CN115612695B - Application of GhGPX5 and GhGPX13 genes in improving salt stress tolerance of plants - Google Patents

Abstract

The invention discloses an application of GhGPX5 and GhGPX13 genes in improving plant salt stress tolerance. The gene sequence numbers of the GhGPX5 and GhGPX13 genes in NCBI are XM_041083558.1 and XM_016881552.2 respectively. The present invention obtains GhGPX5/13 silenced plants by means of gene silencing, and the results show that the leaves of the gene silencing plants are severely wilted and yellowed under high-salt stress, indicating that they are more sensitive to high-salt treatment. Next, the overexpression vectors p35S‑GhGPX5‑GFP and p35S‑GhGPX13‑GFP of GhGPX5/13 were constructed, and wild-type Arabidopsis (Clo‑0, WT) was transformed by Agrobacterium inflorescence infection method to obtain overexpressed plants. The analysis results showed that Under high salt stress, compared with the wild type, GhGPX5/13 can increase the seed germination rate and enhance the salt stress tolerance of Arabidopsis seedlings, thus providing genetic resources for crop salt-tolerant molecular breeding.

Description

GhGPX5 and Application of GhGPX13 Gene in Improving Plant Salt Stress Tolerance

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of GhGPX5 and GhGPX13 genes in improving plant salt stress tolerance.

Background technique

During the whole growth period, plants will be subjected to various biotic and abiotic stress environments, and various stress factors will eventually lead to the accumulation of excessive active oxygen in plants, thereby affecting the growth and development of plants. Existing studies believe that reactive oxygen species are a by-product of plant metabolism and play an important role in cell signal transmission and redox balance. Low concentrations of reactive oxygen species can act as signal molecules in plants, while high concentrations of reactive oxygen species can be toxic to plant cells and oxidize intracellular components such as DNA, protein, and plasma membranes, thereby affecting plant cells. growth and development, thereby reducing crop yield.

During the evolution of plants, complex enzymatic and non-enzymatic systems have been formed to eliminate the oxidative damage caused by reactive oxygen species and maintain the dynamic balance of reactive oxygen species. Among them, the enzymatic system includes various peroxidase families, and the study of the functions of these antioxidant genes can provide theoretical basis and application basis for improving plant stress resistance.

Plant GPXs can scavenge excess reactive oxygen species produced by stressful environments, and they usually use Trx as a reducing agent instead of GSH, so they are considered to have thioredoxin peroxidase activity. Some plant GPXs exhibit both glutathione peroxidase and thioredoxin peroxidase activities, but with thioredoxin as the substrate, the enzymatic activity is higher than that with glutathione as the substrate . But in general, because there are many types of GPXs genes and their corresponding proteins, and the action process is more complicated, further research is needed in order to lay a theoretical foundation for the specific application of genes.

my country's demand for raw cotton is increasing day by day, but it is unrealistic to increase the total output by expanding the area of cultivated land. my country has more than 100 million mu of real estate saline-alkali land and 300 million mu of saline-alkali wasteland, and 60% to 80% of cotton fields are in arid and semi-arid areas. Because cotton requires relatively stronger drought resistance and salt-alkaline tolerance than other crops, it is easily affected by various stress factors during the growth process, so it is easy to accumulate a large amount of active oxygen in the organism, and the GPXs family has the ability to scavenge active oxygen, thereby resisting the external environment the ability to coerce. Therefore, through the study of GPXs genes in cotton, we can more clearly understand the role of GPXs protein family in cotton under biotic and abiotic stress. Furthermore, the use of transgenic technology to cultivate cotton varieties that can resist external stress conditions is of great significance to the long-term stable development of cotton production in my country. To understand and find more genes related to drought resistance and salt-alkali tolerance, and to study genes from the molecular level It is of great significance to the growth and development of cotton and the influence of stress resistance.

Contents of the invention

The purpose of the present invention is to provide the application of GhGPX5 and GhGPX13 genes in improving plant salt stress tolerance.

In order to achieve the above object, the technical scheme adopted in the present invention is summarized as follows:

The GhGPX5 gene used in the present invention has a gene sequence number (Sequence ID) of XM_041083558.1 in NCBI, the messenger RNA (mRNA) sequence length of the GhGPX5 gene is 1313 bp, and the coding sequence length of the GhGPX5 gene is 702 bp, including 233 amino acids;

The gene sequence number (Sequence ID) of GhGPX13 gene in NCBI is XM_016881552.2, the messenger RNA (mRNA) sequence length of GhGPX13 is 1256 bp, and the coding sequence length of GhGPX13 gene is 702 bp, including 233 amino acids. There is a high homology between the amino acid sequences of GhGPX5 and GhGPX13 .

