Use of ghzat10 gene in promoting plant tolerance to low potassium stress

Abstract

The application relates to the field of plant breeding, and particularly relates to application of a GhZAT10 gene in promoting the low potassium stress tolerance of plants, a recombinant carrier, a recombinant microorganism, a method for improving the low potassium stress tolerance of plants and a plant breeding method. The application of the GhZAT10 gene in promoting the low potassium stress tolerance of plants comprises: improving the low potassium stress tolerance of the plants and / or improving the potassium ion absorption capacity of the plants by overexpressing the GhZAT10 in the plants, wherein the nucleotide sequence of the GhZAT10 is shown in SEQ ID NO:1. According to the application of the GhZAT10 gene in promoting the low potassium stress tolerance of plants provided in the embodiments, the ability of the plants to resist low potassium stress can be improved, and the growth of the plants can be significantly promoted in a low potassium environment without adding K+ or adding trace K+ externally.

Description

Technical Field

[0001] This application relates to the field of plant breeding, specifically to the application of the GhZAT10 gene in promoting the ability of plants to tolerate low potassium stress, a recombinant vector, a recombinant microorganism, a method for improving the ability of plants to tolerate low potassium stress, and a plant breeding method. Background Technology

[0002] Potassium (K+) is one of the most abundant nutrients in the soil and also the most abundant cation in plant cells. In recent years, with the continuous increase in crop yields and the reduction in the use of organic fertilizers, the potassium balance in farmland ecosystems has generally shown a deficit. According to statistics, 56% of arable land area is facing potassium deficiency, which poses a serious constraint on my country's agricultural production.

[0003] Cotton (Gossypium hirsutum L.) is a potassium-loving economic crop. Potassium absorption and utilization are closely related to cotton yield and quality. Potassium not only promotes the growth and development of the upper parts and roots of cotton plants but also increases dry matter accumulation. In recent years, with the gradual increase in cotton multiple cropping index and yield, as well as the widespread adoption of genetically modified insect-resistant cotton, potassium deficiency in cotton fields in my country has become increasingly severe. Furthermore, improper fertilizer management further exacerbates potassium deficiency in cotton production, often leading to premature aging and seriously affecting yield and quality.

[0004] Therefore, there is an urgent need for an application that can improve the ability of plants, especially cotton, to tolerate low potassium stress. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems in the related art.

[0006] Therefore, a first aspect of the present invention provides an application of the GhZAT10 gene in promoting plant tolerance to low potassium stress, comprising: improving the plant's ability to promote plant tolerance to low potassium stress and / or enhancing the plant's potassium ion uptake capacity by overexpressing the GhZAT10 in the plant, wherein the nucleotide sequence of the GhZAT10 is as shown in SEQ ID NO:1. The application of the GhZAT10 gene in promoting plant tolerance to low potassium stress according to the embodiments of the present invention can enhance the plant's resistance to low potassium stress in low potassium environments with no exogenous K+ or with trace amounts of added K+, significantly promoting plant growth.

[0007] In some embodiments, the overexpression of GhZAT10 in the plant comprises: inserting a DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector; and introducing the recombinant vector into the plant to overexpress GhZAT10.

[0008] In some embodiments, the RNA of the plant is reverse transcribed to obtain template DNA, and the template DNA is amplified by PCR using primer pairs as shown in SEQ ID NO:2 and SEQ ID NO:3 to obtain the DNA fragment of the GhZAT10 gene.

[0009] In some embodiments, the step of inserting the DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector includes: digesting the pCAMBIA2300::HA vector with restriction endonucleases BamHI and StUI to obtain a vector frame; and ligating the DNA fragment of the GhZAT10 gene to the vector frame to obtain the recombinant vector pCAMBIA2300::HA-GhZAT10.

[0010] In some embodiments, the plant is a monocotyledonous or dicotyledonous plant; preferably, the plant is Arabidopsis thaliana or cotton.

[0011] In some embodiments, the GhZAT10 gene encodes the GhZAT10 protein having the amino acid sequence shown in SEQ ID NO:4.

[0012] A second aspect of the present invention provides a recombinant vector comprising a nucleotide sequence as shown in SEQ ID NO:1.

[0013] In some embodiments, the recombinant vector is pCAMBIA2300::HA-GhZAT10.

[0014] A third aspect of the present invention provides a recombinant microorganism comprising the recombinant vector described in the first aspect of the present invention.

[0015] The fourth aspect of the present invention proposes the application of any one of the following a)-d) in promoting the ability of plants to tolerate low potassium stress;

[0016] a) GhZAT10 protein having the amino acid sequence shown in SEQ ID NO:4 or a variant having a sequence having more than 90% homology with SEQ ID NO:4;

[0017] b) The recombinant vector as described in the second aspect of the present invention;

[0018] c) Recombinant microorganisms as described in the third aspect of the present invention;

[0019] d) Expression cassettes and / or transgenic cell lines containing the nucleotide sequence of the GhZAT10 gene as shown in SEQ ID NO:1 or a sequence that is more than 90% homologous to SEQ ID NO:1.

[0020] A fifth aspect of the present invention provides a method for improving the tolerance of plants to low potassium stress, characterized in that it comprises at least one of the following:

[0021] To overexpress the GhZAT10 gene in the plant;

[0022] To increase the activity of GhZAT10 protein in the plant; and

[0023] Increase the content of GhZAT10 protein in the plant.

[0024] A sixth aspect of the present invention provides a plant breeding method, characterized in that it includes:

[0025] The GhZAT10 gene in the plant is overexpressed to obtain a transgenic plant resistant to low potassium stress. The nucleotide sequence of the GhZAT10 gene is shown in SEQ ID NO:1. The plant is a monocotyledonous or dicotyledonous plant, preferably Arabidopsis thaliana or cotton.

[0026] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0027] This invention provides the application of the GhZAT10 gene in promoting plant tolerance to low potassium stress, and demonstrates that when the GhZAT10 gene is silenced in plants, compared with the control group, the plants are more sensitive to low potassium stress, with a significantly reduced K+ uptake capacity, which in turn affects the accumulation of K+ in the plant and results in more severe potassium deficiency symptoms. Conversely, when the GhZAT10 gene is overexpressed in plants, compared with the control group, it can improve the plant's resistance to low potassium stress in both low potassium environments with no exogenous K+ and with trace amounts of K+, significantly promoting plant growth. Furthermore, this invention is of great value for studying low potassium tolerant plants and exploring the signal regulatory network of plants under stress. Attached Figure Description

[0028] Figure 1 This diagram illustrates the sequence alignment results of the GhZAT10 protein provided in this embodiment of the invention with ZAT10 proteins from other species.

