Application of nucleoside diphosphate kinase GhNDPK9 in promoting salt tolerance of cotton

Abstract

The invention belongs to the technical field of molecular biology breeding, and particularly relates to application of nucleoside diphosphate kinase GhNDPK9 in promoting salt tolerance of cotton. The amino acid sequence of the protein coded by the GhNDPK9 gene is as shown in SEQ ID NO. 2. The invention discloses the effect of the GhNDPK9 gene in positive regulation and control of the salt tolerance of the cotton, enriches functional genes for salt-tolerant cotton breeding, and provides a new effective way for genetic improvement of salt stress resistance of the cotton. By means of transgenic genetic breeding, the expression level of the GhNDPK9 gene is improved, damage symptoms of plants under the salt stress condition are relieved, the salt tolerance of the plants is enhanced, and then the viability of cotton on salinized soil is improved; the method has wide application prospects and good social benefits in the aspects of genetic improvement of salt-tolerant new germplasm, widening of the suitable planting range of cotton and the like.

Description

TECHNICAL FIELD

[0001] The application relates to the field of molecular biology breeding technology, in particular to application of a nucleoside diphosphate kinase GhNDPK9 in promoting salt tolerance of cotton. BACKGROUND

[0002] Cotton is an important economic crop and textile industry raw material, and its fiber is the core raw material of the textile industry, and cotton seeds are also important oil and protein resources. Ensuring the stability and high yield of cotton production is of great significance to the economic development of the textile industry chain and the cotton-producing area.

[0003] With the intensification of global climate change, the problem of water resource shortage, and factors such as unreasonable irrigation mode and seawater backflow, the phenomenon of soil salinization is showing a sustained aggravating trend, and has become one of the key stress factors restricting the high quality and high yield of cotton, bringing great challenges to agricultural production. The inhibition and damage of salt stress on the growth and development of cotton run through the whole growth cycle from seed germination, seedling growth to flowering and boll setting. Under salt stress, the soil soluble salt (mainly Na + and Cl - ) accumulates excessively, and the high salt environment destroys the water absorption, ion balance and root development of plants through osmotic stress, ion toxicity and secondary oxidative damage, etc., leading to a decrease in seed germination rate, wilting, yellowing and growth stagnation of seedlings, etc. After entering the reproductive growth period, salt stress also interferes with the photosynthesis, respiration and material metabolism process of cotton, causing problems such as delay of present budding, bud and boll shedding, boll weight reduction, fiber quality deterioration, etc., and further leading to a substantial reduction in cotton yield. Therefore, analyzing the mechanism of salt tolerance of cotton and cultivating salt-tolerant varieties are key measures to cope with the intensification of soil salinization and ensure the cotton industry.

[0004] Traditional salt-tolerant cotton breeding mainly relies on screening and hybridization of germplasm resources, and high-quality salt-tolerant germplasm resources are scarce, leading to slow breeding process, long cycle, low efficiency, and difficulty in meeting the urgent needs of production. With the development of molecular biology technology, applying salt-tolerant functional genes to cotton resistance breeding through genetic engineering is an efficient and precise way to cultivate new salt-tolerant cotton varieties, and has broad development prospects. Although some plant salt-resistant related genes have been reported, the functional research on cotton salt-tolerant genes is not deep, and key genes suitable for cotton and capable of significantly enhancing its salt tolerance without affecting other agronomic traits are still scarce. Therefore, exploring novel, efficient and clear mechanism cotton salt-resistant functional genes and establishing transgenic genetic breeding technology have important positive significance and application value for improving the salt resistance of cotton, cultivating new salt-tolerant germplasm and promoting the sustainable and healthy development of the cotton industry. SUMMARY

[0005] In view of the above technical problems in the prior art, the application finds that the key functional gene GhNDPK9 of cotton responding to salt stress is discovered, the gene positively regulates salt tolerance of cotton, and the genetic improvement of salt stress resistance of cotton is provided with a new idea by improving the expression level of the GhNDPK9 gene. The application is realized through the following technical solutions:

[0006] The application provides, in a first aspect, the application of the nucleoside diphosphate kinase GhNDPK9 gene in promoting salt tolerance of cotton, wherein the amino acid sequence of the protein encoded by the GhNDPK9 gene is shown in SEQ ID NO. 2.

[0007] Further, the nucleotide sequence of the GhNDPK9 gene is shown in SEQ ID NO. 1.

[0008] Further, the application comprises: enhancing the salt tolerance of cotton by improving the expression amount of the GhNDPK9 gene.