The present invention also constructs a series of plant expression vectors, expression vectors, recombinant vectors or transgenic plant lines containing the above genes and the function of host cells containing the vectors in improving plant salt stress tolerance also fall within the protection scope of the present invention .

The functions of the genes protected in the present invention include not only the above-mentioned GhGPX5 and GhGPX13 genes, but also the functions of homologous genes with high homology (up to 99%) with the GhGPX5 and GhGPX13 genes in terms of salt stress tolerance.

The biological functions of the GhGPX5 and GhGPX13 genes disclosed in the present invention in plant salt-tolerant stress are specifically manifested in: under salt stress, the degree of wilting and yellowing of the leaves of the GhGPX5 and GhGPX13 gene silenced lines is higher than that of the wild type, while the GhGPX5 and GhGPX13 overexpression lines had higher seed germination rate than wild type, and lower leaf yellowing degree than wild type.

According to its function, plants tolerant to salt stress can be obtained by transgenic means. Specifically, GhGPX5 and GhGPX13 genes can be introduced into target plants to obtain transgenic plants whose salt stress tolerance is higher than that of target plants.

Specifically, the GhGPX5 and GhGPX13 genes can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and The transformed plant tissue is grown into plants.

In order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, which is the following (1) or (2) or (3):

(1) By increasing the activity of GhGPX5 and GhGPX13 proteins in the target plant, obtain plants with stronger salt stress tolerance than the target plant;

(2) By promoting the expression of GhGPX5 and GhGPX13 genes in the target plant, obtain plants with stronger salt stress tolerance than the target plant;

(3) By inhibiting the expression of GhGPX5 and GhGPX13 genes in the target plant, plants with lower salt stress tolerance than the target plant are obtained.

The implementation of "promoting the expression of GhGPX5 and GhGPX13 genes in target plants" can be as follows (1) or (2) or (3):

(1) Introducing GhGPX5 and GhGPX13 genes into target plants;

(2) introduction of strong promoters and/or enhancers;

(3) Other common methods in this field.

Wherein, the target plant, the target plant of the present invention is cotton, Arabidopsis.

The target gene, also known as the target gene, is used in gene engineering design and operation to recombine genes, change the traits of recipient cells and obtain expected expression products. It can be from the organism itself or from a different organism.

The method of "regulating the expression of GhGPX5 and GhGPX13 genes in plants" is overexpression, silencing or directed mutation of GhGPX5 and GhGPX13 genes.

Regulating gene expression level includes using DNA homologous recombination technology, virus-mediated gene silencing technology and Agrobacterium-mediated transformation system to regulate the expression of GhGPX5 and GhGPX13 to obtain transgenic plant lines.

In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, floral plants, or forestry plants. The plant may be, for example (not limited to): dicotyledonous plants, monocotyledonous plants or gymnosperms.

As a preferred manner, the "plant" includes, but is not limited to: cotton, Arabidopsis, especially upland cotton ( Gossypium hirsutum ), any gene that has this gene or is homologous to it is applicable.

The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs. Different parts have our target gene or nucleic acid. The "plant" mentioned here also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, where each of the aforementioned objects contains the gene/nucleic acid of interest .

The invention includes any plant cell, or any plant obtained or obtainable by a method therein, and all plant parts and propagules thereof. This patent also covers transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the offspring exhibit the same genotypic or phenotypic characteristics, and the offspring obtained using the method of this patent have the same characteristics.

The invention also extends to harvestable parts of plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It further relates to other derivatives of the harvested plants, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to foods or food additives obtained from related plants.

Advantages of the present invention:

(1) The present invention adopts the method of comparative transcriptomics to innovatively control the glutathione peroxidase (glutathione peroxidase , GPX) GhGPX5 and GhGPX13 (collectively GhGPX5/13 ) was cloned. The prokaryotic expression vector 6P1-GhGPX5/13 was constructed, and the recombinant vector was transformed into Escherichia coli expression competent cells Rosette (DE3), and the massive expression of GST-GhGPX5/13 was induced and purified. The results showed that GhGPX5/13 had peroxidase activity and redox state. The GhGPX5/13 gene silencing vector TRV2-GPX5/13 was further constructed, and the Agrobacterium containing TRV2-GPX5/13 was injected into the cotton leaves by using the method of Agrobacterium infection to obtain GPX5/13 silenced plants. The results showed that the gene silenced plants Under high-salt stress, the leaves wilted and yellowed seriously, indicating that they were more sensitive to high-salt treatment. Next, the overexpression vectors p35S-GhGPX5-GFP and p35S-GhGPX13-GFP of GPX5/13 were constructed, and wild-type Arabidopsis (Clo-0, WT) was transformed by Agrobacterium inflorescence infection method to obtain overexpression plants. The analysis results showed that Under high-salt stress, GhGPX5/13 can increase seed germination rate compared with wild type. Provide genetic resources for crop salt-tolerant molecular breeding.