[0029] Figure 2 The diagram shows the subcellular localization results of the GhZAT10 protein in an embodiment of the present invention.

[0030] Figure 3This diagram illustrates the gene expression levels of the GhZAT10 gene in different tissues of cotton under normal and low potassium stress conditions, as shown in the embodiments of the present invention. * indicates a significant difference analysis result of p < 0.05; ** indicates a significant difference analysis result of p < 0.01; *** indicates a significant difference analysis result of p < 0.001.

[0031] Figure 4 The results show the effects of the GhZAT10 gene on plant growth phenotypes under different potassium ion conditions, wherein... Figure 4 Part A shows the growth phenotypes of wild-type Arabidopsis thaliana (Col-0), T-DNA insertion mutant material of Arabidopsis thaliana AtZAT10 (atzat10), and Arabidopsis thaliana lines overexpressing GhZAT10 (OE#9, OE#14) after 10 days of culture on low-potassium, low-ammonia medium under conditions of no exogenous K+, 0.015 mM K+, and 5 mM K+, respectively. Figure 4 Part B shows a schematic diagram comparing the primary root length of Col-0, atzat10, OE#9, and OE#14 after 10 days of cultivation on low-potassium, low-ammonia medium under conditions of no exogenous K+ addition, addition of 0.015 mM K+, and addition of 5 mM K+, respectively.

[0032] Figure 5 The results of the silencing efficiency of the GhZAT10 gene in plants according to an embodiment of the present invention are shown. VIGS-GhZAT10 represents plants with the GhZAT10 gene silenced, and VIGS-Ctrl represents the control group. * indicates that the difference analysis result is p < 0.05; ** indicates that the difference analysis result is p < 0.01; *** indicates that the difference analysis result is p < 0.001.

[0033] Figure 6 The growth phenotype of GhZAT10 gene-silenced plants in this embodiment of the invention is shown. Figure 6 Part A shows the whole-plant growth phenotypes of the GhZAT10 gene-silenced plant VIGS-GhZAT10 and the control group VIGS-Ctrl under normal and low potassium stress conditions. Figure 6 Part B shows the leaf phenotypes of VIGS-GhZAT10 and VIGS-Ctrl under normal and low potassium stress conditions;

[0034] Figure 7The figures show the potassium accumulation in various parts of the GhZAT10 gene-silenced plant VIGS-GhZAT10 and the control group VIGS-Ctrl under normal and low potassium stress conditions in this embodiment of the invention. * indicates that the difference analysis result is p < 0.05; ** indicates that the difference analysis result is p < 0.01; *** indicates that the difference analysis result is p < 0.001.

[0035] Figure 8 The net K+ uptake rate of the GhZAT10 gene-silenced plant VIGS-GhZAT10 and the control group VIGS-Ctrl in the embodiments of the present invention are shown, where * represents the result of the significance analysis of the difference p < 0.05; ** represents the result of the significance analysis of the difference p < 0.01; *** represents the result of the significance analysis of the difference p < 0.001. Detailed Implementation

[0036] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0037] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0038] With the widespread adoption of Bt gene-modified insect-resistant cotton in the Yellow River and Yangtze River cotton-growing areas, coupled with improper fertilizer and water management, potassium deficiency in cotton in my country has become increasingly severe. Cotton is a potassium-loving crop, and potassium deficiency leads to premature aging, seriously affecting cotton yield and quality. Solving the potassium deficiency problem in cotton is of great significance for improving cotton yield and quality. With the continuous development of molecular biology, it has become possible to cultivate low-potassium-tolerant cotton varieties using biotechnology. Although some progress has been made in cotton gene cloning, it lags far behind that of grain crops such as rice, corn, and wheat.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0040] Virus-induced gene silencing (VIGS) is an effective reverse genetics technique widely used for identifying plant genome function. VIGS can successfully silence endogenous genes in different parts of the plant, providing a practical means to study gene function at different growth stages. This application provides examples of GhZAT10 gene-silenced plants using the VIGS method to demonstrate the function of this gene.

[0041] In this paper, the description of pTRV-GFP and pTRV-RNA1 (pTRV1) by Chun Mu et al. (Chun Mu, Lin Zhou, Libo Shan, Fangjun Li & Zhaohu Li (2019). Phosphatase GhDs PTP 3a interacts with annexin protein Gh ANN 8b to reversely regulate salt tolerance in cotton (Gossypium spp.). New Phytologist, 223(4), 1856-1872) is incorporated herein by reference.

[0042] In this article, Yiru Wang et al. (Yiru Wang, Ye Wang, Bo Li, Changming Xiong, AEgrinya Eneji, Mingcai Zhang, Fangjun Li, Xiaoli Tian & Zhaohu Li (2019). The Cotton High-Affinity K + Transporter,GhHAK5a,Is Essential for Shoot Regulation of K + The records of pTRV2 (pYL156, pTRV-RNA2) and pTRV-GhCLA1 in Uptake in Root under Potassium Deficiency, Plant and Cell Physiology, 60(4), 888-899 are incorporated herein by reference.

[0043] In this paper, the record of Qimeng Zhang et al. (Qimeng Zhang, Ning Luo, Xicheng Dai, Jinhui Lin, Bilal Ahmad, Qingxi Chen, Yan Lei & Zhifeng Wen (2024). Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracose via affecting hormone signaling pathways and lignin production. BMC Genomics, 25(1), 895) on the pCAMBIA2300::HA vector is incorporated herein by reference.

[0044] In this paper, the upland cotton variety “Jin668” is referenced from the description in Xiangqian Zhu et al. (Xiangqian Zhu, Zhongping Xu, Guanying Wang, Yulong Cong, Lu Yu, Ruoyu Jia, Yuan Qin, Guangyu Zhang, Bo Li, Daojun Yuan, Lili Tu, Xiyan Yang, Keith Lindsey, Xianlong Zhang & Shuangxia Jin (2023). Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateralmeristem. Genome Biology, 24(1), 194).

[0045] In this paper, low potassium stress refers to a situation where the amount or concentration of potassium available to the target plant is lower than its requirements for normal growth and development. In some embodiments, this may be a potassium ion concentration ≤0.1 mM. In some embodiments, this may be a potassium ion concentration ≤0.05 mM. In some embodiments, this may be a potassium ion concentration ≤0.03 mM, but not zero.