[0009] Further, the element for improving the expression amount of the GhNDPK9 gene comprises one selected from the following (A)-(C):

[0010] (A), a nucleic acid molecule comprising the full-length coding region of the GhNDPK9 gene;

[0011] (B), a recombinant plant expression vector comprising the nucleic acid molecule in (A);

[0012] (C), a recombinant microorganism comprising the recombinant plant expression vector in (B).

[0013] Further, the method for improving the expression amount of the GhNDPK9 gene comprises the following steps:

[0014] Constructing a GhNDPK9 gene overexpression vector, wherein the GhNDPK9 gene overexpression vector contains the full-length coding region of the GhNDPK9 gene;

[0015] Transforming the GhNDPK9 gene overexpression vector into cotton plants, and culturing to obtain transgenic cotton plants with improved expression level of the GhNDPK9 gene.

[0016] Further, the method for constructing the GhNDPK9 gene overexpression vector comprises the following steps:

[0017] Performing double enzyme digestion on the vector pBI121 by using restriction endonuclease BamH I and Sac I to obtain a linearized vector;

[0018] Obtaining a sequence comprising the full-length coding region of the GhNDPK9 gene by PCR amplification through the primer pair shown in SEQ ID NO. 11-12.

[0019] The linearized vector and the sequence comprising the full-length coding region of the GhNDPK9 gene are connected by a homologous recombination method to construct the GhNDPK9 gene overexpression vector.

[0020] Further, the GhNDPK9 gene overexpression vector is transformed into cotton pollen tissue by a gene gun method.

[0021] Further, the cotton is Gossypium hirsutum.

[0022] The second aspect of the present application provides an application of an element for increasing the expression amount of the GhNDPK9 gene in promoting salt tolerance of cotton, and the element for increasing the expression amount of the GhNDPK9 gene comprises one selected from (A)-(C):

[0023] (A), a nucleic acid molecule comprising the full-length coding region of the GhNDPK9 gene, and the nucleotide sequence of the GhNDPK9 gene is shown as SEQ ID NO. 1;

[0024] (B), a recombinant plant expression vector comprising the nucleic acid molecule in (A);

[0025] (C), a recombinant microorganism comprising the recombinant plant expression vector in (B).

[0026] Further, the cotton is Gossypium hirsutum.

[0027] The third aspect of the present application provides a breeding method for improving salt tolerance of cotton, comprising the following steps:

[0028] The expression amount of the GhNDPK9 gene in the cotton plant is increased, and the nucleotide sequence of the GhNDPK9 gene is shown as SEQ ID NO. 1.

[0029] Further, the cotton is Gossypium hirsutum.

[0030] The present application has the following advantages and positive effects:

[0031] The present application discloses the role of the GhNDPK9 gene in positively regulating salt tolerance of cotton, enriches the functional genes for breeding salt-tolerant cotton, and provides a new effective way for genetic improvement of salt stress resistance of cotton. By transgenic genetic breeding, the expression level of the GhNDPK9 gene is increased, which is conducive to relieving the damage symptoms of plants under salt stress conditions, enhancing the salt tolerance of plants, and further improving the survivability of cotton in saline soil, and has a wide application prospect and good social benefits in genetic improvement of new salt-tolerant germplasm, widening the suitable planting range of cotton, etc. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed in the embodiment description. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0033] Figure 1 Figure is a GhNDPK family gene cluster analysis and cis-acting element map in the promoter of the present application embodiment Gossypium hirsutum L;

[0034] Figure 2 Figure is a transcriptome expression level heat map of the present application embodiment Gossypium hirsutum L. GhNDPK family genes under different stress;

[0035] Figure 3 Figure is a GhNDPK9 gene relative expression level map of the present application embodiment GhNDPK9 gene silenced cotton plants and negative control plants;

[0036] Figure 4 Figure is a phenotype map of the present application embodiment GhNDPK9 gene silenced cotton plants, positive control plants and negative control plants after salt stress treatment;

[0037] Figure 5 Figure is a leaf diamino benzidine staining map of the present application embodiment GhNDPK9 gene silenced cotton plants and negative control plants after salt stress treatment;

[0038] Figure 6 Figure is a physiological index change map of the present application embodiment GhNDPK9 gene silenced cotton plants and negative control plants before and after salt stress treatment, wherein, figures (a)-(e) are malondialdehyde, hydrogen peroxide, ascorbic acid, catalase and ascorbate peroxidase in turn;

[0039] Figure 7 Figure is a GhNDPK9 gene relative expression level map of the present application embodiment GhNDPK9 gene overexpression and editing cotton plants;