(2) Salt-tolerant plants can be obtained through transgenic methods. Specifically, transgenic plants can be obtained by introducing the GhGPX5/13 gene into the target plant. A new way.

Description of drawings

Figure 1 is the comparative analysis of GhGPX5 and GhGPX13 CDS sequences and encoded amino acid sequences; Figure 1A shows the CDS sequences of GhGPX5 and GhGPX13 , and there are only seven differences ; single point difference;

Figure 2 is the enzyme activity and redox state analysis of GhGPX5/13 protein; Figure 3A shows that the expression level of the marker gene GhERF38 induced by high-salt stress increased by more than 10 times at 8 h of salt stress treatment, and at 12 h of salt stress treatment Figure 3B shows that the expression level of GhGPX5/13 increased to 2.5 times when salt stress was treated for 12 h; Figure 4A shows that after 7 days of Agrobacterium infection, the transformed TRV::GhCLA as a positive control group ( TRV::00 ) plants showed a phenotype of leaf albinism; Figure 4B shows the expression levels of GPX5/13 in different plants that were silenced for GPX5/13 ;

Figure 3 is an analysis of the expression levels of GhERF38 and GhGPX5/13 genes under salt stress conditions;

Figure 4 is the analysis of the expression level of GhGPX5/13 in GhGPX5/13 gene silenced plants; Figure 4A shows the expression of leaf albinism in the transformed TRV::GhCLA ( TRV::00 ) plants as a positive control group 7 days after Agrobacterium infection type; Figure 4B shows the expression levels of GPX5/13 in different plants that create GPX5/ 13 silence;

Figure 5 is a comparison of the phenotypes of cotton GhGPX5/13 silenced plants ( TRV::GPX5/13 ) and normal plants ( TRV::00 ) under salt stress;

Figure 6 shows the phenotype, physiological indicators and enzyme activity analysis of cotton GhGPX5/13 silenced plants and normal plants under salt stress; Figure 6A shows that the joint silencing of GhGPX5 and GhGPX13 causes a large number of macules to appear on the leaves, and the severe parts show a large area Yellowing phenomenon; Figure 6B shows that the brown deposits in the leaves after high-salt treatment are significantly more than those in the control group (H ₂ O soaking), and TRV::GPX5/13 leaves have a large area of brown parts, Its staining degree is deeper than TRV::00 ;

Figure 6C shows that high-salt stress causes excess H ₂ O ₂ accumulation in leaves of TRV::GPX5/13 ; Figure 6D shows that blue deposits in leaves after high-salt treatment increased significantly; Figure 6E shows that compared with TRV::00 leaves , the blue parts in leaves of TRV::GPX5/13 plants increased significantly; Figure 6F shows that under high-salt treatment conditions, the contents of H ₂ O ₂ and superoxide anions in leaves increased;

Figure 6G shows that compared with TRV::00 , the content of H ₂ O ₂ and superoxide anion is higher in TRV::GPX5/13 leaves; Figure 6H shows that compared with TRV::00 , TRV::GPX5/13 leaves More MDA accumulated in ; Figure 6I and Figure 6J showed that GPX5/13 gene silencing resulted in lower CAT and SOD activities in leaves than in TRV::00 leaves;

Figure 7 shows the results of expression level analysis and fluorescence detection of GhGPX5 and GhGPX13 transgenic Arabidopsis; Figure 7A shows that the expression level of GhGPX5 is up-regulated by about 40 times in two different lines; Figure 7B shows that the expression level of GhGPX13 in two different lines The line was up-regulated more than 50 times; Figure 7C shows that GFP fluorescence can be detected in the roots of GhGPX5 and GhGPX13 transgenic plants;