[0046] In this paper, the phenotypes of plants sensitive to low potassium stress include reduced plant biomass (root dry weight, stem dry weight, leaf dry weight, etc.) and decreased net potassium ion uptake rate.

[0047] The first aspect of this invention proposes the application of the GhZAT10 gene in promoting plant tolerance to low potassium stress, comprising: improving the plant's ability to promote low potassium stress and / or enhancing its potassium ion uptake capacity by overexpressing the GhZAT10 in the plant, wherein the nucleotide sequence of the GhZAT10 is shown in SEQ ID NO:1. The application of the GhZAT10 gene in promoting plant tolerance to low potassium stress according to this invention can enhance the plant's resistance to low potassium stress and significantly promote plant growth in low potassium environments with no exogenous K+ addition or with trace amounts of K+ addition. Furthermore, this application explores the function of the cotton potassium transporter GhZAT10 in cotton potassium uptake, elucidates the regulatory pathway of GhZAT10 in cotton's response to low potassium stress, studies gene expression and regulation under low potassium stress at the molecular level, improves the signal transduction and gene expression regulation network under stress conditions, and further investigates the plant's response mechanism to low potassium stress signals, laying a good molecular foundation for effectively improving plant tolerance to low potassium stress.

[0048] The nucleotide sequence of GhZAT10 gene is as SEQ ID NO:1 shows: ATGGCGCTTGAAGCTCTGACCTCGCCGGCGACGCCTTTCACCAACAAATACGATGACGTGGACAACAATTACGTCGAGACATGGAAGAAAGGCAAGCGTTCGAAGCGCCAACGTGGCGACTCTCCTGCTGCTGTTGAACTT CAACCCACCACCGAAGAAGAGTACCTCGCTCTTTGTCTCATGCTCGCTCGCGGCTCTCCGGTGCTGATCGTGATGTTATTCGTCGGTCTTCCTCTTCGTCGTCACCGCCTCCGCCGCCGCCTGCTTTGAAGTTGTCTTACAAGT GTAGTGTTTGTGACAAGGCGTTCCCTTCTTATCAAGCTTTGGGCGGTCATAAAGCCAGCCACCGCAAACCCCTTTCCGCCGACGCCGCTACCACCACCGCCGCCGTCAACGTCGATAACCCATCAACAAC CAGCACCGCCACCACCATCACCAGCAGCGGTAGGCTTCACGAGTGTTCCATCTGCCACAAGAGTTTCCCTACGGGCCAAGCCTTGGGTGGTCATAAACGCTGCCACTACGAAGGTGGCAACAACAACACA AAAATAACAACAACAGCGGTAGCGTTAGCGTTAGCGTTAGCGGGGTTACGTCTTCGGATGGGGGGGCGTTGAGCCACAACCACCGTGCAGTCGACTTTGACTTTGACCTCAACTTGCCAGCCTTGCCGGA GTTCAGTCAAATGTACCCAGATGAAGAAGAGGTTCAAAGCCCATTGCCGACCAAGAAACCACGTCTCTTGATCGCCAAGAAAGAGAAACTGGATTCTTCATTAGCACAAGATTTAGAATTAAATTGA(SEQ ID NO: 1).

[0049] In some embodiments, the overexpression of GhZAT10 in the plant comprises: inserting a DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector; and introducing the recombinant vector into the plant to overexpress GhZAT10.

[0050] In some embodiments, the RNA of the plant is reverse transcribed to obtain template DNA, and the template DNA is amplified by PCR using primer pairs as shown in SEQ ID NO:2 and SEQ ID NO:3 to obtain the DNA fragment of the GhZAT10 gene.

[0051] SEQ ID NO: 2: 5'-ctccccttgctccgtggatccATGGCGCTTGAAGCTCTGA-3'(F4);

[0052] SEQ ID NO: 3: 5'-aacgtcgtatgggtaaggcctATTTAATTCTAAATCTTGTGCTAATGAAG-3'(R4).

[0053] In some embodiments, the step of inserting the DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector includes: digesting the pCAMBIA2300::HA vector with restriction endonucleases BamHI and StUI to obtain a vector frame; and ligating the DNA fragment of the GhZAT10 gene to the vector frame to obtain the recombinant vector pCAMBIA2300::HA-GhZAT10.

[0054] In some embodiments, the primer pair used for PCR amplification further includes the following sequences:

[0055] F1:ATGGAACACTCGTGAAGCCTAC (SEQ ID NO: 6);

[0056] R1: CCATTCTAAGGATTGAATCGGT (SEQ ID NO: 7);

[0057] F2: tacaccaaatcgactctagaATGGCGCTTGAAGCTCTGA (SEQ ID NO: 8);

[0058] R2: ataggtacccgggctctaga ATTTAATTCTAAATCTTGTGCTAATGAAG (SEQ ID NO: 9); F3: TCACGAGTGTTCCATCTGCC (SEQ ID NO: 10);

[0059] R3:CGAAGACGTAACCCCGCTAA (SEQ ID NO: 11);

[0060] F5: gtgagtaaggttaccgaattcGCCTTTCACCAACAAATACGAT (SEQ ID NO: 12);

[0061] R5: agacgcgtgagctcggtaccATGGAACACTCGTGAAGCCTAC (SEQ ID NO: 13).

[0062] In some embodiments, the plant is a monocotyledonous or dicotyledonous plant; preferably, the plant is Arabidopsis thaliana or cotton.

[0063] In some embodiments, the GhZAT10 gene encodes the GhZAT10 protein having the amino acid sequence shown in SEQ ID NO:4.

[0064] SEQ ID NO:4:MALEALTSPATPFTNKYDDVDNNYVETWKKGKRSKRQRGDSPAAVELQPTTEEEYLALCLIMLARGSSGADRDVIRRSSSSSSPPPPPPALKLSYKCSVCDKAFPSYQALGGHKASHRKPLSADAATTTAAV NVDNPSTTSTATTITSSGRLHECSICHKSFPTGQALGGHKRCHYEGGNNNNKNNNNSGSVSVSGVTSSDGGALSHNHRAVDFDFDLNLPALPEFSQMYPDEEEVQSPLPTKKPRLLIAKKEKLDSSLAQDLELN.

[0065] A second aspect of the present invention provides a recombinant vector comprising a nucleotide sequence as shown in SEQ ID NO:1.

[0066] In some embodiments, the recombinant vector is pCAMBIA2300::HA-GhZAT10.

[0067] A third aspect of the present invention provides a recombinant microorganism comprising the recombinant vector described in the first aspect of the present invention.

