Application of GhZAT10 gene in promoting low potassium stress resistance of plant

Abstract

The invention relates to the field of plant breeding, in particular to application of a GhZAT10 gene in promoting low potassium stress tolerance of plants, a recombinant vector, a recombinant microorganism, a method for improving the low potassium stress tolerance of the plants and a plant breeding method. According to the application of the GhZAT10 gene in promoting the low potassium stress tolerance of the plant, the GhZAT10 in the plant is over-expressed to improve the low potassium stress tolerance of the plant and / or improve the potassium ion absorption capacity of the plant, and the nucleotide sequence of the GhZAT10 is shown as SEQ ID NO: 1. According to the application of the GhZAT10 gene in promoting the low-potassium stress resistance of the plant, the low-potassium stress resistance of the plant can be improved in a low-potassium environment in which K < + > is not added and a trace amount of K < + > is added in an external source, and the growth of the plant is remarkably promoted.

Description

Technical Field

[0001] The present application relates to the field of plant breeding, and specifically relates 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 Art

[0002] Potassium (K+) is one of the most abundant nutrient elements in the soil circle and is 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 the cultivated land area already faces potassium deficiency problems, which seriously restricts agricultural production in China.

[0003] Cotton (Gossypium hirsutum L.) is a potassium-loving cash crop. The absorption and utilization of potassium are closely related to the yield and quality of cotton. Potassium can not only promote the growth and development of the upper part and roots of cotton but also increase dry matter accumulation. In recent years, with the gradual increase in the multiple cropping index and yield of cotton, as well as the popularization of transgenic insect-resistant cotton, the degree of potassium deficiency in cotton fields in China has become more serious. In addition, due to unreasonable fertilizer management, the potassium deficiency in cotton production is further exacerbated, often leading to premature senescence and seriously affecting yield and quality.

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

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

[0006] To this end, a first aspect embodiment of the present invention provides an application of the GhZAT10 gene in promoting the ability of plants to tolerate low potassium stress, including: improving the ability of the plants to tolerate low potassium stress and / or enhancing the potassium ion absorption ability of the plants by overexpressing the GhZAT10 in the plants, wherein the nucleotide sequence of the GhZAT10 is as shown in SEQ ID NO:1. According to the application of the GhZAT10 gene in promoting the ability of plants to tolerate low potassium stress provided by the embodiments of the present invention, the ability of plants to resist low potassium stress can be improved in a low potassium environment without exogenous addition of K+ and with the addition of a small amount of K+, and the growth of plants can be significantly promoted.

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

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

[0009] In some embodiments, the obtaining of the recombinant vector by inserting the DNA fragment of the GhZAT10 gene into a vector includes: double-digesting the pCAMBIA2300::HA vector with restriction endonucleases BamHI and StuI to obtain a vector framework; ligating the DNA fragment of the GhZAT10 gene with the vector framework, so as to obtain the recombinant vector pCAMBIA2300::HA-GhZAT10.

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

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

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

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

[0014] An embodiment of the third aspect of the present invention provides a recombinant microorganism, comprising the recombinant vector according to the embodiment of the first aspect of the present invention.

[0015] An embodiment of the fourth aspect of the present invention provides the use of any one of a)-d) below in promoting the ability of plants to tolerate low potassium stress;

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

[0017] b) The recombinant vector according to the embodiment of the second aspect of the present invention;

[0018] c) The recombinant microorganism according to the embodiment of the third aspect of the present invention;

[0019] d) An expression cassette and / or a transgenic cell line, which comprises the nucleotide sequence of the GhZAT10 gene shown in SEQ ID NO:1 or a sequence with a homology of more than 90% to SEQ ID NO:1.

[0020] An embodiment of the fifth aspect of the present invention provides a method for improving the low-potassium stress tolerance of plants, which is characterized by including at least one of the following:

[0021] Overexpressing the GhZAT10 gene in the plant;

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

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

[0024] An embodiment of the sixth aspect of the present invention provides a plant breeding method, which is characterized by including:

[0025] Overexpressing the GhZAT10 gene in the plant to obtain a transgenic plant with low-potassium stress tolerance. The nucleotide sequence of the GhZAT10 gene is shown in SEQ ID NO:1. The plant is a monocotyledon or a dicotyledon. Preferably, the plant is Arabidopsis thaliana or cotton.

