Application of GhPEP72 gene in regulation and control of plant salt tolerance

Abstract

The invention belongs to the technical field of plant breeding, and particularly relates to application of a GhPEP72 gene in regulation and control of plant salt tolerance. The nucleotide sequence of the GhPEP72 gene provided by the invention is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein expressed by the GhPEP72 gene is as shown in SEQ ID NO. 2. A research result proves that the growth inhibition of a GhPEP72 gene silencing plant under salt stress treatment is more obvious, the salt damage characteristic is more obvious, sodium ion accumulation in roots, stems and leaves is increased, the potassium ion content is reduced, the ratio of sodium ions to potassium ions in the leaves is increased, and the GhPEP72 gene silencing plant is more sensitive to salt stress; compared with wild arabidopsis thaliana under salt stress treatment, the overground part of a GhPEP72 gene overexpression transgenic homozygous plant grows more vigorously, the number of lateral roots of the underground part is larger, the lateral roots are longer, and the GhPEP72 gene has good stress resistance, so that the GhPEP72 gene plays a positive regulation role in regulating plant salt stress.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically involving GhPEP72 Application of genes in regulating plant salt tolerance. Background Technology

[0002] Saline-alkali land, a widely distributed soil type globally, is mainly concentrated in arid and coastal areas. As a crucial raw material for the textile industry, cotton cultivation is gradually expanding into saline-alkali land to address the dwindling availability of high-quality arable land. However, cotton is not a halophyte, and its salt tolerance is relatively limited. With increasing soil salinity, cotton growth suffers severe salt stress, manifesting as stunted growth, reduced yield, and deterioration of fiber quality. Currently, research on the mechanisms of cotton salt tolerance is relatively lacking, particularly at the molecular level.

[0003] The specific phenotypic manifestations of salt stress threatening cotton growth include: stunted growth, manifested as dwarf plants and reduced, smaller leaves; yellowing of the plant, due to high salt content affecting cellular water absorption and nutrient transport; delayed flowering, reduced flower count, and decreased seed setting rate, significantly impacting cotton yield; and, most importantly, significantly shortening root length, weakening the root system's ability to absorb water and nutrients, further hindering overall cotton growth and yield formation. Therefore, further research is necessary when planting cotton in saline-alkali land to mitigate the adverse effects of salt stress on cotton growth and yield, ensuring the sustainable development of the cotton industry.

[0004] Plant peptides are a novel type of plant peptide growth regulator. Small peptides in plants participate in various processes, including plant growth and development, immune responses, and responses to abiotic stresses. Recent studies have revealed that peptide hormones play important regulatory roles in stress resistance. For example, in Arabidopsis thaliana, when subjected to drought stress, CLE25, a member of the CLE peptide hormone family, is synthesized in large quantities in the roots and transported upwards, accelerating abscisic acid synthesis and regulating stomatal activity to reduce water loss. In Arabidopsis thaliana, overexpression of CLE25... AtPROPEP3 These peptides can significantly improve the plant's tolerance to high-salt environments. However, to date, there are few reports on the relationship between small peptides and cotton's salt stress tolerance, and the functional roles of many new small peptides have not yet been well annotated. Therefore, in-depth research at the molecular level on the response mechanism of cotton peptide hormones to salt stress will help provide excellent targets for regulating cotton salt tolerance, and has important practical significance for improving the utilization efficiency of saline-alkali land and promoting the sustainable development of the cotton industry. Summary of the Invention

[0005] To solve the problems in existing technologies GhPEP72 The present invention addresses the issue that the function of cotton salt tolerance has not been reported. GhPEP72Application of genes in regulating plant salt tolerance. To achieve the above objectives, the present invention adopts the following technical solution.

[0006] The first aspect of the present invention provides GhPEP72 The application of genes in regulating plant salt tolerance, the aforementioned GhPEP72 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] This invention discloses for the first time GhPEP72 This invention provides a positive regulatory effect on salt tolerance in cotton, and offers excellent regulatory targets and theoretical guidance for salt-tolerant cotton cultivation. GhPEP72 The application of genes in regulating plant salt tolerance solves the problems in existing technologies. GhPEP72 The function of cotton in salt tolerance has not yet been reported.

[0008] Preferably, the GhPEP72 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2.

[0009] Preferably, the aforementioned [material] in plants is generated through genetic engineering methods. GhPEP72 Gene overexpression to improve plant salt tolerance.

[0010] Preferably, the genetic engineering method is as follows: using the GhPEP72 Genes are constructed into recombinant expression vectors, which are then introduced into plants to improve their salt tolerance.

[0011] Preferably, the method for introducing the recombinant expression vector into plants includes direct DNA transformation via any one of the Ti plasmid, Ri plasmid, and plant virus vector; or introducing the recombinant expression vector into plants via any one of microinjection, electroporation, and Agrobacterium-mediated transformation. This invention clones cotton. GhPEP72 Genes, and constructed using transgenic technology. GhPEP72 Transgenic Arabidopsis plants overexpressing the virus and constructing a transgenic Arabidopsis using VIGS technology GhPEP72 Silent plants, for GhPEP72 Functional verification was conducted, and the following was clarified. GhPEP72 It can improve the salt tolerance of plants. This facilitates a deeper study of the response mechanisms of plants to salt stress signals, lays a solid molecular foundation for effectively improving plant salt tolerance, and is of great value in exploring the signal regulatory network of plants under salt stress.

[0012] The second aspect of the present invention provides a product containing the aforementioned GhPEP72 Recombinant gene expression vectors.

[0013] Preferably, the recombinant expression vector is used to express the... GhPEP72 DNA fragment insertion of a gene uper1300- GFP carrier XbaI Obtained from a single enzyme digestion site.

[0014] In this process, plant RNA is reverse transcribed to obtain template DNA, and the template DNA is then amplified by PCR using primer pairs shown in SEQ ID NO. 3 and SEQ ID NO. 4 to obtain the template DNA. GhPEP72 A segment of a gene's DNA.