[0040] Figure 8 Figure is a plant and leaf phenotype map of the present application embodiment GhNDPK9 gene overexpression and editing cotton plants after salt stress treatment. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present application more clear, the following will further describe the present application in combination with embodiments. The embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0042] In light of the information contained in this disclosure, changes in the precise descriptions of the present application can be made by those skilled in the art without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the application is not limited to the specific processes, compositions or methods described, since various modifications can occur to those skilled in the art or related arts to which the present application pertains. Indeed, various modifications of the embodiments described may

[0043] For a better understanding of the present application, and not by way of limitation, all the numbers such as those expressing the amounts, percentages, and so forth, are to be understood as modified by the word "about", unless otherwise indicated. It is to be understood that the phrase "about" can be preceded by the word "approximately". Therefore, unless otherwise specifically indicated, the parameters of the description and the appended claims are approximations. It is contemplated that each of the numerical parameters set forth in the description and the claims can be expressed in terms of a different set of numerical limits, and that they could further be approximated, e.g., rounded, to the nearest unit of significant figure.

[0044] In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Therefore, unless otherwise defined, scientific and technical terminologies can be used with the generally accepted and common meanings in the art.

[0045] The use of the terms "including", "containing", "comprising", "having" and variations thereof herein are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0046] The term "and / or" shall be taken as a specific disclosure of each of the individual features recited. For example, "A and / or B" shall be interpreted to mean only A, only B, or A and B.

[0047] In order that the present application can be more clearly understood, the following detailed description and examples are set forth.

[0048] Nucleoside diphosphate kinase (NDPK) is a highly conserved housekeeping enzyme, which is a 70-100 kD protein composed of several 16-20 kD polypeptides. Its classic function is to maintain the dynamic balance of intracellular ATP and other NTP pools through a ping-pong mechanism catalyzing the reversible phosphogroup transfer reaction between nucleoside triphosphate (ATP) and nucleoside diphosphate (NTP), thereby providing a stable energy supply and material basis for basic life processes such as nucleic acid synthesis, protein translation, and cell membrane construction. In recent years, studies have shown that nucleoside diphosphate kinase is involved in various life activities such as plant cell growth and differentiation, photosensitive pigment, hormone response, heat shock response, and oxidative stress response signaling. Even it has nuclease activity. NDPK plays an important role in plant tolerance to abiotic stress, and has more important application value in actual agricultural production. Although the anti-stress function of NDPK in model plants such as Arabidopsis and rice has been preliminarily reported, in cotton, the expression pattern of NDPK gene under abiotic stress and the specific function and mechanism of action of NDPK gene in salt tolerance of cotton are still unclear, and there are few reports on the application of cotton NDPK gene in genetic improvement to improve salt tolerance.

[0049] The present application uses CottonFGD and Pfam databases to systematically identify members of the NDPK gene (GhNDPK) family in Gossypium hirsutum. A total of 11 GhNDPK gene family members are identified in Gossypium hirsutum. Then, using the cotton transcriptome data (PRJNA490626) under different stress (cold, heat, salt, drought) in the NCBI database, the expression levels of these GhNDPK genes under stress are analyzed. The results show that the expression level of GhNDPK9 gene (GH_D09G2594) increases under salt stress, indicating that the gene is induced to express under salt stress environment and may play a key role in the process of cotton responding to salt stress and salt tolerance.

[0050] With GhNDPK9 gene as the research object, the expression of GhNDPK9 gene in cotton plants is inhibited by using virus-induced gene silencing (VIGS) technology and CRISPR / Cas9 gene editing technology, in addition, the GhNDPK9 gene is cloned from cotton, the expression of GhNDPK9 gene in cotton plants is improved by using overexpression vector pBI121, and the GhNDPK9 gene overexpression strain is constructed. The growth and development phenotypes of the transgenic cotton strain and the wild type cotton plant under normal conditions and salt stress (400mM NaCl) conditions are evaluated, and the results show that the strain overexpressing GhNDPK9 gene has the lightest injury symptom under salt stress, and the leaves are not obviously wilted after 24h of salt stress treatment, while the cotton strain silencing or knocking out GhNDPK9 gene has serious leaf wilting after 24h of salt stress treatment, and shows salt sensitivity. Through mechanism research, it is found that the activities of catalase (CAT) and ascorbate peroxidase (APX) in the silencing plant are reduced, the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) in the cell is increased, part of the defense genes is down-regulated, and the content of ascorbic acid (AsA) is down-regulated. These results prove that GhNDPK9 gene is positively correlated with the salt tolerance of cotton. The present application firstly reveals that GhNDPK9 plays an important role in the response process of plants to salt stress, and has important practical significance for studying the response mechanism of cotton to salt stress and improving the salt tolerance of cotton in breeding.