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

[0027] The present invention provides the application of the GhZAT10 gene in promoting the low-potassium stress tolerance of plants, and it is proved that when the GhZAT10 gene is silenced in the plant, compared with the control group, it is more sensitive to low-potassium stress, the K+ absorption ability is significantly reduced, which further affects the accumulation of K+ in the plant, showing more serious potassium deficiency symptoms; while when the GhZAT10 gene is overexpressed in the plant, compared with the control group, in a low-potassium environment without adding exogenous K+ and adding trace amounts of K+, the ability of the plant to resist low-potassium stress can be improved, and the growth of the plant can be significantly promoted; and it has important value for studying low-potassium tolerant plants and exploring the signal regulation network of plants under adversity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shows a schematic diagram of the alignment result of the GhZAT10 protein provided by the embodiment of the present invention with the ZAT10 protein sequences of other species;

[0029] Figure 2 Shows the subcellular localization result map of the GhZAT10 protein of the embodiment of the present invention;

[0030] Figure 3Schematic diagram of the gene expression levels of the GhZAT10 gene of the embodiments of the present invention in different tissue parts of cotton under normal and low potassium stress conditions, where * represents the result of differential significance analysis with p < 0.05; ** represents the result of differential significance analysis with p < 0.01; *** represents the result of differential significance analysis with p < 0.001;

[0031] Figure 4 Shows the influence results of the GhZAT10 gene of the embodiments of the present invention on the plant growth phenotype under different potassium ion conditions, where Figure 4 Part A shows the growth phenotype results of Arabidopsis wild type (Col-0), T-DNA insertion mutant material of Arabidopsis AtZAT10 (atzat10), and Arabidopsis lines overexpressing GhZAT10 (OE#9, OE#14) on a low potassium and low ammonium medium, cultured for 10 days under the conditions of no exogenous addition of K+, addition of 0.015 mM K+, and addition of 5 mM K+ respectively; Figure 4 Part B shows a schematic diagram of the comparison results of the primary root lengths of Col-0, atzat10, OE#9, and OE#14 cultured for 10 days on a low potassium and low ammonium medium under the conditions of no exogenous addition of K+, addition of 0.015 mM K+, and addition of 5 mM K+ respectively;

[0032] Figure 5 Shows the detection results of the silencing efficiency of the GhZAT10 gene of the embodiments of the present invention in plants, where VIGS-GhZAT10 is a plant with the GhZAT10 gene silenced, and VIGS-Ctrl is a control group, where * represents the result of differential significance analysis with p < 0.05; ** represents the result of differential significance analysis with p < 0.01; *** represents the result of differential significance analysis with p < 0.001;

[0033] Figure 6 Shows the growth phenotype of the plant with the GhZAT10 gene silenced in the embodiments of the present invention, Figure 6 Part A shows the whole plant growth phenotypes of the plant with the GhZAT10 gene silenced 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 7Shows the potassium accumulation in various parts of the GhZAT10 gene-silenced plants VIGS-GhZAT10 and the control group VIGS-Ctrl in the present invention embodiments under normal and low potassium stress conditions, where * represents the result of significant difference analysis as p < 0.05; ** represents the result of significant difference analysis as p < 0.01; *** represents the result of significant difference analysis as p < 0.001;

[0035] Figure 8 Shows the net K+ uptake rate of the GhZAT10 gene-silenced plants VIGS-GhZAT10 and the control group VIGS-Ctrl in the present invention embodiments, where * represents the result of significant difference analysis as p < 0.05; ** represents the result of significant difference analysis as p < 0.01; *** represents the result of significant difference analysis as p < 0.001. Detailed implementation manners

[0036] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and should not be construed as a limitation to the present invention.

[0037] The present invention is made based on the inventor's discovery and recognition of the following facts and problems:

[0038] With the popularization of Bt transgenic insect-resistant cotton in the Yellow River Basin cotton area and the Yangtze River Basin cotton area, as well as unreasonable fertilizer and water management, the potassium deficiency phenomenon of cotton in China is becoming more and more serious. Cotton is a potassium-loving crop, and potassium deficiency leads to an increasingly serious premature senescence phenomenon of cotton, which seriously affects the yield and quality of cotton. Solving the potassium deficiency problem of cotton is of great significance for improving the cotton yield and quality. With the continuous development of molecular biology, it has become possible to cultivate cotton varieties resistant to low potassium by using biotechnology means. Although certain achievements have been made in the gene cloning work of cotton currently, the gene cloning work of cotton lags far behind that of food crops such as rice, corn, and wheat.