Figure 8 is an analysis of the seed germination rate and seedling growth status of Arabidopsis overexpressing GhGPX5 and GhGPX13 under high salt stress; Figure 8A shows that the seed germination rate of overexpressing GhGPX13 is greater than 70%, while the seed germination rate of WT does not exceed 30%; Fig. 8B shows that the germination rate of overexpressed GhGPX5 and GhGPX13 is more than 90%, while that of WT is about 70% . However, the germination rate of WT seeds was less than 70%; Figure 8D shows that the germination rate of overexpressed GhGPX5 seeds is close to 80%, and the germination rate of overexpressed GhGPX13 is about 60%, while the germination rate of WT seeds is less than 20%; Figure 8E shows that Under the conditions of different concentrations of salt treatment, the growth of seedlings overexpressing GhGPX5 and GhGPX13 was significantly better than that of WT, showing that their growth was not sensitive to high-salt stress compared with WT;

Figure 9 is an analysis of the growth status of Arabidopsis plants overexpressing GhGPX5 and GhGPX13 under high-salt stress.

Detailed ways

The present invention will be described in detail through specific examples below. These embodiments are provided for a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA and bioinformatics, techniques apparent to those skilled in the art. These techniques have been fully explained in the published literature. In addition, the DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition and other methods used in the present invention, except for the following Except for the method adopted in the embodiment, it can be realized by adopting the methods already disclosed in the existing documents.

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 (eg, messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited thereto. 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 the genomic sequence, and/or include the coding sequence in the cDNA, and/or include the cDNA and its regulatory sequences. In particular embodiments, eg in relation to an isolated nucleic acid sequence, it is preferentially assumed that it is cDNA.

biomaterials

Cotton TM-1 seeds are preserved in the laboratory; Arabidopsis Col-0 seeds are preserved in the laboratory;

The prokaryotic expression vector pGEX6P1; the gene silencing vector empty vector and the positive control vector TRV::GhCLA are kept in the laboratory; the overexpression vector pSuper-1300-GFP is kept in the laboratory;

Escherichia coli DH5α and Agrobacterium GV3101 were kept in the laboratory;

Primer synthesis and sequencing were completed by Zhengzhou Qingke Biological Company.

experimental reagent

RNA extraction kits, reverse transcription kits and fluorescence quantitative kits were purchased from Novozyme Biotechnology Co., Ltd.;

Common reagents such as NaCl were purchased from Suo Lai Bao Company;

Hygromycin was purchased from Suleibao Biological Company;

MS medium was purchased from Beijing Kulaibo Technology Co., Ltd.;

Various endonucleases were purchased from Mona Biotechnology Co., Ltd.;

One-step cloning enzyme was purchased from Nuoweizan Biotechnology Co., Ltd.;

Plasmid mini-extraction kit and gel recovery kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.

Laboratory equipment

The PCR instrument was purchased from Bio-rad company;

The refrigeration centrifuge was purchased from Eppendorf;

Quantitative PCR instrument was purchased from Bio-rad company;

Laser confocal microscope was purchased from Zeiss;

The high temperature and high pressure sterilizer MLS-3750 was purchased from Japan Sanyo Company;

Nucleic acid detector Nanodrop 2000C was purchased from Thermo Scientific;

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

Example 1 Cloning of GhGPX5/13 gene and its amino acid sequence analysis

The RNA of upland cotton TM-1 grown for 15 days was extracted, and the cDNA obtained from the reverse transcription reaction was used as a template. The gene sequence obtained from the NCBI database was designed with Primer Premier 5.0 and specific primers were used to clone GhGPX5/13 by PCR. The coding sequences of the two genes were transformed into protein sequences through the Translate (https://www.expasy.org/resources/ online tool). Further, the GhGPX5/13 gene sequence and the encoded protein sequence were analyzed using DNAMAN software. Comparison.

The results showed that the coding sequence of cotton glutathione peroxidase GhGPX5/13 gene contained 702 bp bases. The encoded proteins each consist of 233 amino acids. The CDS sequences of GhGPX5 and GhGPX13 have only seven differences (Fig. 1A), and the encoded amino acids have only three differences (Fig. 1B), showing high homology.

Example 2 Construction of expression vector pGEX6P1-GhGPX5/13

In order to explore the characteristics of the GhGPX5/13 protein, the inventors respectively constructed the prokaryotic expression vector pGEX6P1- GhGPX5/13 , and the specific process is briefly introduced as follows.

At first, design the primer that has restriction endonuclease Eco R Ⅰ restriction site, sequence is as follows:

6P1-X5-F: 5'-GGGATCCCCGGAATTCATGCTCGTTCGACGAAAT-3'

6P1-X5-R: 5'-GTCGACCCGGGAATTCGCCAAGCAGTTTCTTTAT-3'

6P1-X13-F: 5'-GGGATCCCCGGAATTCATGCTCGTTCGACGAAATCT-3'

6P1-X13-R: 5'-GTCGACCCGGGAATTCCGTATCCACTCCCAATGCTT-3'

Then, using the cDNA sample prepared in Example 1 as a template, carry out PCR amplification, and purify and recover the amplified product;

Thirdly, the PGEX6P1 vector was digested with Eco RI, and the digested products were purified.