[0068] The fourth aspect of the present invention proposes the application of any one of the following a)-d) in promoting the ability of plants to tolerate low potassium stress;

[0069] a) GhZAT10 protein having the amino acid sequence shown in SEQ ID NO:4 or a variant having a sequence having more than 90% homology with SEQ ID NO:4;

[0070] b) The recombinant vector as described in the second aspect of the present invention;

[0071] c) Recombinant microorganisms as described in the third aspect of the present invention;

[0072] d) Expression cassettes and / or transgenic cell lines containing the nucleotide sequence of the GhZAT10 gene as shown in SEQ ID NO:1 or a sequence that is more than 90% homologous to SEQ ID NO:1.

[0073] A fifth aspect of the present invention provides a method for improving the tolerance of plants to low potassium stress, characterized in that it comprises at least one of the following:

[0074] To overexpress the GhZAT10 gene in the plant;

[0075] To increase the activity of GhZAT10 protein in the plant; and

[0076] Increase the content of GhZAT10 protein in the plant.

[0077] A sixth aspect of the present invention provides a plant breeding method, characterized in that it includes: overexpressing the GhZAT10 gene in the plant to obtain a transgenic plant resistant to low potassium stress, wherein the nucleotide sequence of the GhZAT10 gene is shown in SEQ ID NO:1, and the plant is a monocotyledonous plant or a dicotyledonous plant, preferably, the plant is Arabidopsis thaliana or cotton.

[0078] This invention also provides a protein obtained from cotton (Gossypium hirsutum), specifically from the cotton variety "Jin668", named GhZAT10 protein, which is (a) a protein composed of the amino acid sequence shown in SEQ ID NO: 4; or (b) a protein derived from SEQ ID NO: 1 with one or more amino acid residues substituted and / or deleted and / or added compared to the amino acid sequence shown in SEQ ID NO: 4 and associated with the plant's tolerance to low potassium stress and / or potassium ion uptake capacity.

[0079] This invention also provides a gene encoding the GhZAT10 protein, named the GhZAT10 gene. In some embodiments, the nucleotide sequence of the GhZAT10 gene is as shown in SEQ ID NO: 1 or a sequence with more than 90% homology to SEQ ID NO: 1.

[0080] This invention also provides the application of substances that inhibit GhZAT10 gene expression in the cultivation of gene-silencing plants.

[0081] In some embodiments, the expression level of GhZAT10 protein or GhZAT10 gene in plants is reduced, thereby reducing the plant's tolerance to low potassium stress and / or its potassium ion uptake capacity.

[0082] In some embodiments, increased expression of the GhZAT10 protein or the GhZAT10 gene in plants enhances the plant's tolerance to low potassium stress and / or its potassium ion uptake capacity.

[0083] This invention also provides a method for cultivating gene-silencing plants, which involves silencing the GhZAT10 gene in a target plant to obtain a silenced plant with a lower dry matter accumulation capacity under low potassium stress than the target plant.

[0084] And / or, silent plants whose potassium ion uptake capacity is lower than that of the target plant under low potassium stress;

[0085] And / or, silent plants with potassium content lower than that of the target plant under low potassium stress.

[0086] In some embodiments, the substance that inhibits GhZAT10 gene expression includes an interference vector. In some specific embodiments, the interference vector may be a recombinant plasmid obtained by inserting a nucleotide sequence of a double-stranded DNA molecule, as shown in SEQ ID NO: 5, between the KpnⅠ and EcoRI restriction sites of the pTRV2 vector. The GhZAT10 gene is silenced by introducing the above-described interference vector into the target plant.

[0087] SEQ ID NO: 5: GCCTTTCACCAACAAATACGATGACGTGGACAACAATTACGTCGAGACATGGAAGAAAGGCAAGCGTTCGAAGCGCCAACGTGGCGACTCTCCTGCTGCTGTTGAACTTCAACCCACCACCGAAGAAGAGTACCTCGCTCTTTGTCTCATCATGCTCGCTCGCGGCTCTTCCGGTGCTGATCGTGATGTTATTCGTCGGTCTTCCTCTTCGTCGTCA CCGCCTCCGCCGCCGCCTGCTTTGAAGTGTTCTTACAAGTGTAGTGTTTGTGACAAGGCGTTCCCTTCTTATCAAGCTTTGGGCGGTCATAAAGCCAGCCACCGCAAACCCCTTTCCGCCGACGCCGCTACCACCACCGCCGCCGTCAACGTCGATAACCCATCAACAACCAGCACCGCCACCACCATCACCAGCAGCGGTAGGCTTCACGAGTGTTCCAT.

[0088] In some embodiments, the substances that inhibit GhZAT10 gene expression also include pTRV-RNA1 and Agrobacterium GV3101.

[0089] In some embodiments, the silencing of the GhZAT10 gene in the target plant can be achieved using the VIGS system.

[0090] In some embodiments, silencing the GhZAT10 gene in the target plant is specifically achieved by injecting an infection solution into the leaves of the target plant. The infection solution may contain two types of recombinant Agrobacterium: recombinant Agrobacterium obtained by introducing an interference vector into Agrobacterium GV3101 and recombinant Agrobacterium obtained by introducing pTRV RNA1 into Agrobacterium GV3101.

[0091] In summary, this invention provides the application of the GhZAT10 gene in promoting plant tolerance to low potassium stress. It demonstrates that when the GhZAT10 gene is silenced in plants, compared to the control group, the plants are more sensitive to low potassium stress, with significantly reduced K+ uptake, thus affecting K+ accumulation and exhibiting more severe potassium deficiency symptoms. Conversely, when the GhZAT10 gene is overexpressed in plants, compared to the control group, it enhances the plant's resistance to low potassium stress in both low-potassium environments without exogenous K+ and with trace amounts of K+, significantly promoting plant growth. Furthermore, this invention is of significant value for studying low-potassium tolerant plants and exploring the signal regulatory network of plants under stress.

[0092] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0093] In the embodiments described herein, the conventional MS (Murashige and Skoog) medium for Arabidopsis thaliana was prepared as follows: 2.215 g MS salt and 15.0 g sucrose were added, the pH was adjusted to 5.8 using 5M NaOH, 4.0 g agar was added, and the volume was brought up to 500 mL with water. The medium was then sterilized at 115°C under high temperature and autoclave for 20 min and poured into sterile plastic square dishes.