[0051] Therefore, the present application provides an application of nucleoside diphosphate kinase GhNDPK9 gene in promoting salt tolerance of cotton, wherein the amino acid sequence of the protein encoded by the GhNDPK9 gene is shown in SEQ ID NO. 2.

[0052] The present application mines a new salt-tolerant functional gene, GhNDPK9 gene, reveals the role of GhNDPK9 gene in positively regulating the salt tolerance of cotton, enriches the functional genes for salt-tolerant cotton breeding, and provides a new effective way for genetic improvement of salt stress resistance of cotton. By transgenic genetic breeding, the expression level of GhNDPK9 gene is improved, which is beneficial to alleviate the injury symptom of plants under salt stress conditions, enhance the salt tolerance of plants, and further improve the survivability of cotton in saline soil, and has wide application prospect and good social benefit in genetic improvement of new salt-tolerant germplasm, widening the suitable planting range of cotton, etc.

[0053] The sequence of GhNDPK9 gene can be obtained by conventional means such as codon coding rules, central rules, etc. according to the amino acid (AA) sequence of the protein. The full length of GhNDPK9 gene sequence or its fragment can be obtained by PCR amplification, recombination or artificial synthesis.

[0054] In an alternative embodiment, the nucleotide sequence of the GhNDPK9 gene is shown as SEQ ID NO. 1.

[0055] Alternatively, the application comprises enhancing the salt tolerance of cotton by increasing the expression level of the GhNDPK9 gene.

[0056] Alternatively, the element for increasing the expression level of the GhNDPK9 gene comprises one selected from (A)-(C) below:

[0057] (A) a nucleic acid molecule comprising the full-length coding region of the GhNDPK9 gene;

[0058] (B) a recombinant plant expression vector comprising the nucleic acid molecule of (A);

[0059] (C) a recombinant microorganism comprising the recombinant plant expression vector of (B).

[0060] The vector backbone of the recombinant expression vector described above can be selected from commonly used plant expression vectors, such as plant binary expression vectors pBI121 and pCambia1302. The microorganism can be Agrobacterium or other microorganisms that can mediate transformation or transfection, such as Rhizobium, etc.

[0061] Alternatively, the method for increasing the expression level of the GhNDPK9 gene comprises the following steps:

[0062] constructing a GhNDPK9 gene overexpression vector containing the full-length coding region of the GhNDPK9 gene;

[0063] transforming the GhNDPK9 gene overexpression vector into cotton plants to obtain transgenic cotton plants with increased expression level of the GhNDPK9 gene.

[0064] Alternatively, the method for constructing the GhNDPK9 gene overexpression vector comprises the following steps:

[0065] linearizing the vector pBI121 by double digestion with restriction endonuclease BamH I and Sac I;

[0066] obtaining a sequence comprising the full-length coding region of the GhNDPK9 gene by PCR amplification using the primer pair shown as SEQ ID NO. 11-12;

[0067] connecting the linearized vector and the sequence comprising the full-length coding region of the GhNDPK9 gene by homologous recombination to construct the GhNDPK9 gene overexpression vector.

[0068] Specifically, the GhNDPK9 gene overexpression vector is constructed using a seamless cloning method (in-fusion technology) based on homologous recombination. The transformation of cotton plant cells or tissues (e.g., cotyledons, shoot tips, pollen) with the aforementioned GhNDPK9 gene overexpression vector can be performed using conventional techniques well-known to those skilled in the art, such as calcium phosphate co-precipitation, Ti plasmid method, Ri plasmid method, viral vector method, gene gun method, microinjection method, electroporation method, or Agrobacterium-mediated transformation. In a preferred embodiment, the gene gun method is used, and the recipient tissue is pollen.

[0069] Another embodiment of the present invention provides the application of an element that increases the expression level of the GhNDPK9 gene in promoting salt tolerance in cotton. The element that increases the expression level of the GhNDPK9 gene includes one selected from (A)-(C) below:

[0070] (A) A nucleic acid molecule containing the full-length coding region of the GhNDPK9 gene, wherein the nucleotide sequence of the GhNDPK9 gene is shown in SEQ ID NO.1;

[0071] (B) A recombinant plant expression vector containing the nucleic acid molecules described in (A);

[0072] (C) Recombinant microorganisms containing the recombinant plant expression vector described in (B).

[0073] This invention also provides a breeding method for improving the salt tolerance of cotton, comprising the following steps:

[0074] To increase the expression level of the GhNDPK9 gene in cotton plants, the nucleotide sequence of the GhNDPK9 gene is shown in SEQ ID NO.1.