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

[0040] Virus-induced gene silencing (VIGS), as an effective reverse genetics technique, is widely used for the identification of plant genome functions. Virus-induced gene silencing can successfully silence endogenous genes in different parts of plants, providing a practical means for studying gene functions at different growth stages. In the embodiments of this application, VIGS method was used to provide GhZAT10 gene-silenced plants to prove the function of this gene.

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

[0042] In this article, the descriptions of pTRV2 (pYL156, pTRV-RNA2) and pTRV-GhCLA1 by 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 + Uptake in Root under Potassium Deficiency, Plant and Cell Physiology, 60(4), 888-899) are incorporated herein by reference.

[0043] In this article, the description of the pCAMBIA2300::HA vector by 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 anthracnose via affecting hormone signaling pathways and lignin production. BMC genomics, 25(1), 895) is incorporated herein by reference.

[0044] In this article, the upland cotton variety "Jin668" refers to 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 lateral meristem. Genome Biology, 24(1), 194).

[0045] In this article, low - potassium stress refers to the amount or concentration of potassium available to the target plant being lower than the amount or concentration required for its normal growth and development. In some embodiments, it can be a potassium ion concentration ≤ 0.1 mM or less. In some embodiments, it can be a potassium ion concentration ≤ 0.05 mM or less. In some embodiments, it can be a potassium ion concentration ≤ 0.03 mM. But it is not zero.

[0046] In this article, the biological phenotypes sensitive to low - potassium stress include a decrease in the biomass of plants (such as root dry weight, stem dry weight, leaf dry weight, etc.) and a decrease in the net potassium ion absorption rate.

[0047] The application of the GhZAT10 gene in promoting the ability of plants to tolerate low potassium stress in the first aspect of the present invention includes: improving the ability of the plant to tolerate low potassium stress and / or enhancing the potassium ion absorption ability of the plant by overexpressing the GhZAT10 in the plant, wherein the nucleotide sequence of the GhZAT10 is as shown in SEQ ID NO: 1. According to the application of the GhZAT10 gene provided by the embodiments of the present invention in promoting the ability of plants to tolerate low potassium stress, the ability of plants to resist low potassium stress can be improved in a low potassium environment without adding K+ externally and with a trace amount of K+ added, and the growth of plants can be significantly promoted. In addition, this application explores the function of the cotton potassium transporter GhZAT10 in cotton potassium absorption, analyzes the regulatory pathway of GhZAT10 in the process of cotton responding to low potassium stress, studies the gene expression and regulation at the molecular level under low potassium stress, improves the signal transmission and gene expression regulation network under stress conditions, and conducts a more in-depth study on the response mechanism of plants to low potassium stress signals, laying a good molecular foundation for effectively improving the low potassium stress tolerance of plants.

[0048] The nucleotide sequence of the GhZAT10 gene is shown in SEQ ID NO:1: ATGGCGCTTGAAGCTCTGACCTCGCCGGCGACGCCTTTCACCAACAAATACGATGACGTGGACAACAATTACGTCGAGACATGGAAGAAAGGCAAGCGTTCGAAGCGCCAACGTGGCGACTCTCCTGCTGCTGTTGAACTTCAACCCACCACCGAAGAAGAGTACCTCGCTCTTTGTCTCATCATGCTCGCTCGCGGCTCTTCCGGTGCTGATCGTGATGTTATTCGTCGGTCTTCCTCTTCGTCGTCACCGCCTCCGCCGCCGCCTGCTTTGAAGTTGTCTTACAAGT GTAGTGTTTGTGACAAGGCGTTCCCTTCTTATCAAGCTTTGGGCGGTCATAAAGCCAGCCACCGCAAACCCCTTTCCGCCGACGCCGCTACCACCACCGCCGCCGTCAACGTCGATAACCCATCAACAACCAGCACCGCCACCACCATCACCAGCAGCGGTAGGCTTCACGAGTGTTCCATCTGCCACAAGAGTTTCCCTACGGGCCAAGCCTTGGGTGGTCATAAACGCTGCCACTACGAAGGTGGCAACAACAACAACAAAAATAACAACAACAGCGGTAGCGTTAGCGTTAGCGTTAGCGGGGTTACGTCTTCGGATGGGGGGGCGTTGAGCCACAACCACCGTGCAGTCGACTTTGACTTTGACCTCAACTTGCCAGCCTTGCCGGAGTTCAGTCAAATGTACCCAGATGAAGAAGAGGTTCAAAGCCCATTGCCGACCAAGAAACCACGTCTCTTGATCGCCAAGAAAGAGAAACTGGATTCTTCATTAGCACAAGATTTAGAATTAAATTGA(SEQ ID NO:1).