[0094] In this embodiment, the preparation method of Arabidopsis thaliana low-potassium, low-ammonia culture medium is as follows: 50 mL of low-potassium, low-ammonia macronutrient stock solution (10×), 10 mL of low-potassium, low-ammonia micronutrient stock solution (50×), 5 g of sucrose, and 0.2 M KCl according to the required K... + To achieve the final concentration, adjust the pH to 5.8 using 5M NaOH, then add 4.0g of agarose, and bring the volume to 500mL with water. After sterilizing at 115℃ under high pressure for 20 minutes, pour the mixture into sterile plastic square dishes.

[0095] In the embodiments described herein, the method for preparing the macro-element stock solution of 10× low potassium and low ammonium culture medium is as follows: MgSO4·7H2O 3.70g, NH4H2PO4 1.44g, Ca(NO3)2·4H2O 7.06g, diluted with water to 500mL.

[0096] In this embodiment, the method for preparing a trace element stock solution for a 50× low potassium and low ammonium culture medium is as follows: 2.45 g of trace element powder is diluted with water to a final volume of 500 mL.

[0097] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0098] Example

[0099] Example 1: Cloning and localization of GhZAT10 protein and its encoding gene

[0100] 1.1 GhZAT10 gene screening

[0101] This embodiment uses transcriptome differential analysis on cotton "Jin668" subjected to low potassium stress to screen significant genes and performs sequence alignment using a cotton database. The sequence alignment was generated by CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw), and the visualization was created using ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi).

[0102] This embodiment obtained a significantly upregulated gene and aligned it to the protein sequence of Gh_A13G2112. Homologous gene sequences of proteins from other species were searched, generating multiple sequence alignment results for ZAT10 proteins from multiple species. The results are as follows: Figure 1 The diagram illustrates the sequence alignment results of the GhZAT10 protein provided in this embodiment of the invention with ZAT10 proteins from other species. Figure 1 In the diagram, green shading indicates residues with high homology across all species. C2H2-type zinc finger domains (zf-C2H2) and potential nuclear localization signal sequences (Putative NLS) are indicated by black boxes. Pink or blue shading indicates less preserved residuals. Amino acid sequence alignment showed that Gh_A13G2112 has the highest homology with Arabidopsis thaliana AtZAT10 (AT1g27730), therefore this gene was named GhZAT10. A novel protein was obtained from the cotton variety “Jin668” and named GhZAT10.

[0103] 1.2GhZAT10 gene cloning

[0104] (1) Based on the GhZAT10 gene sequence obtained from the cotton database, two specific primers were designed: F1: ATGGAACACTCGTGAAGCCTAC (SEQ ID NO: 6) and R1: CCATTCTAAGGATTGAATCGGT (SEQ ID NO: 7).

[0105] (2) RNA was extracted from cotton leaves using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Beijing Qingke Biotechnology Co., Ltd., Catalog No.: TSP412), and first-strand cDNA was synthesized using the TRUEscript 1st Strand cDNA Synthesis Kit (Beijing Adley Biotechnology).

[0106] (3) The full-length GhZAT10 gene was amplified using a high-fidelity DNA polymerase kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: 10154ES03). The amplification system is shown in Table 1 and Table 2.

[0107] Table 1

[0108]

[0109] Table 2

[0110]

[0111] (4) Electrophoresis of the PCR product on a 1.5% agarose gel. After electrophoresis, cut the target band under UV light and purify it using an agarose gel DNA recovery kit (Suzhou Youyilandi Biotechnology Co., Ltd., catalog number: UE-GX-250). Refer to the instruction manual of the kit for the operation steps.

[0112] (5) Add A to the end of the recovered fragment. The 10 μL reaction system used is: 10×Buffer 1 μL, dATP 1 μL, Taq enzyme 0.5 μL, recovered fragment 7.5 μL, react at 72℃ for 30 min.

[0113] (6) The recovered fragment after adding A was ligated with the PMD18-T vector (purchased from Baori Biotechnology (Beijing) Co., Ltd., catalog number: D101A). The operation was performed according to the instructions. 4.5 μL of the fragment after adding A, 0.5 μL of PMD18-T and 5 μL of Solution I were added to the PCR tube in sequence, with a total volume of 10 μL; the ligation was carried out overnight at 16℃.

[0114] (7) Take 5 μL of the ligation product and transform it into Escherichia coli DH5α (Beijing Qingke Biotechnology Co., Ltd.) using the heat shock method (refer to J. Sambrook et al., translated by Huang Peitang et al., Molecular Cloning: A Laboratory Manual (3rd Edition), Science Press, 2002). Screen positive clones in LB solid plates containing 50 mg / L ampicillin, pick 5 clones for sequencing (sequencing work was done by Beijing Qingke Biotechnology Co., Ltd.), obtain the required full-length gene cDNA and perform sequencing.

[0115] Sequencing results showed that the full-length gene sequence is 807 bp, encoding a complete ORF reading frame of 269 amino acids. The nucleotide sequence of the GhZAT10 gene is shown in SEQ ID NO: 1 (807 bp). The amino acid sequence of the GhZAT10 protein is shown in SEQ ID NO: 4 (269 amino acids).

[0116] 1.3 Subcellular localization of the GhZAT10 protein

[0117] (1) Total RNA was extracted from the roots of “Jin668” using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Beijing Qingke Biotechnology Co., Ltd., Catalog No.: TSP412), and the first strand cDNA was synthesized using the TRUEscript 1st Strand cDNA Synthesis Kit (Beijing Adley Biotechnology).

[0118] (2) PCR amplification was performed using F2 (tacaccaaatcgactctagaATGGCGCTTGAAGCTCTGA, SEQ ID NO: 8) and R2 (ataggtacccgggctctaga ATTTAATTCTAAATCTTGTGCTAATGAAG, SEQ ID NO: 9) as primers. The primers were ligated using homologous recombination, with lowercase letters representing homologous arms and uppercase letters representing gene sequences.

[0119] (3) The obtained PCR amplification product was digested with restriction endonucleases BamHI and StuI, and the pSuper1300::GFP vector was digested with restriction endonuclease XbaI. The product was recovered and homologous recombination was performed. The cDNA sequence of GhZAT10 was constructed on the pSuper1300::GFP expression vector and transiently transformed in tobacco leaves. The results were observed under a microscope.

[0120] Microscopic observation results as follows Figure 2 The diagram shows the subcellular localization results of the GhZAT10 protein according to an embodiment of the present invention. GhZAT10 co-localizes with the nuclear localization marker, indicating that the GhZAT10 protein is a nuclear-localized protein.