Fourth, the PCR amplified product and the digested vector were subjected to homologous recombination to construct a 6p1-GhGPX5/13 expression vector;

Fifth, use the heat shock transformation method to transform the ligation product into E. coli DH5α , perform A (ampicillin, 50 μg/mL) resistance screening, select positive colonies for PCR detection, and amplify the correct colonies identified by PCR detection. Send for sequencing, and extract the plasmid from the bacteria solution with correct sequencing for future use.

Sixth, the extracted plasmid was transformed into Escherichia coli expression competent cells Rosette (DE3) to induce massive expression of GST-GhGPX5/13 and purified to obtain GhGPX5/13 protein.

In the presence of H ₂ O ₂ , Trx was used as a substrate to detect whether the purified GhGPXs had peroxidase activity. It was found that GhGPX5/13 has peroxidase activity (Figure 2). In order to further analyze whether GhGPX5/13 has the characteristics of redox state, the purified GhGPX5/13 protein was treated with β-mercaptoethanol (β-ME), dithiothreitol (DTT) and H ₂ O ₂ , passed through polypropylene Amide gel electrophoresis (SDS-PAGE) analysis showed that GhGPX5/13 with peroxidase had a redox state.

Example 3 Silencing the GhGPX5/13 gene to verify its function in cotton salt stress tolerance

In order to analyze whether GhGPX5/13 is involved in the process of cotton response to high-salt stress, first, the expression pattern of GhGPX5/13 under high-salt stress was analyzed. The wild-type cotton plant TM-1 was immersed in 400 mM NaCl solution for 18 days, and the leaves were sampled at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h, and real-time fluorescent quantitative PCR (qRT- PCR) to detect the expression level of GhGPX5/13 ( GhGPX5/13 has a high homology, and a pair of primers are designed according to its conserved sequence to detect the expression levels of the two genes at the same time).

It was found that the expression level of the marker gene GhERF38 induced by high salt stress increased by more than 10 times at 8 h of salt stress, and 15 times at 12 h of salt stress (Fig. 3A), indicating that cotton seedlings were indeed affected by high salt stress. Salt stress treatment; the expression level of GhGPX5/13 increased to 2.5 times after 12 h of salt stress treatment (Fig. 3B). This result suggested that GhGPX5/13 may be involved in regulating the response of cotton to high-salt stress.

In order to further study the function of GhGPX5/13 in the response of upland cotton to high salt, the inventors constructed the GhGPX5 /13 GhGPX5 /13 conserved region using the virus-induced gene silencing (VIGS) system. The gene silencing vector TRV2-GPX5/13 obtained GPX5/13 silenced cotton plants ( GhGPX5/13 has high homology, and the constructed TRV2-GPX5/13 vector can simultaneously silence two genes.). The specific process is briefly introduced as follows.

First, design primers with restriction endonucleases Eco R Ⅰ and Kpn Ⅰ restriction sites, the sequences are as follows:

TRV2-GPX5/13-F: GTGAGTAAGGTTACCGAATTCGAGTCCTCCAAGGGGTCAGTT

TRV2-GPX5/13-R: GAGACGCGTGAGCTCGGTACCACCTTTGCTAGCCTTCAGGAA

First, use the cDNA sample prepared in Example 1 as a template to perform PCR amplification, and purify and recover the amplified product;

Thirdly, the TRV2 vector was digested with Eco R Ⅰ and Kpn Ⅰ, and the digested product was purified;

Fourth, the PCR amplified product and the digested vector were subjected to homologous recombination to construct a TRV2-GhGPX5/13 expression vector;

Fifth, use the heat shock transformation method to transform the ligation product into E. coli DH5α , carry out K ⁺ (kanamycin, 50 μg/mL) resistance screening, select positive colonies for PCR detection, and amplify the correct colonies identified by PCR detection. Amplify and send for sequencing, and extract plasmids from bacteria liquid with correct sequencing for future use.

Sixth, the extracted plasmid was transformed into Agrobacterium competent cell GV3101 , and stored at -80°C for future use.

Seventh, the method of using Agrobacterium to infect cotton leaves Inject the Agrobacterium containing TRV2-GPX5/13 , TRV2 , TRV-CLA , and TRV1 into cotton leaves to obtain GPX5/13 silenced plants.