[0015] The primer in the primer pair shown in SEQ ID NO.3 is the upstream primer F1, and its nucleotide sequence is shown below: 5'-TACACCAAATCGACTCTAGAATGGCAGAGCTTTGCTTCTC-3'.

[0016] As shown in SEQ ID NO.4, the primer in the primer pair is the downstream primer R1, and its nucleotide sequence is shown below: 5'-ATAGGTACCCGGGCTCTAGAAATGTACTCAACCCAGTGCTTC-3'.

[0017] Furthermore, containing the aforementioned GhPEP72 The method for constructing a recombinant gene expression vector includes the following steps: Amplification was performed using the correctly sequenced PGADT7-GhPEP72 plasmid as a template.

[0018] The obtained amplification product was digested with two enzymes and then combined with... XbaI single The enzyme-digested plant binary transformation vector Super1300-GFP was ligated, transformed, and the positive clone plasmids that were verified to be correct were extracted and used as the recombinant expression vector 35S::GhPEP72-GFP.

[0019] Among them, based on the multiple cloning site of the plant binary transformation vector super1300-GFP and the aforementioned GhPEP72 The coding region sequence of the gene was designed to amplify the aforementioned GhPEP72 The forward primer F1 and the reverse primer R1 for the entire coding region of the gene were used to obtain the 35S::GhPEP72-GFP recombinant expression vector. The specific construction method is as follows: As stated GhPEP72 Using the gene as a template, PCR amplification was performed using forward and reverse primers to obtain the gene containing the specified gene. GhPEP72 The product of genes; utilization XbaI Enzymatic digestion includes the above GhPEP72The gene product and super1300 were digested to obtain enzyme digestion products and vector frameworks, which were then recovered. The enzyme digestion products and vector frameworks were ligated to obtain the recombinant expression vector 35S::GhPEP72-GFP, which is the expression vector for super1300. XbaI The DNA fragments between the restriction enzyme sites are replaced with those described in SEQ ID NO.1. GhPEP72 The vector was obtained by reproducing the gene while keeping the other sequences of super1300 unchanged.

[0020] Among them, the plant binary transformation vector super1300-GFP is a recombinant expression vector linking the GFP gene. This recombinant expression vector is recorded in the doctoral dissertation "Metabolic characteristics of Cuscuta chinensis during in vitro growth and study of Agrobacterium tumefaciens mis gene in its genome, Zhang Yuexia, China Agricultural University, 2020". With the author's consent, the public can obtain it from the Li Zhaohu research group of China Agricultural University to repeat the experiments in this invention, but it cannot be used for other purposes. In this invention, it is referred to as "super1300".

[0021] The structure of the recombinant expression vector 35S::GhPEP72-GFP is as follows: Figure 3 As shown.

[0022] A third aspect of the present invention provides a method for improving the salt tolerance of plants, such that the salt tolerance of plants is... GhPEP72 Gene overexpression; and / or enhancement of the gene expression in plants as described above GhPEP72 The activity of the gene-expressed protein; and / or the enhancement of the activity of the protein described in the plant. GhPEP72 The level of proteins expressed by genes is adjusted to improve the salt tolerance of plants.

[0023] In the above method, increasing the expression level of the gene for protein GhPEP72 and / or the activity and / or content of protein GhPEP72 in the target plant means overexpressing protein GhPEP72 in the plant.

[0024] Preferably, the plant contains the GhPEP72 Gene overexpression involves introducing the recombinant expression vector into plants via Agrobacterium-mediated transformation to improve the plants' salt tolerance.

[0025] Preferably, the plant is a monocotyledonous or dicotyledonous plant. More preferably, the plant is a plant of the Brassicaceae and / or Malvaceae families. Even more preferably, the plant is a plant of the genus *Gossypium* and / or *Arabidopsis*. Most preferably, the plant is *Arabidopsis* and / or cotton.

[0026] A fourth aspect of the present invention provides a method for breeding salt-tolerant plants, wherein the plant contains... GhPEP72 Overexpression of the gene yields salt-tolerant transgenic plants.

[0027] The plant in question is M1, M2, M3, M4, or M5.

[0028] M1) Monocotyledons or dicotyledons.

[0029] M2) Brassicaceae (Fructus Cruciferae).

[0030] Arabidopsis thaliana M3.

[0031] M4) Plants of the genus Gossypium.

[0032] M5) Cotton.

[0033] The fifth aspect of the present invention provides a protein, named cotton polypeptide GhPEP72, or simply GhPEP72 protein, derived from cotton, and is any of the following proteins: A1) Contains or is a protein with the amino acid sequence SEQ ID NO.2.

[0034] A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.2.

[0035] A3) A protein that has more than 90% identity and function with the protein shown in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0036] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0037] In the aforementioned proteins, a protein tag refers to a polypeptide or protein that is fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracking, or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag.

[0038] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0039] In the aforementioned proteins, the 90% or more identity can be at least 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0040] Biomaterials related to the aforementioned GhPEP72 protein are also within the scope of protection of this invention, and this invention also provides new uses for biomaterials related to the GhPEP72 protein.

[0041] This invention relates to the application of biomaterials related to the GhPEP72 protein in regulating plant stress resistance, wherein the biomaterials are any of the following: C1) is a nucleic acid molecule that encodes the GhPEP72 protein.

[0042] C2) An expression cassette containing the nucleic acid molecule described in C1).

[0043] C3) A recombinant expression vector containing the nucleic acid molecule described in C1), or a recombinant expression vector containing the expression cassette described in C2).

[0044] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant expression vector described in C3).

[0045] C5) A transgenic plant cell line containing the nucleic acid molecule described in C1), or a transgenic plant cell line containing the expression cassette described in C2), or a transgenic plant cell line containing the recombinant expression vector described in C3).