[0075] The elements and methods for increasing the expression level of the GhNDPK9 gene have been described previously and will not be repeated in this embodiment.

[0076] The cotton mentioned above is upland cotton, specifically Zhong 9807 and TM-1.

[0077] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or as recommended by the manufacturer. Furthermore, all materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0078] Example

[0079] 1. Identification and transcriptome data analysis of the GhNDPK gene family in upland cotton

[0080] To identify the cotton nucleoside diphosphate kinase (NDPK) family of genes, cotton genome data were downloaded from CottonFGD (https: / / cottonfgd.org / ). The hidden Markov model (HMM) PF00334 for the conserved domains of NDPK was downloaded from the Pfam (http: / / pfam.xfam.org / ) database. NDPK family members were retrieved using hmmer software and further screened using NCBI-CD Search. A phylogenetic tree was constructed using MEGA 7.0. A 2000 bp DNA sequence upstream of the start codon of the GhNDPK gene was selected, and cis-regulatory elements related to stress and hormone responses in the GhNDPK gene promoter region were extracted using the PlantCARE website.

[0081] like Figure 1 As shown, 11 members of the GhNDPK gene family (GhNDPK1-11) were identified in upland cotton. Cluster analysis revealed that the NDPK family members were divided into three subgroups: the first subgroup contained 6 members, the second subgroup contained 4 members, and the third subgroup contained 1 member. Gene functional analysis showed that the GhNDPK gene family possesses corresponding functional modules for biotic and abiotic stresses, including auxin-responsiveness, abscisic acid responsiveness, defense and stress responsiveness, light responsiveness, low-temperature responsiveness, methyl jasmonic acid responsiveness, salicylic acid responsiveness, and gibberellin responsiveness.

[0082] Transcriptome sequencing (RNA-Seq) data (PRJNA490626) of cotton under different abiotic stresses (cold, heat, salinity, drought) were downloaded from NCBI to analyze the expression patterns of the GhNDPK gene family under these abiotic stresses. Results are as follows: Figure 2As shown, 1h, 3h, 6h, or 12h cold represents cold treatment; 1h, 3h, 6h, or 12h hold represents heat treatment; 1h, 3h, 6h, or 12h salt represents salt treatment; and 1h, 3h, 6h, or 12h PEG represents simulated drought treatment. It can be seen that the expression level of the GhNDPK9 gene (GH_D09G2594) first decreases and then increases under salt stress, while the expression levels of GhNDPK2 and GhNDPK8 show no change under adverse conditions.

[0083] The nucleotide sequence of the GhNDPK9 gene is shown below:

[0084] (See SEQ ID NO.1).

[0085] The amino acid sequence of the protein encoded by the GhNDPK9 gene is shown below:

[0086] METITLSTSLTTSSFGASAAAAAAATAKRSSATCLSYTSHGNLNVNHLAAFHKQSHLFTKSPTRSFAFTKTRANKSTHGIFLPHLVASLEQVEQTYIMVKPDGVQRGLVGEIISRFERKGFK LTGLKLFQCHKELAEEHYKDLKTKSFYPTLIDYITSGPVVCMVWEGVGVVASARKLIGSTNPLQAEPGTIRGDLAVQTGRNVVHGSDSPENGKRETALWFKEGELCEWTPAQAPWLME (see SEQ ID NO.2).

[0087] 2. Studying the function of GhNDPK9 using virus-mediated gene silencing technology (VIGS).

[0088] This embodiment employs virus-induced gene silencing (VIGS) technology to silence gene expression. By inserting the nucleic acid fragment of the target gene GhNDPK9 into a VIGS-based gene silencing vector, pYL156, a recombinant expression vector pYL156:GhNDPK9 for silencing the target gene was obtained. The recombinant expression vector pYL156:GhNDPK9 was then transformed into upland cotton 9807 plants via Agrobacterium-mediated transformation, specifically inducing the degradation of the target gene mRNA, thereby inhibiting the expression of the endogenous GhNDPK9 gene.

[0089] Based on the VIGS primer design principle, specific primers for amplifying the GhNDPK9 gene were designed. The nucleotide sequences (5'-3') of the primer pairs are shown below, where F represents the upstream primer and R represents the downstream primer:

[0090] VIGS-F: AGAAGGCCTCCATGGGGATCCAACAAATCCACCCATGGCATC (see SEQ ID NO.3);

[0091] VIGS-R: GGCCTCGAGACGGCGTGAGCTCTGCAACAACACCAACACCCTC (see SEQ ID NO. 4).