The results showed that 7 days after Agrobacterium infection, the transformed TRV::GhCLA ( TRV::00 ) plants used as a positive control group had a leaf albino phenotype (Fig. 4A), indicating that the gene silencing system played a role. The expression levels of GPX5 /13 in different plants silenced by the VIGS system were detected by qRT-PCR. The results showed that among the six cotton seedlings detected, the expression levels of GPX5/13 in plants 1, 2, 3 and 6 The expression level was 1/5 of that of the control group, while the expression level of GPX5/13 in plants 3 and 4 was only 1/10 of that of the control group (Figure 4B). These results indicated that GPX5/13 silenced plants were successfully created.

On the basis of the analysis of the results of the aforementioned example 4, the gene silencing plants TRV::GPX5/13 and TRV::00 were treated with an aqueous solution containing 400 mM NaCl. After 8 days of treatment, the leaves of TRV::00 plants were still green , while TRV::GPX5 The leaves of /13 plants wilted severely (Fig. 5). This result indicated that GhGPX5/13 positively regulated the tolerance of cotton to high-salt stress.

Based on the analysis of the aforementioned results, the second true leaves of the created gene silencing plants TRV::GPX5/13 and TRV::00 were respectively soaked in H ₂ O and an aqueous solution containing 400 mM NaCl, and soaked in water after 24 h. The leaves were still green, while the leaves soaked in high-salt solution showed different degrees of macula, which showed that TRV::00 leaves had a small number of macula, while TRV::GPX5/13 had a large number of macula, severe The site showed extensive yellowing (Fig. 6A). This indicated that co-silencing of GhGPX5 and GhGPX13 significantly reduced the ability of leaves to tolerate high-salt stress.

In order to analyze the level of reactive oxygen species in leaves of TRV::GPX5/13 and TRV::00 plants under high-salt stress, the second true leaves of TRV::GPX5/13 and TRV::00 were subjected to high-salt treatment for 12 h. DAB (3,3-diaminobenzidine tetrahydrochloride) staining, it was found that the brown deposits in the leaves treated with high salt were significantly more than those in the control group (H ₂ O soaked), and, TRV: :GPX5/13 leaves had a large area of tan, and the staining degree was deeper than that of TRV::00 (Fig. 6B), indicating that TRV::GPX5/13 leaves accumulated more H ₂ O ₂ under high-salt stress.

The same parts of different leaves were taken by Image J, and the quantitative results of leaf staining also showed that high-salt stress caused excess H ₂ O ₂ accumulation in TRV::GPX5/13 leaves (Fig. 6C). In addition, NBT (nitrotetrazolium blue chloride) staining was used to analyze the accumulation of superoxide anion in the leaves, and it was found that the blue deposits in the leaves after high-salt treatment increased significantly (Figure 6D), and compared with TRV: :00 leaves, the blue parts in the leaves of TRV::GPX5/13 plants were significantly increased, and the quantitative analysis of the stained parts also verified this result (Fig. 6E). These results indicated that the silencing of the three GPX5/13 genes led to the accumulation of more reactive oxygen species in TRV::GPX5/13 plants under high-salt treatment, which caused more damage to the leaves, manifested as a deeper yellowing of the leaves. High salt treatment is more sensitive.

To analyze the levels of H ₂ O 2 _and superoxide anion in leaves of TRV::GPX5/13 and TRV::00 plants under high-salt treatment, we detected the contents of H ₂ O ₂ and superoxide anion under high-salt stress . It was found that the contents of H ₂ O ₂ and superoxide anion in leaves increased under high-salt treatment, while the contents of H ₂ O ₂ and superoxide anion in leaves of TRV::GPX5/13 were higher than those in TRV::00. The content was higher (Fig. 6F, G), which was consistent with the results of DAB and NBT staining.

In order to further analyze the level of reactive oxygen species in cells under high-salt stress, we detected the content of malondialdehyde (MDA) in leaves. than, more MDA accumulated in TRV:: GPX5/13 leaves (Fig. 6H). MDA is one of the commonly used indicators to measure the degree of oxidative stress, which can reflect the degree of membrane lipid peroxidation in plants. The excessive accumulation of MDA indicates that under high-salt stress conditions, GPX5/13 gene silencing leads to a higher degree of membrane lipid peroxidation in leaves. Furthermore, in order to analyze the effects of other ROS-scavenging enzymes on intracellular ROS levels, we detected the activities of catalase (CAT) and superoxide dismutase (SOD) in leaves, respectively. The results showed that, compared with the control group, high-salt treatment reduced the activities of CAT and SOD, and GPX5/13 gene silencing resulted in lower enzyme activities of CAT and SOD in leaves than in TRV::00 leaves (Fig. 6I, J). This result indicated that TRV:: GPX5/13 leaves accumulated excessive reactive oxygen species under high-salt stress conditions, which was caused by down-regulation of GPX5/13 expression on the one hand, and decreased activities of CAT and SOD on the other hand. of.