[0049] In some embodiments, overexpressing the GhZAT10 in the plant includes: 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 the GhZAT10.

[0050] In some embodiments, the RNA of the plant is reverse transcribed to obtain template DNA, and PCR amplification is performed based on the template DNA using the primer pairs shown in SEQ ID NO:2 and SEQ ID NO:3, thereby obtaining 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, inserting the DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector includes: using restriction endonucleases BamHI and StuI to double digest the pCAMBIA2300::HA vector to obtain a vector framework; ligating the DNA fragment of the GhZAT10 gene with the vector framework, thereby obtaining the recombinant vector pCAMBIA2300::HA-GhZAT10.

[0054] In some embodiments, the primer pairs for PCR amplification further include the sequences shown below:

[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 monocotyledon or a dicotyledon; preferably, the plant is Arabidopsis thaliana or cotton.

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

[0064] SEQ ID NO: 4: MALEALTSPATPFTNKYDDVDNNYVETWKKGKRSKRQRGDSPAAVELQPTTEEEYLALCLIMLARGSSGADRDVIRRSSSSSSPPPPPPALKLSYKCSVCDKAFPSYQALGGHKASHRKPLSADAATTTAAVNVDNPSTTSTATTITSSGRLHECSICHKSFPTGQALGGHKRCHYEGGNNNNKNNNNSGSVSVSVSGVTSSDGGALSHNHRAVDFDFDLNLPALPEFSQMYPDEEEVQSPLPTKKPRLLIAKKEKLDSSLAQDLELN.

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

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

[0067] An embodiment of the third aspect of the present invention provides a recombinant microorganism comprising the recombinant vector according to the embodiment of the first aspect of the present invention.

[0068] An embodiment of the fourth aspect of the present invention provides the use of any one of a)-d) below in promoting the ability of plants to tolerate low potassium stress;

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

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

[0071] c) a recombinant microorganism as described in the embodiment of the third aspect of the present invention;

[0072] d) an expression cassette and / or a transgenic cell line, which contains the nucleotide sequence of the GhZAT10 gene shown in SEQ ID NO:1 or a sequence with a homology of more than 90% to SEQ ID NO:1.

[0073] The embodiment of the fifth aspect of the present invention provides a method for improving the ability of a plant to tolerate low potassium stress, which is characterized by including at least one of the following:

[0074] overexpressing the GhZAT10 gene in the plant;

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

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

[0077] The embodiment of the sixth aspect of the present invention provides a plant breeding method, which is characterized by including: overexpressing the GhZAT10 gene in the plant to obtain a transgenic plant tolerant to low potassium stress, the nucleotide sequence of the GhZAT10 gene is as shown in SEQ ID NO:1, the plant is a monocotyledon or a dicotyledon, and preferably, the plant is Arabidopsis thaliana or cotton.

[0078] The embodiment of the present invention also provides a protein, obtained from cotton (Gossypium hirsutum), specifically obtained 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 residue substitutions and / or deletions and / or additions and related to the ability of the plant to tolerate low potassium stress and / or the ability to absorb potassium ions.

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

[0080] The embodiment of the present invention also provides the application of a substance that inhibits the expression of the GhZAT10 gene in cultivating gene-silenced plants

[0081] In some embodiments, the expression level of the GhZAT10 protein or the GhZAT10 gene in a plant is decreased, and the low-potassium stress tolerance of the plant is decreased, and / or the potassium ion absorption capacity is decreased.

[0082] In some embodiments, the expression level of the GhZAT10 protein or the GhZAT10 gene in a plant is increased, and the low-potassium stress tolerance of the plant is increased, and / or the potassium ion absorption capacity is increased.