[0046] C6) Transgenic plant tissue containing the nucleic acid molecule described in C1), or transgenic plant tissue containing the expression cassette described in C2), or transgenic plant tissue containing the recombinant expression vector described in C3).

[0047] C7) A transgenic plant organ containing the nucleic acid molecule described in C1), or a transgenic plant organ containing the expression cassette described in C2), or a transgenic plant organ containing the recombinant expression vector described in C3).

[0048] C8) A transgenic plant containing the nucleic acid molecule described in C1), or a transgenic plant containing the expression cassette described in C2), or a transgenic plant containing the recombinant expression vector described in C3).

[0049] C9) Tissue cultures produced from the regenerative cells of the transgenic plant described in C8).

[0050] C10) Protoplasts produced from the tissue culture described in C9).

[0051] C11) A recombinant expression vector or recombinant microorganism that inhibits the expression level of the gene for the GhPEP72 protein and / or inhibits the activity of the GhPEP72 protein and / or reduces the content of the GhPEP72 protein.

[0052] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA. The nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0053] In the above-mentioned biological materials, the nucleic acid molecule described in C1) is any one of the following: B1) The DNA molecule shown in SEQ ID NO.1.

[0054] B2) The coding sequence is the DNA molecule shown in SEQ ID NO.1.

[0055] B3) DNA molecules that hybridize under strict conditions with DNA molecules defined by B1) or B2) and encode the GhPEP72 protein.

[0056] The above stringent conditions were: hybridization in a 2×SSC, 0.1% SDS solution at 68°C with two washes of 5 min each, followed by hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C with two washes of 15 min each.

[0057] In the aforementioned biological materials, the expression cassette described in C2) refers to DNA capable of expressing the GhPEP72 protein in host cells, and this DNA may include not only the promoter but also... GhPEP72 The promoter of gene transcription may also include a terminator. GhPEP72 Transcription terminator.

[0058] Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: the... GhPEP72Gene promoters themselves, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by jasmonic acid methyl ester); heat shock promoters (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (Chinese Patent CN101063139B); and promoters specific to seed storage proteins (e.g., beta-glucan, napin, oleosin, and soybean beta-glucan). The promoters of conglycin (Beach et al. (1985) EMBO J. 4:3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: GhPEP72 Terminators of genes themselves, Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tmL terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example, Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev, 5:141; Mogen et al. (1991) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627.

[0059] In the aforementioned biological materials, the recombinant expression vector described in C3) may contain the DNA molecule encoding the protein GhPEP72 as shown in SEQ ID NO.1. Recombinant expression vectors containing the gene for the GhPEP72 protein or the gene expression cassette of the GhPEP72 protein can be constructed using existing plant expression vectors. The plant expression vector may be a Gateway system vector or a binary expression vector, such as super1300, pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. GhPEP72 When constructing recombinant expression vectors, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0060] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0061] In a specific embodiment of the present invention, the recombinant expression vector in C3) is 35S:: GhPEP72 -GFP recombinant expression vector, the 35S:: GhPEP72 -GFP recombinant expression vector will express super1300 XbaI single The DNA fragment between the restriction enzyme sites is replaced with that of SEQ ID NO.1. GhPEP72 The vector was obtained by reproducing the gene while keeping the other sequences of super1300 unchanged.

[0062] In the above-mentioned biological materials, the recombinant expression vector described in C11) is the recombinant expression vector pYL156- GhPEP72 The pYL156- GhPEP72 For the pYL156 vector EcoR I and Kpn The recombinant expression vector was obtained by replacing the DNA fragment between the I restriction sites with the DNA molecule shown in positions 1-240 of SEQ ID NO.1, while keeping the other sequences of the pYL156 vector unchanged.

[0063] In the above-mentioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi; for example, the bacteria may be Agrobacterium GV3101.

[0064] In the aforementioned biological materials, the transgenic plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0065] In the above-mentioned biological materials, the tissue culture can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0066] Among the aforementioned biological materials, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs do not include propagation materials.

[0067] The present invention further provides a product for regulating plant stress resistance, containing the above-mentioned GhPEP72 protein or related biological materials.

[0068] The application of the aforementioned GhPEP72 protein or related biological materials in any of the following is also within the scope of protection of this invention: D1) Application in the cultivation of transgenic plants with enhanced stress resistance.

[0069] D2) Application in the preparation and cultivation of transgenic plant products with enhanced stress resistance.

[0070] Application of D3 in the cultivation of gene-silenced plants with reduced stress resistance.

[0071] D4) Application in the preparation of gene-silenced plant products with reduced stress resistance.

[0072] Application of D5 in plant breeding.

[0073] In the above applications, the specific application in plant breeding can be the use of biological materials containing the GhPEP72 protein or related materials (such as the gene encoding the GhPEP72 protein). GhPEP72 Plants are hybridized with other plants for plant breeding.

[0074] Compared with the prior art, the present invention has the following beneficial effects: This invention provides GhPEP72The application of genes in regulating plant salt tolerance. The results of this invention confirm... GhPEP72 Gene-silenced plants (especially cotton) showed more pronounced growth inhibition and salt damage characteristics under salt stress treatment. They exhibited increased sodium ion accumulation and decreased potassium ion accumulation in roots, stems, and leaves, with a higher sodium-to-potassium ion ratio in leaves, making them more sensitive to salt stress. Furthermore, compared to wild-type Arabidopsis, they showed greater sensitivity to salt stress under salt stress treatment. GhPEP72 The homozygous transgenic plants with overexpressed genes exhibited more vigorous aboveground growth, more numerous and longer lateral roots, and better resistance to adverse conditions, thus indicating... GhPEP72 Genes play a positive regulatory role in regulating salt stress in plants. This invention reveals for the first time... GhPEP72 This invention provides a positive regulatory effect on salt tolerance in cotton, and offers excellent regulatory targets and theoretical guidance for salt-tolerant cotton cultivation. GhPEP72 The application of genes in regulating plant salt tolerance solves the problems in existing technologies. GhPEP72 The function of cotton in salt tolerance has not yet been reported. Attached Figure Description

[0075] Figure 1 Phylogenetic tree of the cotton GhPEP72 family.

