00 leaf enzyme activity (FIGS. 8F, 8G). This result shows that under high salt stress conditions, the TRV GhTSD leaves accumulate excessive amounts of active oxygen, on the one hand, due to the down-regulation of GhTSD7 expression levels and, on the other hand, due to the reduced activity of CAT and SOD in the leaves. Finally, we also examined the content of Proline (PRO) in leaves three days after salt treatment, and the results showed that high salt stress resulted in more PRO accumulated in leaves, whereas TRV:: ghTSD accumulated more proline in leaves than TRV: 00 (fig. 8H). Excessive accumulation of proline indicates that GhTSD gene silencing leads to a higher degree of leaf damage under high salt and drought stress.

In conclusion, the salt stress tolerance of the arabidopsis is improved after the arabidopsis is overexpressed, which indicates that GhTSD gene positively regulates the tolerance of the arabidopsis to high salt stress; and the salt stress tolerance of cotton is reduced after the GhTSD gene is silenced, which also indicates that the GhTSD gene positively regulates the tolerance of cotton to high salt stress. Therefore, ghTSD gene plays an important role in regulating and controlling high-salinity stress of plants, and has important significance for cultivating cotton varieties capable of resisting external stress conditions.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art through substitution or modification according to the technical disclosure in the present specification, so that all changes and modifications made in the principle of the present invention shall be included in the scope of the present invention.

Claims

The application of the GhTSD7 gene in improving the salt stress tolerance of plants is characterized in that a GhTSD over-expression vector is constructed to obtain a transgenic plant with high salt stress tolerance, the nucleotide sequence of the GhTSD gene is shown as SEQ ID NO.1, the salt stress tolerance is high-concentration salt stress tolerance, and the high-concentration salt concentration is 400 mM; the plant is cotton or Arabidopsis thaliana.
2. The use according to claim 1, wherein the salt stress tolerance is manifested as: under salt stress, seed germination rate of GhTSD7 over-expression strain is higher than that of wild type, yellowing degree of leaf is lower than that of wild type, damage degree of leaf is lower than that of wild type, and plant height of GhTSD gene silencing strain is lower than that of wild type, yellowing and withering phenomenon of leaf is higher than that of wild type, and damage degree of leaf is higher than that of wild type.
3. A plant breeding method is characterized in that plants with salt stress tolerance higher or lower than that of wild plants are obtained by regulating and controlling the expression of GhTSD gene in target plants, wherein the mode of regulating and controlling the expression of GhTSD gene in target plants is over-expression or silencing GhTSD gene, the nucleotide sequence of GhTSD gene is shown as SEQ ID NO.1, and the target plants are cotton.
4. A plant breeding method is characterized in that plants with salt stress tolerance stronger than that of wild plants are obtained by regulating and controlling the expression of GhTSD gene in target plants, wherein the mode of regulating and controlling the expression of GhTSD gene in target plants is over-expression GhTSD gene, the nucleotide sequence of GhTSD gene is shown as SEQ ID NO.1, and the target plants are Arabidopsis thaliana.