[0083] The embodiments of the present invention further provide a method for cultivating a gene-silenced plant, which is to silence the GhZAT10 gene in a target plant to obtain a silenced plant with a lower dry matter accumulation capacity than the target plant under low-potassium stress;

[0084] and / or, a silenced plant with a lower potassium ion absorption capacity than the target plant under low-potassium stress;

[0085] and / or, a silenced plant with a lower potassium content than the target plant under low-potassium stress.

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

[0087] SEQ ID NO: 5: GCCTTTCACCAACAAATACGATGACGTGGACAACAATTACGTCGAGACATGGAAGAAAGGCAAGCGTTCGAAGCGCCAACGTGGCGACTCTCCTGCTGCTGTTGAACTTCAACCCACCACCGAAGAAGAGTACCTCGCTCTTTGTCTCATCATGCTCGCTCGCGGCTCTTCCGGTGCTGATCGTGATGTTATTCGTCGGTCTTCCTCTTCGTCGTCACCGCCTCCGCCGCCGCCTGCTTTGAAGTTGTCTTACAAGTGTAGTGTTTGTGACAAGGCGTTCCCTTCTTATCAAGCTTTGGGCGGTCATAAAGCCAGCCACCGCAAACCCCTTTCCGCCGACGCCGCTACCACCACCGCCGCCGTCAACGTCGATAACCCATCAACAACCAGCACCGCCACCACCATCACCAGCAGCGGTAGGCTTCACGAGTGTTCCAT。

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

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

[0090] In some embodiments, silencing the GhZAT10 gene in the target plant is specifically achieved by injecting the infiltration solution into the leaves of the target plant. The infiltration solution can have the bacterial solutions of the following two recombinant Agrobacterium tumefaciens: the recombinant Agrobacterium tumefaciens obtained by introducing the interference vector into Agrobacterium tumefaciens GV3101 and the recombinant Agrobacterium tumefaciens obtained by introducing pTRV RNA1 into Agrobacterium tumefaciens GV3101.

[0091] In summary, the present invention provides the application of the GhZAT10 gene in promoting the ability of plants to tolerate low potassium stress, and proves that when the GhZAT10 gene is silenced in plants, compared with the control group, it is more sensitive to low potassium stress, the K+ absorption ability is significantly reduced, thereby affecting the accumulation of K+ in the plant body, showing more serious potassium deficiency symptoms; while when the GhZAT10 gene is overexpressed in plants, compared with the control group, it can improve the ability of plants to resist low potassium stress and significantly promote the growth of plants in a low potassium environment without exogenous addition of K+ and with trace addition of K+; and it has important value for studying low potassium-tolerant plants and exploring the signal regulation network of plants under adversity.

[0092] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0093] In the examples of this article, the conventional method for preparing Arabidopsis thaliana MS (Murashige and Skoog) medium: 2.215 g of MS salts, 15.0 g of sucrose, adjust the pH to 5.8 with 5 M NaOH, then add 4.0 g of agar, and make up the volume to 500 mL with water. After autoclaving at 115 °C and high pressure for 20 min, pour it into a sterile plastic square dish.

[0094] In the examples of this article, the method for preparing Arabidopsis thaliana low-potassium and low-ammonium medium: 50 mL of low-potassium and low-ammonium macroelement stock solution (10×), 10 mL of low-potassium and low-ammonium microelement stock solution (50×), 5 g of sucrose, 0.2 M KCl added according to the required final concentration of K + Adjust the pH to 5.8 with 5 M NaOH, then add 4.0 g of agarose, and make up the volume to 500 mL with water. After autoclaving at 115 °C and high pressure for 20 min, pour it into a sterile plastic square dish.

[0095] In the examples of this article, the method for preparing the 10× low-potassium and low-ammonium macroelement stock solution of the medium: 3.70 g of MgSO 4 ·7H 2 O, 1.44 g of NH 4 H 2 PO 4 1, 7.06 g of Ca(NO 3 ) 2 ·4H 2 O, and make up the volume to 500 mL with water.

[0096] In the examples of this article, the method for preparing the 50× low-potassium and low-ammonium microelement stock solution of the medium: 2.45 g of trace element powder, and make up the volume to 500 mL with water.

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

[0098] Example

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

[0100] 1.1 Screening of GhZAT10 gene

[0101] In this example, transcriptome differential analysis was performed on cotton "Jin668" under low potassium stress treatment to screen for significant genes, and sequence alignment was performed using the cotton database. The sequence alignment was generated by CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw), and visualization was created using ESPript3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi).