[0076] Figure 2 As described in the embodiments of the present invention GhPEP72 Agarose gel electrophoresis image of the gene amplification product.

[0077] Figure 3 The recombinant expression vector 35S:: described in the embodiments of the present invention GhPEP72 Schematic diagram of the structure of -GFP.

[0078] Figure 4 This is a subcellular localization map of the GhPEP72 protein described in this embodiment of the invention.

[0079] Figure 5 As described in the embodiments of the present invention GhPEP72 Agarose gel electrophoresis image of the amplification products from positions 1 to 240 of the gene.

[0080] Figure 6 VIGS- in the embodiments of the present invention GhPEP72 The silencing efficiency and salt stress phenotype of silent plants; among them, Figure 6 Figure (A) in the figure is described in an embodiment of the present invention. GhPEP72 Figure showing the results of gene silencing efficiency assessment in cotton; Figure 6 Figure (B) in the figure is as described in the embodiment of the present invention. GhPEP72 The growth phenotype results of the gene-silenced plant in verifying its stress resistance are shown in the figure.

[0081] Figure 7 As described in the embodiments of the present invention GhPEP72 A schematic diagram showing the sodium ion content in the leaves, stems, and roots of gene-silenced plants; among them, Figure 7 Figure (A) shows VIGS after salt treatment. GF P and VIGS- GhPEP72 Sodium ion content in the root; Figure 7 Figure (B) shows VIGS after salt treatment. GFP and VIGS- GhPEP72 Potassium ion content in roots; Figure 7 Figure (C) shows VIGS after salt treatment. GF P and VIGS- GhPEP72 The ratio of sodium ion content to potassium ion content in the root; Figure 7 Figure (D) shows VIGS after salt treatment. GF P and VIGS- GhPEP72 Sodium ion content in the stem; Figure 7 Figure (E) shows VIGS after salt treatment. GFP and VIGS- GhPEP72 Potassium ion content in the stem; Figure 7 Figure (F) shows VIGS after salt treatment. GF P and VIGS- GhPEP72 The ratio of sodium ion content to potassium ion content in the stem; Figure 7 The (G) diagram shows VIGS after salt treatment. GF P and VIGS- GhPEP72 Sodium ion content in leaves; Figure 7 The (H) diagram shows VIGS after salt treatment. GFP and VIGS- GhPEP72 Potassium ion content in leaves Figure 7 Figure (I) shows VIGS after salt treatment. GFP and VIGS- GhPEP72 The ratio of sodium ion content to potassium ion content in leaves.

[0082] Figure 8 As described in the embodiments of the present invention GhPEP72 Western blotting results of homozygous transgenic Arabidopsis thaliana overexpression lines.

[0083] Figure 9 As described in the embodiments of the present invention GhPEP72 A comparison of the growth of homozygous overexpressing transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana under salt stress conditions; among them, Figure 9 The plants in the image, from left to right, are wild-type Arabidopsis thaliana (WT). GhPEP72 Overexpression of transgenic Arabidopsis homozygous line OE1, GhPEP72OE2, a homozygous transgenic Arabidopsis thaliana line, was overexpressed.

[0084] Figure 10 As described in the embodiments of the present invention GhPEP72 A schematic diagram showing the statistical results of the number of lateral roots of overexpressing transgenic Arabidopsis homozygous lines and wild-type Arabidopsis under salt stress conditions. Detailed Implementation

[0085] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0086] Virus-induced gene silencing, as an effective reverse genetics technique, is widely used for identifying plant genome function. Virus-induced gene silencing can successfully silence endogenous genes in different parts of the plant, providing a practical means to study gene function at different growth stages. In the embodiments of this invention, a virus-induced gene silencing method is provided... GhPEP72 Gene-silencing plants are used to demonstrate the function of a gene. Virus-induced gene silencing (VIGS) is a technique used to study gene silencing.

[0087] This invention confirms GhPEP72 Silent cotton plants exhibited more pronounced growth inhibition and salt damage characteristics under salt stress treatment, with increased sodium ion accumulation in leaves and roots, making them more sensitive to salt stress. Furthermore, under salt stress treatment... GhPEP72 Overexpressing homozygous transgenic Arabidopsis plants had more lateral roots than wild-type Arabidopsis and exhibited better stress resistance, thus indicating... GhPEP72 Genes play a positive regulatory role in regulating salt stress in plants.

[0088] Therefore, an embodiment of the first aspect of the present invention proposes an application of the GhPEP72 gene in regulating plant salt tolerance. Example 1: GhPEP72 The discovery, cloning, localization, and... GhPEP72 Construction of overexpression recombinant vector 1.1 GhPEP72 The discovery of genes This embodiment screened for salt-related genes in the VIGS cDNA library using a cotton database. A protein was obtained from the cotton variety "Xinshi 17" and named GhPEP72 protein. The amino acid sequence of the GhPEP72 protein is shown in SEQ ID NO.2.

[0089] MAELCFSKGIMIMFVAVVLSLTTTASAKDSAMAPAPSMDTGAAFSPAVSG IAVMFSLIVSLVALLKHWVEYI.

[0090] The gene encoding the GhPEP72 protein mentioned above is named GhPEP72 The gene, whose open reading frame nucleotide sequence is shown in SEQ ID NO.1: ATGGCAGAGCTTTGCTTCTCTAAAGGCATTATGATCATGTTTGTTGCCGTTGTGCTCTCACTCACCACCACTGCATCAGCTAAGGACAGTGCAATGGCGCCTGCGCCGTCAAATGGATACCGGAGCTGCTTTCTCGCCGGCGGTTTCCGGAATTGCGGTTATGTTCTCTCTGATTGTTTCCCTTGTTGCGCTTTTGAAGCA CTGGGTTGAG TACATTTGA.