Example 4 Overexpression of Arabidopsis to verify the function of GhGPX5/13 gene in Arabidopsis salt stress tolerance

In order to further analyze the function of GPX5/13 , the inventors respectively constructed GPX5/13 overexpression vectors p35S- GhGPX5-GFP and p35S-GhGPX13-GFP , and obtained overexpressed Arabidopsis plants. The specific process is briefly introduced as follows.

First, design primers with restriction endonuclease Sal Ⅰ restriction site, the sequence is as follows:

1300-GPX5-F: 5'-GGGGCCCGGGGTCGACATGCTCGTTCGACGAAAT-3'

1300-GPX5-R: 5'-GTATTTAAATGTCGACGGCCAAGCAGTTTCTTTAT-3'

1300-GPX13-F: 5'-GGGGCCCGGGGTCGACATGCTCGTTCGACGAAATCT-3'

1300-GPX13-R: 5'-GTATTTAAATGTCGACGGCCAAGCAGTTTCTTTATAT-3'

Then, using the cDNA sample prepared in Example 1 as a template, carry out PCR amplification, and purify and recover the amplified product;

Thirdly, the 1300-GFP vector was digested with Sal I, and the digested product was purified.

Fourth, the PCR amplified product and the digested vector were connected by homologous recombination to construct a 1300-GhGPX5/13 overexpression vector;

Fifth, use the heat shock transformation method to transform the ligation product into E. coli DH5α , carry out K ⁺ (kanamycin, 50 μg/mL) resistance screening, select positive colonies for PCR detection, and amplify the correct colonies identified by PCR detection. Amplify and send for sequencing, and extract plasmids from bacteria liquid with correct sequencing for future use.

Sixth, the extracted plasmid was transformed into Agrobacterium competent cell GV3101 , and stored at -80°C for future use.

Seventh, transform wild-type Arabidopsis thaliana (Clo-0, WT) using the Agrobacterium inflorescence infection method, screen the harvested seeds on MS medium containing hygromycin, and harvest seeds from individual potential transgenic plants, Select again on the medium containing hygromycin until the potential homozygous transgenic plants are obtained by screening in the T ₃ generation.

The expression levels of GhGPX5 and GhGPX13 in potential transgenic plants were analyzed by qRT-PCR. The results showed that in the GhGPX5 potential transgenic plants, the expression level of GhGPX5 was up-regulated by about 40 times in two different lines (Fig. 7A), and in the GhGPX13 potential transgenic plants, the expression level of GhGPX13 It was upregulated more than 50-fold in 2 different lines (Fig. 7B). The GFP fluorescence in the roots of overexpressed Arabidopsis thaliana was observed by laser confocal microscopy, and the results showed that GFP fluorescence could be detected in the roots of the screened GhGPX5 and GhGPX13 transgenic plants (Fig. 7C). These results indicated that the constructed GhGPX5 and GhGPX13 transgenic Arabidopsis were GhGPX5-GFP and GhGPX13-GFP overexpression plants, respectively.

High-salt stress can inhibit seed germination. In order to further analyze whether GhGPX5 and GhGPX13 are involved in regulating the process of high-salt stress inhibiting seed germination, we sowed WT, GhGPX5 and GhGPX13 overexpressed Arabidopsis seeds in different concentrations of NaCl (0 mM , 50 mM, 100 mM and 150 mM) MS medium, treated at 4°C for 3 days, then placed in a 12h light/12h dark greenhouse (21°C), and placed under a stereo microscope every 12 hours Seed germination statistics.

The results showed that the germination rate of overexpressed seeds grown on MS medium was significantly higher than that of WT at 24 h and 36 h, showing that the germination rate of overexpressed GhGPX5 seeds was close to 100% at 36 h, and the germination rate of overexpressed GhGPX13 seeds was The seed germination rate of WT was more than 70%, while the seed germination rate of WT was not more than 30% (Fig. 8A); different concentrations of NaCl treatment significantly inhibited seed germination, and, with the increase of NaCl concentration in MS medium, the effect of inhibiting seed germination was more significantly. Seeds grown on MS medium containing 50 mM for 36 h, the germination rate of overexpressed GhGPX5 and GhGPX13 was more than 70%, while the germination rate of WT seeds was less than 20%, after 60 h of growth, the germination rate of overexpressed GhGPX5 and GhGPX13 The seed germination rate of WT was more than 90%, while that of WT was about 70% (Fig. 8B).