[0092] Total RNA was extracted from leaves of the salt-tolerant variety 9807 and reverse transcribed into cDNA. Using the cDNA as a template, the target fragment of the GhNDPK9 gene was amplified. The GhNDPK9 gene was ligated into the pEASY-Blunt Cloning Vector (purchased from TransGen Biotech) to construct the GhNDPK9-T cloning vector. The plasmid was extracted and preserved as a template for subsequent vector construction.

[0093] Using GhNDPK9-T as a template, the target fragment was amplified using the primer pairs described above. The pYL156 vector was double-digested with restriction endonucleases BamHI and SacI, and the digestion products were recovered to obtain the pYL156 linear vector. The digestion products of the pYL156 vector and the target fragment were ligated using DNA ligase to obtain the recombinant expression vector pYL-156:GhNDPK9. The recombinant expression vector pYL156:GhNDPK9 was then sequenced for verification.

[0094] The recombinant expression vector pYL156:GhNDPK9 was transformed into Agrobacterium competent cells LBA4404 using the freeze-thaw method. After colony PCR verification, the culture was expanded. The empty pYL156 vector was used as a negative control, and pYL156:PDS was used as a positive control (after the PDS gene was silenced, the leaves showed bleaching).

[0095] Recombinant Agrobacterium containing different types of pYL156 vectors were mixed with helper bacteria 192 at a volume ratio of 1:1 and allowed to stand. The mixed bacterial solution was then injected into the lower surface of the cotyledons of cotton variety Zhong9807 using a 1 mL needleless syringe, resulting in pYL156:GhNDPK9 and pYL156:PDS silent plants, as well as VIGS negative control plants. After injection treatment, the plants were cultured in the dark for 24 h, and then placed under normal light at 25°C.

[0096] When the pYL156:PDS control plants exhibited an albino phenotype, leaf samples were collected. Total RNA was extracted from cotton seedling leaves using the EASYspin Plus Plant RNA Kit (RN38) from Beijing Adley Co., Ltd., and reverse transcribed into cDNA using the reverse transcription kit (AT311-02) from Beijing TransGen Biotech Co., Ltd. Gene silencing efficiency was analyzed by quantitative real-time PCR (qRT-PCR). The primer pairs used included qRT-GhNDPK9-F and qRT-GhNDPK9-R, and GhUBQ7-F and GhUBQ7-R. GhUBQ7 (GenBank accession No. DQ116441) was used as an internal control gene, and the relative expression level of the GhNDPK9 gene was calculated using a 2-1 ratio. -ΔΔCt The method was used for calculation, and the results are shown below. Figure 3The x-axis represents grouping, and the y-axis represents the relative expression level of the GhNDPK9 gene. The nucleotide sequences of the primer pairs used for detection are shown below:

[0097] qRT-GhNDPK9-F: GTGTACAACGTGGCCTTGTTGG (see SEQ ID NO.5);

[0098] qRT-GhNDPK9-R:CTGTCACTACCATGGACAAC (see SEQ ID NO.6);

[0099] GhUBQ7-F: GAAGGCATTCCACCTGACCAAC (see SEQ ID NO. 7);

[0100] GhUBQ7-R: CCGCATTAGGGCACTCTTTTC (see SEQ ID NO. 8).

[0101] Plants with good silencing effects were selected. When the cotton seedlings reached the three-leaf stage, they were subjected to salt stress treatment with 400mM NaCl and placed in a 25℃ greenhouse under normal light conditions. Leaf phenotypes were observed after 24 hours. The results are as follows: Figure 4 As shown, the left image shows pYL156:PDS silenced plants, and the right image, from left to right, shows: a positive control (treated with water), a VIGS negative control plant treated with salt stress for 24 h (transformed into the empty vector pYL-156), and a pYL156:GhNDPK9 silenced plant treated with salt stress for 24 h (transformed into the pYL156:GhNDPK9 vector). Figures 3-4 It is evident that the gene expression level in the GhNDPK9 gene-silenced lines was significantly reduced, and the silent lines exhibited obvious wilting.

[0102] When the leaves show obvious wilting, the surge of reactive oxygen species in the cotton leaves is detected. The cotton leaves are stained with diaminobenzidine (DAB). DAB reacts with peroxidase to produce an insoluble brown precipitate, such as... Figure 5 As shown, studies on reactive oxygen species bursts revealed more brown deposits in the leaves of GhNDPK9 gene-silenced plants, indicating a stronger reactive oxygen species burst and more cell necrosis that could be observed in the silenced plants.