[0091] The screening method can be found in "Study on the function of cotton stress resistance genes using VIGS technology, Li Fangjun, China Agricultural University, 2014".

[0092] For details on the cotton variety "Guoxin No. 3", please refer to "Cultivation Techniques of Insect-Resistant Cotton No. 3", Feng Sulian, Hebei Agriculture, 2011, 06.

[0093] 1.2 GhPEP72 Cloning of genes This embodiment uses reverse transcription PCR to... GhPEP72 The gene was cloned and amplified, confirmed by electrophoresis, and the obtained clone was further sequenced.

[0094] (1) RNA was extracted from cotton leaves using the Adley kit and first-strand cDNA was synthesized using the M-mLV reverse transcription kit. The obtained first-strand cDNA was used as a template for amplification. GhPEP72 Full-length gene.

[0095] The Adley reagent kit was purchased from Beijing Adley Biotechnology Co., Ltd. The M-mLV reverse transcription kit was purchased from Takara.

[0096] (2) Design two specific primers: upstream primer F1 and downstream primer R1, perform PCR amplification, and obtain PCR products.

[0097] Among them, the Planta® Max Super-Fidelity DNA Polymerase kit from Novizan was used to amplify... GhPEP72 Full-length gene.

[0098] The nucleotide sequence of upstream primer F1 is shown in SEQ ID NO.3: 5'-TACACCAAATCGACTCTAGAATGGCAGAGCTTTGCTTCTC-3'.

[0099] The nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO.4: 5'-ATAGGTACCCGGGCTCTAGAAATGTACTCAACCCAGTGCTTC-3'.

[0100] The PCR amplification reaction system is shown in Table 1: Table 1 PCR reaction system The PCR amplification reaction procedure is shown in Table 2: Table 2 PCR amplification program (3) The PCR amplification products were subjected to electrophoresis on a 1.5% agarose gel. The agarose gel electrophoresis results are as follows: Figure 2 As shown.

[0101] (4) After electrophoresis, the target band was cut under UV light and purified using an agarose gel DNA recovery kit. The agarose gel DNA recovery kit was purchased from Kangwei Biotechnology Co., Ltd.

[0102] (5) The recovered fragments were recombined with the pGADT7 vector. The recombination reaction system is shown in Table 3. The reaction was carried out at 37℃ for 30 min.

[0103] Table 3 Connection Reaction System Note: " / " indicates that this item is not present.

[0104] (6) Take 5 μL of the ligation product and transform it into Escherichia coli DH5α by heat shock method. Screen positive clones in LB solid plates containing 50 mg / L ampicillin. The heat shock method is described in "J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition), Science Press, 2002".

[0105] (7) Select 5 clones and send them to Beijing Qingke Sequencing Company for sequencing to obtain the required full-length gene CDS, thus obtaining the full-length gene CDS. GhPEP72 Genes. Cotton GhPEP72 Family system evolution tree such as Figure 1 As shown.

[0106] Sequencing results showed that... GhPEP72 The gene sequence is 240 bp in length and encodes a complete ORF reading frame of 80 amino acids.

[0107] 1.4 GhPEP72 Subcellular localization of genes Tobacco epidermal cells were used to study the subcellular localization of the GhPEP72 protein.

[0108] The expression will have super100- GhPEP72 Agrobacterium with the GFP plasmid was cultured overnight in 10 ml of YEP liquid medium containing 50 mg / ml Kan and 25 mg / ml Gen. The culture was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended to OD=0.6. The resuspended bacterial solution was injected into tobacco leaves. Two days later, the injected tobacco leaves were observed under a laser confocal microscope; the excitation light for GFP was 488 nm.

[0109] Figure 4 This is a subcellular localization map of the GhPEP72 protein described in this embodiment of the invention. It shows that GhPEP72 is located around the cell membrane.

[0110] Example 2: Obtaining by the VIGS method GhPEP72 Silent plants and their phenotypic characteristics under salt stress This embodiment constructs VIGS- using the VIGS method. GhPEP72 Silent carrier, further utilizing this VIGS- GhPEP72 Silent carrier acquisition GhPEP72 Silent plants were identified, and their phenotypes under salt stress were investigated.

[0111] 2.1 VIGS- GhPEP72 Construction of silent carriers (1) Total RNA was extracted from the leaves of the cotton variety “Guoxin No. 3” according to the steps in Example 1 and reverse transcribed into cDNA.

[0112] (2) Using the cDNA obtained in step (1) as a template, perform PCR amplification with the following primer pair to obtain the PCR amplification product.

[0113] The nucleotide sequence of primer F1 is shown in SEQ ID NO.5: 5'-gtgagtaaggttaccgaattcATGGCAGAGCTTTGCTTCTC-3'. Where the underscores in the sequence represent... EcoR I restriction enzyme site.

[0114] The nucleotide sequence of primer R1 is shown in SEQ ID NO.6: 5'-gagacgcgtgagctcggtaccAATGTACTCAACCCAGTGCTTC-3'. The underscores in the sequence represent... Kpn I restriction enzyme site.

[0115] (3) Use restrictive internal incision EcoR I and Kpn The PCR amplification product obtained from the double enzyme digestion step (2) was recovered.

[0116] (4) Using restriction endonucleases EcoR I and Kpn The pYL156 vector was digested with two enzymes, and the vector backbone was recovered. The pYL156 vector is also known as the pTRV2:RNA2 vector, which is described in the non-patent literature "Gao X, 2013, Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced genesilencing."

[0117] (5) Ligate the enzyme digestion product from step (3) with the vector backbone from step (4) to obtain the recombinant plasmid pYL156- GhPEP72 The connection system is described in the steps outlined in the embodiments.