Seeds grown on MS medium containing 100 mM for 48 h, the germination rate of overexpressed GhGPX5 and GhGPX13 was more than 60%, while the germination rate of WT seeds was about 10%, after 72 h of growth, the germination rate of overexpressed GhGPX5 seeds Close to 100%, the germination rate of overexpressed GhGPX13 was about 90%, while the germination rate of WT seeds was less than 70% (Fig. 8C); Seeds grown for 60 h on MS medium containing 150 mM, overexpressed GhGPX5 and GhGPX13 The germination rate of GhGPX5 overexpressed seeds was about 30%, while that of WT seeds was about 5%. The germination rate was less than 20% (Fig. 8D).

These results suggest that overexpression of GhGPX5 and GhGPX13 in Arabidopsis can promote seed germination under high-salt stress. Observing the seeds after 8 days of growth, it was found that under different concentrations of salt treatment, the growth of the seedlings overexpressing GhGPX5 and GhGPX13 was significantly better than that of WT, showing that their growth was not sensitive to high-salt stress compared with WT (Figure 8E).

In addition, 25-day-old Arabidopsis WT and transgenic plants overexpressing GhGPX5 and GhGPX13 were treated with an aqueous solution containing 200 mM NaCl. It was found that after 7 days of high-salt stress, in the control group (water treatment without NaCl), overexpression Transgenic plants expressing GhGPX5 and GhGPX13 showed no significant difference from WT, while high-salt stress significantly inhibited the growth of transgenic plants overexpressing GhGPX5 and GhGPX13 and WT, showing smaller rosette leaves, and the leaves of WT showed obvious yellowing Phenomenon, compared with WT, transgenic plants overexpressing GhGPX5 and GhGPX13 did not appear yellow leaves (Fig. 9), showing stronger high salt tolerance.

In summary, the salt stress tolerance of cotton decreased after silencing the GhGPX5/13 gene, indicating that the GhGPX5/13 gene positively regulates the tolerance of cotton to high salt stress; while overexpressing Arabidopsis, its salt stress tolerance increased, It was also shown that the GhGPX5/ 13 gene positively regulates the tolerance of Arabidopsis to high-salt stress. It can be seen that the GhGPX5/13 gene plays an important role in regulating high-salt stress in plants, and is of great significance for breeding cotton varieties that can resist external stress conditions.

The embodiments described above are only preferred embodiments of the present invention, and are only used to explain the present invention, not to limit the implementation scope of the present invention. Technical content, other implementation modes can be easily made through replacement or change, so all changes and improvements made on the principle of the present invention should be included in the patent scope of the present invention.

Claims (4)

1.GhGPX5AndGhGPX13use of a gene for increasing salt stress tolerance in plants, characterized in that the gene comprisesGhGPX5AndGhGPX13the gene sequence numbers of the genes in NCBI are XM_041083558.1 and XM_016881552.2 respectively, by constructingGhGPX5AndGhGPX13and (3) over-expressing the vector to obtain a transgenic plant with high salt stress tolerance, wherein the plant is cotton or Arabidopsis thaliana.

2. The use of claim 1, wherein the salt stress tolerance is a high concentration salt stress tolerance, the high concentration salt concentration being 400 mM.

3. The use according to claim 1, wherein the salt stress tolerance is manifested as: under the stress of salt, the water-soluble polymer is subjected to the stress of salt,GhGPX5andGhGPX13leaf wilting and yellowing of the gene silencing lines are higher than that of wild typeGhGPX5AndGhGPX13the seed germination rate of the over-expression strain is higher than that of the wild type, and the yellowing degree of the leaf blade is lower than that of the wild type.

4. A plant breeding method characterized in that the method is (1) or (2) below:

(1) By promoting the growth of the target plantsGhGPX5AndGhGPX13the expression of the gene can obtain a plant with salt stress tolerance stronger than that of the target plant;

(2) By inhibition in plants of interestGhGPX5AndGhGPX13the expression of the gene, obtaining a plant with salt stress tolerance lower than that of the target plant;

the saidGhGPX5AndGhGPX13the gene sequence numbers of the genes in NCBI are XM_041083558.1 and XM_016881552.2 respectively, and the target plant is cotton or Arabidopsis thaliana.

Images