[0121] Example 2: Low-KJ Response Patterns of the GhZAT10 Gene in Different Tissues of "Jin668"

[0122] This example uses real-time quantitative PCR to analyze the effects of normal potassium supply treatment (always containing 2.5 mM K) on potassium levels. + Cotton plants cultured in nutrient solution and those treated with low potassium (normally cultured to the three-leaf stage and then placed in a solution containing 0.03 mM K) were compared. + The expression levels of the GhZAT10 gene in the roots, stems, and leaves of cotton plants “Jin668” (which were nutrient-treated for 24 hours) were analyzed.

[0123] (1) Referring to steps 1.3-(1), samples were taken from the roots, stems and leaves of cotton plants under normal potassium supply and low potassium stress, and total RNA was extracted and reverse transcribed to obtain cDNA.

[0124] (2) Then, real-time quantitative PCR was performed using an ABI 7500Fast (Applied Biosystem). The primer pairs used were: F3:TCACGAGTGTTCCATCTGCC (SEQ ID NO: 10) and R3:CGAAGACGTAACCCCGCTAA (SEQ ID NO: 11). The PCR program was: 94℃ denaturation for 30s; 94℃ denaturation for 5s, 60℃ annealing for 35s, 40 cycles; the relative expression level was calculated using 2... -ΔΔCt The method was calculated using the cotton GhUBQ7 gene as a control (the primer pair used to identify the GhUBQ7 gene was: F6: GAAGGCATTCCACCTGACCAAC (SEQ ID NO: 14) and R6: CTTGACCTTCTTCTTCTTGTGCTTG (SEQ ID NO: 15).

[0125] The relative expression levels of the GhZAT10 gene in cotton roots, stems, and leaves at the three-leaf stage under normal potassium supply and low potassium treatments (with the expression level of GhZAT10 in roots under normal potassium supply conditions set as 1) are shown in [reference needed]. Figure 3 GhZAT10 was expressed in the roots, stems, and leaves of cotton plants, and its expression increased after exposure to low potassium stress, indicating that GhZAT10 is induced at the transcriptional level by low potassium stress.

[0126] Example 2: Obtaining and phenotypic identification of transgenic Arabidopsis thaliana

[0127] 2.1 Construction of recombinant plasmids

[0128] (1) Step 1.3-(1) Extract total RNA from the roots of “Jin668” and reverse transcribe it into cDNA.

[0129] (2) Using the cDNA obtained in step (1) as a template, PCR amplification was performed using primers composed of F4 and R4 to obtain PCR amplification products.

[0130] F4: 5'-ctccccttgctccgtggatccATGGCGCTTGAAGCTCTGA-3'; (SEQ ID NO: 2)

[0131] R4: 5'-aacgtcgtatgggtaaggcctATTTAATTCTAAATCTTGTGCTAATGAAG-3'. (SEQ IDNO: 3)

[0132] (3) The PCR amplification product obtained in step (2) was digested with restriction endonucleases BamHI and StUI, and the digested product was recovered.

[0133] (4) The pCAMBIA2300::HA vector was digested with restriction endonucleases BamHI and StuI, and the vector backbone was recovered.

[0134] (5) Connect the enzyme digestion product from step (3) with the vector backbone from step (4) to obtain the recombinant plasmid pCAMBIA2300::HA-GhZAT10.

[0135] Based on the sequencing results, the structure of the recombinant plasmid pCAMBIA2300::HA-GhZAT10 is as follows: A double-stranded DNA molecule as shown in SEQ ID NO: 1 was inserted between the BamHI and StUI restriction sites of the pCAMBIA2300::HA vector.

[0136] 2.2 Preparation of Arabidopsis transgenic plants

[0137] (1) After Arabidopsis thaliana bolts one week after being transplanted into the soil, remove the top and cultivate until it reaches the flowering stage with multiple branches.

[0138] (2) The plasmid pCAMBIA2300::HA-GhZAT10 obtained in 2.1 was transformed into Agrobacterium competent cells GV3101 and spread on YEP solid medium containing the corresponding antibiotics (500 mL YEP solid medium contains 50 μg / mL kanamycin and 50 μg / mL gentamicin) and cultured for 2-3 days.

[0139] (3) Select the correct positive clones and inoculate them into 5 mL of YEP liquid culture medium containing the corresponding antibiotics. Incubate overnight at 28°C with shaking at 200 rpm.

[0140] (4) Transfer the culture medium to 25 mL of YEP liquid medium containing the corresponding antibiotic at a ratio of 1:50, and incubate at 28°C with shaking at 200 rpm until OD. 600 =1.2~1.8.

[0141] (5) Centrifuge the bacterial culture from (4) at 4000 rpm for 15 min, discard the supernatant, and resuspend the bacterial culture in Arabidopsis thaliana osmotic conversion buffer (1 L of osmotic conversion buffer contains 2.2 g MS powder, 50 g sucrose, 200 μ L Silwet L-77, 200 μ L 6-BA (5 mg·mL–1), pH = 5.60), and adjust the OD value. 600 =0.8~1.0, to obtain the conversion solution.

[0142] (6) Pour the conversion solution into a 9cm diameter plastic round petri dish. Place the Arabidopsis thaliana plants (with flowers, siliques, and exposed flower buds removed) sideways in the conversion solution and soak for 1–3 minutes. After conversion, place the Arabidopsis thaliana plants sideways in a tray and cover them with a black plastic bag to protect them from light. After 16–24 hours, stand the flowerpots upright, label them to prevent cross-contamination with other materials, and cultivate them normally in a greenhouse. After about one month, harvest the seeds from the converted material, which are the T1 generation.

[0143] (7) After disinfection, T1 generation seeds that have been dried for more than 7 days are sown on MS solid medium containing antibiotics and cultured in a light incubator. Seedlings with green leaves and normal growth are selected and transplanted into the soil. T2 generation seeds are harvested from each seedling.

[0144] (8) Statistical analysis of the segregation ratio of seeds in the T2 generation: Prepare 100-200 Arabidopsis thaliana seeds, disinfect them, and sow them on a resistant culture medium for culture and observation. If the ratio of positive seedlings to negative seedlings is 3:1 and the chi-square test value is less than 3.841, this line is a single copy insertion. Propagate this line and harvest T3 generation seeds. Generally, about 15 plants are planted for each line.

[0145] (9) Take about 100 seeds from the T3 generation and screen for resistance again. If all seedlings are positive, the line is a single-copy insertion homozygous overexpression line.

[0146] (10) The homozygous overexpression lines were grown on low potassium and low ammonium medium for 10 days, and the plant growth phenotype under low potassium stress was identified.