[0118] (6) For the recombinant vector pYL156- GhPEP72 Sequencing verification was performed.

[0119] Sequencing results showed that the recombinant plasmid pYL156- GhPEP72 To transport the pYL156 vector EcoR I and Kpn The DNA fragment between the I restriction sites is replaced with the portion shown in positions 1-240 of SEQ ID NO.1. GhPEP72 The vector obtained by separating the gene fragments and keeping other sequences of the pYL156 vector unchanged is called VIGS- GhPEP72 A silent vessel. GhPEP72 The agarose gel electrophoresis image of the amplification products from positions 1 to 240 of the gene is shown below. Figure 5 As shown.

[0120] Figure 6 Figure (A) in the figure is described in an embodiment of the present invention. GhPEP72 Results of gene silencing efficiency assessment in cotton.

[0121] Depend on Figure 6 It can be seen that VIGS- GhPEP72 Plants and VIGS- GFP Compared to other plants, GhPEP72 Genes have indeed been silenced.

[0122] 2.2 VIGS- GhPEP72 Obtaining Silent Plants (1) pYL156- GhPEP72 pYL156-GFP, pTRV-RNA1, and pYL156-GhCLA1 were electroporated to transform Agrobacterium GV3101, resulting in recombinant strains pYL156-GhPEP72 / GV3101, pYL156-GFP / GV3101, pTRV1 / GV3101, and pYL156-GhCLA1 / GV3101.

[0123] Among them, pYL156- GhPEP72 This refers to the VIGS constructed in step 2.1 of this embodiment. GhPEP72 A silent vessel.

[0124] pYL156-GFP, pTRV1 (pTRV-RNA1), and pYL156-GhCLA1 are all described in the non-patent literature “Gao X, 2013, Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.”

[0125] (2) The recombinant bacteria were cultured in LB liquid medium containing antibiotics at 28°C in a shaker.

[0126] The formulation of the LB liquid culture medium containing antibiotics is as follows: 50 μg / mL kanamycin, 25 μg / mL gentamicin, 10 mM MES at pH 5.6, 20 μM acetylsuccinone, and water as the solvent.

[0127] (3) Resuspend the recombinant bacteria in VIGS solution and adjust the bacterial concentration to OD. 600 =1.5.

[0128] The VIGS solution was formulated as follows: 10 mM MES, 10 mM MgCl2, and 200 μM acetylsuccinone at pH 5.6, with water as the solvent. (4) Mix the specific concentrations of recombinant bacteria pYL156-GhPEP72 / GV3101, pYL156-GFP / GV3101 and pYL156-GhCLA1 / GV3101 with the bacterial solution of recombinant bacteria pTRV1 / GV3101 at a volume ratio of 1:1 to obtain mixture 1, mixture 2 and mixture 3.

[0129] (5) Using a 1mL needleless syringe, spray the lower surface of the cotyledons of different cotton plants, specifically "Xinshi 17", with mixtures 1, 2, and 3. Incubate for 2 weeks to obtain VIGS- GhPEP72 Silent plants, VIGS-GFP control plants, and VIGS-GhCLA1 indicator plants.

[0130] (6) Approximately two weeks after the VIGS-GhCLA1 indicator plants injected with mixture 3 showed an albino phenotype, RNA was extracted from leaf tissues of plants injected with mixture 1 and mixture 2, and cDNA was reverse transcribed. Gene silencing efficiency was analyzed by real-time quantitative PCR, and relative expression levels were calculated using a 2-1... -ΔΔCt Method calculation.

[0131] The primer pairs and PCR procedure used are shown below: The nucleotides of primer F2 are shown in SEQ ID NO.7: 5'-TGTGCTCTCACTCACCACC-3'.

[0132] The nucleotides of primer R2 are shown in SEQ ID NO. 8: 5'-GCAACAAGGGAAACAATCAG-3'.

[0133] PCR program: 94℃ denaturation for 30s; 94℃ denaturation for 5s, 60℃ annealing for 35s, 40 cycles.

[0134] Among them, cotton Actin9 Genes were used as a control to identify cotton. Actin9 The primer pairs for the gene are as follows: The nucleotides of primer F3 are shown in SEQ ID NO. 9: 5'-GCCTTGGACTATGAGCAGGA-3'.

[0135] The nucleotides of primer R3 are shown in SEQ ID NO.10: 5'-AAGAGATGGCTGGAAGAGGA-3'.

[0136] Figure 6 Figure (A) in the figure is described in an embodiment of the present invention. GhPEP72 The results of gene silencing efficiency assessment in cotton.

[0137] Figure 6 The results in Figure (A) show that the plants injected with mixture 1... GhPEP72 The gene expression level was significantly lower than that of plants injected with mixture 2, indicating that silencing was successfully achieved using the above method. GhPEP72VIGS of genes GhPEP72 Silent plants and VIGS-GFP control plants.

[0138] Mixture 1 contains bacterial cultures of pYL156-GhPEP72 / GV3101 and pTRV1 / GV3101.

[0139] Mixture 2 contains bacterial cultures of pYL156-GFP / GV3101 and pTRV1 / GV3101.

[0140] 2.3 VIGS- GhPEP72 Phenotypic Study of Silent Plants under Salt Stress (1) The VIGS obtained in step 2.2 of this embodiment GhPEP72 Silent plants and VIGS-GFP control plants were subjected to 250 mM NaCl stress treatment, and their growth phenotypes were observed.

[0141] The stress treatment method is as follows: when the cotton reaches the two-leaf stage, treat VIGS- with 250mM NaCl. GhPEP72 Silent plants and VIGS-GFP control plants.

[0142] (2) Regarding the VIGS obtained in step 2 of this embodiment. GhPEP72 Silent plants and VIGS-GFP control plants were subjected to 250 mM NaCl stress treatment, and leaves and roots of the plants were collected to detect the sodium ion content in the leaves and roots.