[0103] The results of changes in the contents of MDA, H2O2, and AsA, and the changes in the enzyme activities of CAT and APX in the leaves are as follows: Figure 7As shown, measurements of malondialdehyde (MDA), hydrogen peroxide (H2O2), and ascorbic acid (AsA) in GhNDPK9 gene-silenced plants revealed that under salt stress, the MDA and H2O2 contents in GhNDPK9 gene-silenced cotton seedlings were higher than those in control plants, and the AsA content in silenced plants was significantly reduced. Furthermore, under salt stress, the activities of catalase (CAT) and ascorbate peroxidase (APX) in silenced plants were significantly reduced. This confirms that the GhNDPK9 gene has a positive regulatory effect on cotton salt tolerance.

[0104] 3. Knockout of the GhNDPK9 gene using CRISPR / Cas9 gene editing technology and phenotypic analysis of knockout plants.

[0105] To further verify the function of the GhNDPK9 gene, primer pairs for CRISPR / Cas9 gene editing were designed based on the GhNDPK9 gene sequence. These primer pairs contain sgRNA target sites. New cotton materials were created using CRISPR / Cas9 gene editing technology, and their salt tolerance was assessed. The nucleotide sequences (5'-3') of the primer pairs are shown below:

[0106] ndpk9-F:

[0107] AGAGTCGAAGTAGTGATTGTCAGGGAGGCTGATTGCTGGTTTTAGAGCTAGAAATA (see SEQ IDNO.9);

[0108] ndpk9-R:

[0109] TATTTCTAGCTCTAAAACCCTGCTTATCCTACACCTCTAATCACTACTTCGACTC (see SEQ ID NO. 10).

[0110] Using GhNDPK9-T as a template, PCR amplification was performed using primer pairs containing the sgRNA target site to obtain a single-stranded guide RNA (sgRNA) targeting the GhNDPK9 gene. The sgRNA was ligated into the pEASY-T1 vector to obtain the editing vector pEASY-T1, which was successfully constructed by sequencing. The editing vector pEASY-T1 was transformed into the shoot tip tissue of upland cotton TM-1 (as recipient material) via shoot tip genetic transformation. Transformed plants were obtained after resistance selection and culture. Three independent positive GhNDPK9 gene knockout transgenic lines were successfully obtained, named ndpk9-cr (cr2, cr6, and cr9). Salt tolerance was analyzed after culturing the gene-edited plants to the T3 generation.

[0111] 4. Phenotypic analysis of transgenic plants overexpressing the GhNDPK9 gene using transgenic technology.

[0112] To positively verify the salt tolerance function of GhNDPK9, the GhNDPK9 gene was ligated into the plant expression vector pBI121, which was double-digested with SacI and BamHI, using homologous recombination. Using GhNDPK9-T as a template, PCR amplification was performed using the primer pairs for overexpressing the GhNDPK9 gene, as shown below:

[0113] GhNDPK9-pBI121-F:

[0114] ACGGGGGACTCTAGAGGATCCATGGAGACAATAACATTATCCACTTCG (see SEQ ID NO. 11);

[0115] GhNDPK9-pBI121-R:

[0116] CGATCGGGGAAATTCGAGCTCCTCCATTAACCATGGTGCTTGA (see SEQ ID NO. 12).

[0117] Using the ClonExpress II One Step Cloning Kit (C112) from Nanjing Novizan Biotechnology Co., Ltd., the sequence including the full-length coding region of the GhNDPK9 gene, obtained by PCR amplification, and the double-digested linearized expression vector pBI121 were ligated by homologous recombination to obtain the GhNDPK9 gene overexpression vector.

[0118] The GhNDPK9 gene overexpression vector was transformed into pollen of upland cotton TM-1 (as recipient material) using the gene gun in vivo transformation method. The specific operation was as follows: (1) Gold powder was washed with 70% ethanol and a gold powder suspension of 60 mg / mL was prepared and stored at 4℃ for later use; (2) The gold powder suspension and GhNDPK9 gene overexpression vector were mixed at a ratio of 10:1, and 40 μL spermidine (0.1M) was added and mixed, and 100 μL CaCl2 (2.5M) was shaken for 1 min, centrifuged at 13000 rpm for 40 s, and the supernatant was discarded; (3) 300 μL anhydrous ethanol was added to wash the gold powder, shaken for 40 s, centrifuged at 13000 rpm for 40 s, and the supernatant was discarded; (4) 100 μL anhydrous ethanol was added and mixed to obtain the gold powder / overexpression vector mixture, which was stored at -20℃ for later use; (5) The collected TM -1. Remove impurities from pollen. Add 10 μL of the gold powder / overexpression vector mixture to the wells of a GDS-80 gene gun to bombard the pollen. Apply the bombarded pollen to the stigma of the stamen during the peak pollen viability period (9-11 AM). Replace the stigma with a wax tube. Harvest the seeds after they mature in the field. Propagate the harvested seeds by multiple generations. Verify each generation using kanamycin, PCR, and qRT-PCR until the T3 generation. Obtain lines with high GhNDPK gene expression levels, named GhNDPK9-OE (OE-3, OE-8, and OE-11), and conduct salt tolerance assessments.