[0147] As a result of the breeding, two Arabidopsis thaliana lines (OE#9 and OE#14) overexpressing GhZAT10 were obtained. The T-DNA of Arabidopsis thaliana AtZAT10 was inserted into the mutant atzat10 material.

[0148] The results of plant growth phenotypic identification are shown below Figure 4 . Figure 4 The results show the effects of the GhZAT10 gene on plant growth phenotypes under different potassium ion conditions, wherein... Figure 4 Part A shows the growth phenotypes of wild-type Arabidopsis thaliana (Col-0), the T-DNA insertion mutant of Arabidopsis thaliana AtZAT10 (atzat10), and Arabidopsis thaliana lines overexpressing GhZAT10 (OE#9 and OE#14) after 10 days of cultivation on low-potassium, low-ammonia medium under conditions of no exogenous K+, 0.015 mM K+, and 5 mM K+, respectively. The root length photographs show the growth phenotypes under conditions of no exogenous K+, 0.015 mM K+, and 5 mM K+. + And add 0.015mM K +On the culture medium, OE#9 and OE#14 were significantly greater than Col-0, indicating that GhZAT10 overexpression is beneficial to the growth of Arabidopsis thaliana in low potassium and low ammonium culture and is more conducive to Arabidopsis thaliana resisting low potassium stress.

[0149] Figure 4 Part B shows a schematic diagram comparing the primary root lengths of Col-0, atzat10, OE#9, and OE#14 cultured on low-potassium, low-ammonia medium for 10 days under conditions of no exogenous K+ addition, addition of 0.015 mM K+, and addition of 5 mM K+, respectively. The root length statistics show that without exogenous K+ addition... + And add 0.015mM K + On the culture medium, the main root lengths of OE#9 and OE#14 were significantly greater than those of Col-0, indicating that GhZAT10 overexpression is beneficial to the growth of Arabidopsis thaliana in low potassium and low ammonium culture and is more conducive to Arabidopsis thaliana resisting low potassium stress.

[0150] Example 3: Identification of growth phenotypes in VIGS-GhZAT10 silent plants

[0151] 3.1 Construction of GhZAT10 recombinant plasmid

[0152] (1) Total RNA was extracted from the leaves of cotton variety “Jin668” and reverse transcribed into cDNA.

[0153] (2) Using the cDNA obtained in step (1) as a template, PCR amplification was performed using primers consisting of F5 and R5 to obtain the PCR amplification product.

[0154] F5: gtgagtaaggttaccgaattcGCCTTTCACCAACAAATACGAT (SEQ ID NO: 12)

[0155] R5: gagacgcgtgagctcggtaccATGGAACACTCGTGAAGCCTAC (SEQ ID NO: 13)

[0156] (3) The PCR amplification product obtained in step (2) was digested with restriction endonucleases EcoRI and KpnI, and the digested product was recovered.

[0157] (4) The pTRV2 vector was digested with restriction endonucleases EcoRI and KpnI, and the vector backbone was recovered.

[0158] (5) The enzyme digestion product from step (3) and the vector backbone from step (4) are linked to obtain the recombinant plasmid pTRV2-GhZAT10.

[0159] Based on the sequencing results, the recombinant plasmid pTRV2-GhZAT10 was described as follows: a double-stranded DNA molecule, as shown in SEQ ID NO: 5, was inserted between the EcoRI and KpnI restriction sites of the pTRV2 vector.

[0160] SEQ ID NO: 5: GCCTTTCACCAACAAATACGATGACGTGGACAACAATTACGTCGAGACATGGAAGAAAGGCAAGCGTTCGAAGCGCCAACGTGGCGACTCTCCTGCTGCTGTTGAACTTCAACCCACCACCGAAGAAGAGTACCTCGCTCTTTGTCTCATCATGCTCGCTCGCGGCTCTTCCGGTGCTGATCGTGATGTTATTCGTCGGTCTTCCTCTTCGTCGTCA CCGCCTCCGCCGCCGCCTGCTTTGAAGTGTTCTTACAAGTGTAGTGTTTGTGACAAGGCGTTCCCTTCTTATCAAGCTTTGGGCGGTCATAAAGCCAGCCACCGCAAACCCCTTTCCGCCGACGCCGCTACCACCACCGCCGCCGTCAACGTCGATAACCCATCAACAACCAGCACCGCCACCACCATCACCAGCAGCGGTAGGCTTCACGAGTGTTCCAT.

[0161] 3.2 Obtaining VIGS-GhZAT10 Silent Plants

[0162] (1) The recombinant plasmid pTRV2-GhZAT10 from 3.1 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium.

[0163] (2) The recombinant Agrobacterium was resuspended in VIGS solution (500 mL YEP liquid medium containing 50 μg / mL kanamycin, 50 μg / mL gentamicin, 10 mM 2-(N-morpholine)ethanesulfonic acid, and 20 μM acetylsuccinone) to obtain OD. 600nm =1.5 bacterial solution.

[0164] (2) The pTRV1 vector was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was suspended in VIGS solution to obtain OD. 600nm =1.5 bacterial solution.

[0165] (3) Mix the bacterial solution obtained in step (1) and the bacterial solution obtained in step (2) in equal volumes to obtain mixture A.

[0166] (4) pTRV-GhCLA1, pTRV2-GhZAT10, and pTRV-GFP were introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was suspended in VIGS solution, and OD was obtained. 600nm =1.5 bacterial solution.

[0167] (5) Mix the pTRV-GhCLA1, pTRV2-GhZAT10 and pTRV-GFP bacterial solutions obtained in step (4) with the bacterial solution obtained in step 2 in equal volumes to obtain mixtures B, C and D respectively.

[0168] (6) The mixture was injected in groups, and the specific grouping was as follows:

[0169] Experimental group: The Xinshi 17 plants in the cotyledon stage were operated on by injecting mixed solution A into the lower surface of the cotyledons (two cotyledons of each plant were injected until the cotyledons were filled).

[0170] Positive control group: The Xinshi 17 plants in the cotyledon stage were operated on by injecting a mixture of B and C into the lower surface of the cotyledons (two cotyledons of each plant were injected until the cotyledons were completely filled).

[0171] Negative control group: The Xinshi 17 plants at the cotyledon stage were operated on by injecting the mixed solution D into the lower surface of the cotyledons (two cotyledons of each plant were injected until the cotyledons were filled).