[0143] Figure 6 Figure (B) in the figure is as described in the embodiment of the present invention. GhPEP72 The growth phenotype results of VIGS- gene-silenced plants verifying their stress resistance are shown in the figure. The results indicate that under salt stress, VIGS- GhPEP72 Compared with the VIGS-GFP control plants, the silent plants showed more significant inhibition of plant growth, more pronounced salt damage characteristics such as wilting and curling of leaf margins, and were more sensitive to salt stress; in the control group without NaCl treatment, VIGS-GFP... GhPEP72 There was no difference in growth characteristics between the silent plants and the VIGS-GFP control plants.

[0144] Figure 7 As described in the embodiments of the present invention GhPEP72 A schematic diagram showing the sodium ion content in the leaves and roots of gene-silenced plants. The results indicate that VIGS- GhPEP72 Compared to the VIGS-GFP control plants, the silent plants showed increased sodium ion accumulation and decreased potassium ion content in the roots, stems, and leaves, and an increased sodium to potassium ion ratio in the leaves, making them more sensitive to salt stress.

[0145] Based on the above results, VIGS gene silencing technology can be used in cotton to... GhPEP72 Gene expression levels were significantly reduced, with a gene silencing efficiency of 61.2%; compared with the VIGS-GFP control plants, VIGS- GhPEP72 Silent plants showed more pronounced growth inhibition and salt damage characteristics under salt stress treatment, with increased sodium ion accumulation in leaves and roots, making them more sensitive to salt stress.

[0146] The above experimental results show that: GhPEP72 Genes play a positive regulatory role in cotton salt stress and other conditions.

[0147] Example 3: GhPEP72 Construction of overexpression vectors, acquisition of overexpression transgenic plants and their phenotypic study under salt stress 3.1 GhPEP72 Construction of overexpression vectors (1) with GhPEP72 Using the gene as a template, PCR amplification was performed using forward and reverse primers to obtain a sample containing... GhPEP72 For the amplification of the gene product, please refer to Example 1. The forward primer is the upstream primer F1; the reverse primer is the downstream primer R1.

[0148] (2) Utilization XbaI Single enzyme digestion includes GhPEP72 The gene product and the super1300-GFP vector were digested to obtain enzyme digestion products and vector frames, which were then recovered.

[0149] (3) The recombinant expression vector 35S:: was obtained by ligating the enzyme digestion product and the vector framework. GhPEP72 -GFP recombinant vector, i.e. GhPEP72 For the overexpression vector and ligation reaction, please refer to Example 1.

[0150] Obtained Super 1300 XbaI The DNA fragment between the single enzyme cleavage sites is replaced with the one in SEQ ID NO.1. GhPEP72 Following the gene, and keeping other sequences of super1300 unchanged, the recombinant expression vector 35S:: was obtained. GhPEP72 -GFP. The recombinant expression vector 35S:: GhPEP72 The structural diagram of GFP is shown below. Figure 3 As shown.

[0151] 3.2 GhPEP72 Obtaining transgenic plants by overexpression (1) The recombinant expression vector 35S:: obtained in step 3.1 was used. GhPEP72 -GFP was introduced into Agrobacterium strain GV3101 to obtain recombinant Agrobacterium.

[0152] (2) The recombinant Agrobacterium obtained in (1) was cultured in LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL gentamicin at 28℃ for 24 h in a shaker.

[0153] (3) Centrifuge at 4000 rpm for 10 min, collect the recombinant Agrobacterium and resuspend it in a resuspension solution, and adjust the concentration of the resuspension solution to OD. 600 =0.8. The resuspension was formulated as follows: 50 mM MES at pH 5.6, 5 wt% sucrose, and water as the solvent.

[0154] (4) Add 500 μl / L of silwetL-77 to the bacterial suspension, then use it to wet the Arabidopsis thaliana inflorescences that have not yet turned white, and wrap the Arabidopsis thaliana in a black plastic bag to maintain humidity. Place it flat and incubate in the dark at 20˚C for 24 hours. Remove the plastic bag, restore light, and cultivate the plants according to the conventional method until they bear fruit. Harvest mature T0 generation seeds.

[0155] The silwetL-77 was purchased from Beijing Coollab Technology Co., Ltd.; item number: CS9791.

[0156] (5) T0 generation seeds were cultured in 1 / 2 MS medium containing 50 mg / L hygromycin and positive plants were selected. Positive plants were characterized by healthy, dark green true leaves and roots extending into the culture medium.

[0157] (6) Self-pollinate the obtained positive plants to obtain T1 generation seeds, then culture the T1 generation seeds in 1 / 2 MS medium containing 25 mg / L hygromycin and select positive plants from them. The selection criterion is that the proportion of positive plants is greater than 3:1.

[0158] (7) Self-pollinate the positive plants obtained in (6) to obtain T2 generation seeds. Culture the T2 generation seeds in 1 / 2 MS medium containing 25 mg / L hygromycin and select positive plants from them. The selection criterion is that all plants in the line are positive.

[0159] (8) Self-pollinate the positive plants obtained in (7) to obtain T3 generation seeds, cultivate T3 generation seeds, and obtain T3 generation transgenic plants. GhPEP72 Arabidopsis thaliana plant.

[0160] (9) T3 Substitute GhPEP72 Leaf samples were taken from Arabidopsis plants in the fourth week of growth using a perforator. The leaf tissue was ground with liquid nitrogen and then SDS plant total protein extraction solution (250mM Tris-HCl pH 6.8, 4% SDS, 40% glycerol, 0.1% bromophenol blue, 4% β-mercaptoethanol) was added. After mixing, the mixture was denatured at 95 degrees Celsius or higher for 10 minutes. The supernatant was collected by centrifugation and used as the denatured total protein for SDS-PAGE analysis.