[0119] Figure 7 The relative expression levels of the GhNDPK9 gene in cotton plants edited with the GhNDPK9 gene and those overexpressing it are shown. The horizontal axis represents the grouping, the vertical axis represents the relative expression level of the GhNDPK9 gene, and WT represents the wild-type recipient material without the transformed vector. It can be seen that the embodiments of the present invention successfully obtained GhNDPK9 gene knockout transgenic lines (cr2, cr6, and cr9) and three lines with high gene expression levels (OE-3, OE-8, and OE-11).

[0120] The cotton recipient material, along with the overexpression and gene-editing materials, were planted in a plant light incubator. When the seedlings reached the three-leaf stage, they were subjected to salt stress treatment with 400 mM NaCl and then placed in a 25°C greenhouse under normal light conditions. After 24 hours, the wilting of the plant leaves was observed. Figure 8 As shown, the wilting of leaves in the gene-edited lines was more severe than that in the recipient material and the overexpression lines, and the overexpression lines showed better salt tolerance than the recipient material.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the nucleoside diphosphate kinase GhNDPK9 gene in promoting salt tolerance in cotton, characterized in that, The amino acid sequence of the protein encoded by the GhNDPK9 gene is shown in SEQ ID NO.

2.

2. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to claim 1 in promoting salt tolerance in cotton, characterized in that, The nucleotide sequence of the GhNDPK9 gene is shown in SEQ ID NO.

1.

3. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to claim 1 in promoting salt tolerance in cotton, characterized in that, The application includes enhancing the salt tolerance of cotton by increasing the expression level of the GhNDPK9 gene.

4. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to claim 3 in promoting salt tolerance in cotton, characterized in that, Elements that increase the expression level of the GhNDPK9 gene include one selected from (A)-(C) below: (A) A nucleic acid molecule containing the full-length coding region of the GhNDPK9 gene; (B) A recombinant plant expression vector containing the nucleic acid molecules described in (A); (C) Recombinant microorganisms containing the recombinant plant expression vector described in (B).

5. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to claim 3 in promoting salt tolerance in cotton, characterized in that, The method for increasing the expression level of the GhNDPK9 gene includes the following steps: Construct a GhNDPK9 gene overexpression vector, wherein the GhNDPK9 gene overexpression vector contains the full-length coding region of the GhNDPK9 gene; The GhNDPK9 gene overexpression vector was transformed into cotton plants to obtain transgenic cotton plants with increased GhNDPK9 gene expression levels.

6. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to claim 5 in promoting salt tolerance in cotton, characterized in that, The method for constructing the GhNDPK9 gene overexpression vector includes the following steps: The vector pBI121 was double-digested with restriction endonucleases BamHI and SacHI to obtain a linearized vector; Using the primer pair shown in SEQ ID NO.11-12, the sequence including the full-length coding region of the GhNDPK9 gene was amplified by PCR; The GhNDPK9 gene overexpression vector was constructed by connecting the linearized vector and the sequence including the full-length coding region of the GhNDPK9 gene using homologous recombination.

7. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to claim 5 in promoting salt tolerance in cotton, characterized in that, The GhNDPK9 gene overexpression vector was transformed into cotton pollen tissue using the gene gun method.

8. The application of the nucleoside diphosphate kinase GhNDPK9 gene according to any one of claims 1-7 in promoting salt tolerance in cotton, characterized in that, The cotton in question is upland cotton.

9. The application of elements that increase the expression level of the GhNDPK9 gene in promoting salt tolerance in cotton, characterized in that... Elements that increase the expression level of the GhNDPK9 gene include one selected from (A)-(C) below: (A) A nucleic acid molecule containing the full-length coding region of the GhNDPK9 gene; (B) A recombinant plant expression vector containing the nucleic acid molecules described in (A); (C) Recombinant microorganisms containing the recombinant plant expression vector described in (B); The nucleotide sequence of the GhNDPK9 gene is shown in SEQ ID NO.1, and the cotton is upland cotton.

10. A breeding method for improving the salt tolerance of cotton, characterized in that, Includes the following steps: To increase the expression level of the GhNDPK9 gene in cotton plants, wherein the nucleotide sequence of the GhNDPK9 gene is shown in SEQ ID NO.1, and the cotton is upland cotton.

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