[0172] All groups of plants were treated with Hoagland's nutrient solution (2.5 mM KNO3, 2.5 mM Ca(NO3)2, 1 mM MgSO4). 4, 0.5 mM (NH4)H2PO4, 0.1 mM FeNa-EDTA, and trace elements (2 × 10⁻⁶) -4 mM CuSO4, 1×10 -3 mM ZnSO4,2×10 -2 mM H3BO3, 5×10 -6 mM(NH4)6Mo7O 24 and 1×10 -3 Hydroponics are carried out using mM MnSO4, with the nutrient solution changed weekly.

[0173] Thus, VIGS-GhZAT10 silenced plants (experimental group), VIGS-GhCLA1 control plants (positive control group), and VIGS-Ctrl control plants (negative control group) were obtained. Among them, the CLA1 gene encodes 1-deoxyxylulose 5-phosphate synthase, which is involved in chloroplast development and is highly conserved in evolution. Silencing the CLA1 gene results in white leaves, which is an easily identifiable marker trait. Therefore, VIGS-GhCLA1 can be used as a positive control.

[0174] 3.3 Phenotypic determination of VIGS-GhZAT10 silent plants

[0175] Cotton plants with silenced GhZAT10 genes were obtained using the method described in 3.2. Control plants injected with pTRV2-GhCLA1 bacterial solution 7 days after injection showed an albino phenotype. The VIGS-GhZAT10 silenced plants and the control VIGS-Ctrl were then treated with normal nutrient solution (CK, 2.5 mM K). + ) and low-potassium nutrient solution (LK, 0.03mM K) + Incubate for 21 days.

[0176] (1) Detect the gene silencing efficiency of silent plants.

[0177] (2) Phenotypic observation. The dry weight was measured by drying the plants at 80℃ to constant weight and weighing them; the dry weight of the leaves / stems / roots was measured. Twelve plants were sampled from each group under each treatment, and the average value was taken.

[0178] Figure 5 The results of the silencing efficiency of the GhZAT10 gene in plants according to an embodiment of the present invention are shown, where VIGS-GhZAT10 represents plants with the GhZAT10 gene silenced, and VIGS-Ctrl represents the control group. It can be seen that the GhZAT10 gene was successfully silenced.

[0179] Figure 6 The growth phenotype of GhZAT10 gene-silenced plants in this embodiment of the invention is shown. Figure 6 Part A shows the whole-plant growth phenotypes of the GhZAT10 gene-silenced plant VIGS-GhZAT10 and the control group VIGS-Ctrl under normal and low potassium stress conditions. Figure 6 Part B shows the leaf phenotypes of VIGS-GhZAT10 and VIGS-Ctrl under normal and low potassium stress conditions. Under low potassium conditions, the experimental group plants showed more severe leaf yellowing compared to the negative control group.

[0180] Figure 7This illustration shows the potassium accumulation in various parts of the GhZAT10 gene-silenced plant VIGS-GhZAT10 and the control group VIGS-Ctrl under normal and low potassium stress conditions in this embodiment of the invention. Specifically, under low potassium treatment conditions, compared with the negative control group, the potassium accumulation in various parts (roots, stems, and leaves) of the experimental group plants was [not specified]. + The content was lower. Under normal potassium supply conditions, compared with the control plants, the K content in various parts (roots, stems, and leaves) of the experimental group plants was lower. + There were no significant differences in content.

[0181] The above results indicate that the experimental group plants (i.e., GhZAT10 gene-silenced plants) are more sensitive to low potassium.

[0182] 3.4 Determination of root potassium ion uptake rate in VIGS-GhZAT10 silent plants

[0183] (1) After obtaining VIGS-GhZAT10 silent plants according to the method in 3.2, they were grown to the three-leaf stage under culture conditions with sufficient potassium supply.

[0184] (2) VIGS-GhZAT10 silent plants were divided into two groups, one group receiving normal potassium supply (CK, 2.5 mM K). + Another group was cultured under low potassium stress (LK, 0.03 mM K), while the other group was cultured under low potassium stress (LK, 0.03 mM K). + Cultured for 8 days under the following conditions.

[0185] (3) Select plants of similar size for K testing. + The absorption rate was determined by testing all VIGS plants for K... + After 2 days of starvation treatment, the initial K is replaced. + The K in the depleted solution with a concentration of 0.08 mM was measured after 10 hours of depletion. + Content, converted to K per unit fresh weight per unit time + Absorption rate.

[0186] Figure 8 The net K+ uptake rate of the GhZAT10 gene-silenced cotton seedlings VIGS-GhZAT10 and the control group VIGS-Ctrl is shown in this embodiment of the invention. Specifically, the K+ uptake rate of the GhZAT10 gene-silenced cotton seedlings... + The absorption capacity is significantly reduced, which in turn affects the potassium content in the plant. + The accumulation of these substances leads to more severe potassium deficiency symptoms.

[0187] The above results indicate that the experimental group plants (i.e., GhZAT10 gene-silenced plants) K + Absorption capacity is significantly reduced.

[0188] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0189] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0190] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0191] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. GhZAT10 The application of genes in promoting plant tolerance to low potassium stress is characterized by, include: By making the plant GhZAT10 Overexpression to improve the plant's ability to tolerate low potassium stress and / or enhance the plant's potassium uptake capacity, wherein... GhZAT10 The nucleotide sequence is shown in SEQ ID NO:

1. The plant in question is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The plant GhZAT10 Overexpression includes: Will GhZAT10 Recombinant vectors are obtained by inserting DNA fragments of genes into vectors; and The recombinant vector was introduced into the plant, so that the GhZAT10 Overexpression.

3. The application according to claim 1, characterized in that, GhZAT10 The gene encodes the GhZAT10 protein, which has the amino acid sequence shown in SEQ ID NO:

4.

4. Any one of the following (a)-d) can be used to promote plant tolerance to low potassium stress; a) GhZAT10 protein having the amino acid sequence shown in SEQ ID NO: 4; b) A recombinant vector containing the nucleotide sequence shown in SEQ ID NO: 1; c) Recombinant microorganisms containing the recombinant vector shown in b); d) Expression cassettes and / or transgenic cell lines containing the contents shown in SEQ ID NO: 1 GhZAT10 The nucleotide sequence of a gene The plant in question is Arabidopsis thaliana.

5. A method for improving the tolerance of plants to low potassium stress, characterized in that, Includes at least one of the following: The plant GhZAT10 Gene overexpression, wherein GhZAT10 The nucleotide sequence is shown in SEQ ID NO: 1; To enhance the activity of the GhZAT10 protein in the plant, wherein the GhZAT10 protein has the amino acid sequence shown in SEQ ID NO: 4; and To increase the content of GhZAT10 protein in the plant, The plant in question is Arabidopsis thaliana.

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