[0161] The formulation of the SDS plant total protein extract is as follows: 250mM Tris-HCl at pH 6.8, 4wt% SDS, 40wt% glycerol, 0.1wt% bromophenol blue, and 4wt% β-mercaptoethanol.

[0162] change GhPEP72 Arabidopsis thaliana plants GhPEP72 Overexpression of transgenic Arabidopsis thaliana.

[0163] Figure 8 As described in the embodiments of the present invention GhPEP72 Western blotting results of the homozygous transgenic Arabidopsis thaliana overexpression lines. The results indicate that this example successfully obtained... GhPEP72 Overexpression of transgenic Arabidopsis homozygous line OE1 and GhPEP72 OE2, a homozygous transgenic Arabidopsis thaliana line, was overexpressed.

[0164] 3.3 GhPEP72 Phenotypic studies of transgenic plants overexpressing salt stress (1) The information obtained in step 3.2 GhPEP72 Overexpression of transgenic Arabidopsis homozygous line OE1, GhPEP72 Seeds of the overexpressing transgenic Arabidopsis homozygous line OE2 and wild-type Arabidopsis (WT) were vernalized at 4℃ for 72 hours and then transferred to a 20℃ greenhouse with 16 hours of light / 8 hours of darkness and a light intensity of 60 μmol / m². 2 Cultured for 4 days in a culture room with a humidity of 65±5% for 1 second.

[0165] (2) Then the Arabidopsis seedlings were transferred to a culture medium containing 0 mM or 100 mM NaCl and continued to grow for 8 days. Their growth phenotypes were observed and the changes in their roots were recorded.

[0166] Figure 9 As described in the embodiments of the present invention GhPEP72 A comparison of the growth of homozygous overexpressing transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana under salt stress conditions; among them, Figure 9 The plants in the image, from left to right, are wild-type Arabidopsis thaliana (WT). GhPEP72 Overexpression of transgenic Arabidopsis homozygous line OE1, GhPEP72 OE2, a homozygous transgenic Arabidopsis thaliana line, was overexpressed.

[0167] Figure 10 As described in the embodiments of the present invention GhPEP72 A schematic diagram showing the statistical results of the number of lateral roots of overexpressing transgenic Arabidopsis homozygous lines and wild-type Arabidopsis under salt stress conditions.

[0168] The results showed that, on MS medium used as a normal control group, wild-type Arabidopsis thaliana (WT) and GhPEP72 Overexpression of the transgenic homozygous line OE1, GhPEP72 There was no significant difference in the number of lateral roots among the homozygous transgenic Arabidopsis line OE2. However, under salt stress treatment, GhPEP72 Overexpression of the transgenic homozygous line OE1, GhPEP72 The number of lateral roots in the overexpressing transgenic homozygous line OE2 was significantly higher than that in wild-type Arabidopsis thaliana (WT). This indicates that... GhPEP72 Overexpression of the transgenic homozygous line OE1, GhPEP72 The OE2 line, a homozygous transgenic Arabidopsis thaliana overexpressing transgenic strains, exhibits good stress resistance. GhPEP72 Genes play a positive regulatory role in regulating salt stress in plants. GhPEP72 Genes can be used to improve the salt tolerance of plants.

[0169] This invention clones cotton. GhPEP72 Genes, and constructed using transgenic technology. GhPEP72 Transgenic Arabidopsis plants overexpressing the virus and constructing a transgenic Arabidopsis using VIGS technology GhPEP72 Silent plants, for GhPEP72 Functional verification was conducted, and the following was clarified. GhPEP72 It can improve the salt tolerance of plants, which is conducive to a deeper study of the response mechanism of plants to salt stress signals, lays a good molecular basis for effectively improving the salt tolerance of plants, and has great value for exploring the signal regulation network of plants under salt stress.

[0170] This invention discloses for the first time GhPEP72 This invention provides a positive regulatory effect on salt tolerance in cotton, and offers excellent regulatory targets and theoretical guidance for salt-tolerant cotton cultivation. GhPEP72 The application of genes in regulating plant salt tolerance solves the problems in existing technologies. GhPEP72 The function of cotton in salt tolerance has not yet been reported.

[0171] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0172] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. GhPEP72 The application of genes in regulating plant salt tolerance is characterized by, The GhPEP72 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The GhPEP72 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, Using genetic engineering methods to make the aforementioned in plants GhPEP72 Gene overexpression to improve plant salt tolerance.

4. The application according to claim 3, characterized in that, The genetic engineering method is as follows: utilizing the... GhPEP72 Genes are constructed into recombinant expression vectors, which are then introduced into plants to improve their salt tolerance.

5. A device comprising the form described in claim 1 GhPEP72 Recombinant gene expression vectors.

6. The recombinant expression vector according to claim 5, characterized in that, The recombinant expression vector is used to express the... GhPEP72 Gene insertion Super1300-GFP carrier Xba I Obtained from a single enzyme digestion site.

7. A method for improving the salt tolerance of plants, characterized in that, To make the plant as described in claim 1 GhPEP72 Gene overexpression; and / or enhancement of the gene expression as described in claim 2 in plants GhPEP72 The activity of the gene-expressed protein; and / or the enhancement of the activity of the protein as described in claim 2 in plants. GhPEP72 The level of proteins expressed by genes is adjusted to improve the salt tolerance of plants.

8. The method according to claim 7, characterized in that, The plants described GhPEP72 Gene overexpression is achieved by introducing the recombinant expression vector as described in claim 6 into plants in a manner mediated by Agrobacterium to improve the salt tolerance of plants.

9. The method according to claim 8, characterized in that, The plants mentioned include plants of the genera *Gossypium* and / or *Arabidopsis*.

10. A method for breeding salt-tolerant plants, characterized in that, To make the plant as described in claim 1 GhPEP72 Overexpression of genes yields salt-tolerant transgenic plants; The plant in question is cotton.

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