[0102] In this example, significantly up-regulated genes were obtained, and their protein sequences were aligned to the protein sequence of Gh_A13G2112. Homologous gene sequences of proteins from other species were retrieved to generate a multiple sequence alignment result of ZAT10 proteins from multiple species. The results are as Figure 1 shown, which shows a schematic diagram of the alignment result of the GhZAT10 protein provided in the example of the present invention with the ZAT10 protein sequences of other species. In Figure 1 it, the green shadow indicates residues with high identity in all species. The C2H2-type zinc finger domain (zf-C2H2) and the putative nuclear localization signal sequence (Putative NLS) are indicated by black boxes. Pink or blue shadows indicate fewer conserved residues. The amino acid sequence alignment result shows that Gh_A13G2112 has the highest homology with Arabidopsis thaliana AtZAT10 (AT1g27730), so this gene is named GhZAT10. A new protein was obtained from the cotton variety "Jin668" and named GhZAT10.

[0103] 1.2 Cloning of the GhZAT10 gene

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

[0105] (2) The total RNA of cotton leaves was extracted using a polysaccharide polyphenol plant total RNA extraction kit (Beijing Tsingke Biotechnology Co., Ltd., product number: TSP412), and the first-strand cDNA was synthesized using a TRUEscript 1st Strand cDNA Synthesis Kit reverse transcription kit (Beijing Aidlab Biotechnologies).

[0106] (3) The high-fidelity DNA polymerase kit (Novoprotein Scientific Inc., Nanjing, product number: 10154ES03) was used to amplify the full length of the GhZAT10 gene from the obtained first-strand cDNA, and the amplification system is shown in Tables 1 and 2.

[0107] Table 1

[0108]

[0109] Table 2

[0110]

[0111] (4) The PCR products were electrophoresed on a 1.5% agarose gel. After electrophoresis, the target band was cut under ultraviolet light and purified using an agarose gel DNA recovery kit (UYLANDBIO Co., Ltd., Suzhou, product number: UE-GX-250), and the operation steps were referred to the instruction manual of this kit.

[0112] (5) To add A to the ends of the recovered fragments, the 10 μL reaction system used was: 1 μL of 10× Buffer, 1 μL of dATP, 0.5 μL of Taq enzyme, and 7.5 μL of the recovered fragments, and the reaction was carried out at 72 °C for 30 min.

[0113] (6) The recovered fragments after adding A were ligated to the PMD18-T vector (purchased from Takara Biotechnology (Beijing) Co., Ltd., product number: D101A), and the operation was carried out according to the instruction manual. In the PCR tube, 4.5 μL of the fragment after adding A, 0.5 μL of PMD18-T, and 5 μL of Solution I were added in sequence, and the total volume was 10 μL; the ligation was carried out overnight at 16 °C.

[0114] (7) Take 5 μL of the ligation product and transform Escherichia coli DH5α (Beijing Tsingke Biotechnology Co., Ltd.) by the heat shock method (refer to J. Sambrook, et al., translated by Huang Peitang, et al., Molecular Cloning: A Laboratory Manual (Third Edition), Science Press, 2002 edition). Screen positive clones on an LB solid plate containing 50 mg / L ampicillin, pick 5 clones for sequencing (the sequencing work was carried out by Beijing Tsingke Biotechnology Co., Ltd.), and obtain the required full-length gene cDNA and carry out sequencing.

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

[0116] 1.3 Subcellular localization of the GhZAT10 protein

[0117] (1) The total RNA of the roots of "Jin668" was extracted using a polysaccharide polyphenol total plant RNA extraction kit (Beijing Tsingke Biotechnology Co., Ltd., product number: TSP412), and the first-strand cDNA was synthesized using a TRUEscript 1st Strand cDNA Synthesis Kit reverse transcription kit (Beijing Aidlab Biotechnologies).

[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. The lowercase letters in the sequence are homologous arms, and the uppercase letters are gene sequences.

[0119] (3) The obtained PCR amplification product was double-digested with restriction enzymes BamHI and StuI, and the pSuper1300::GFP vector was single-digested with restriction enzyme XbaI. The recovered products were subjected to homologous recombination, and the cDNA sequence of GhZAT10 was constructed on the pSuper1300::GFP expression vector, transiently transformed in tobacco leaves, and observed using a microscope.

[0120] The results of microscope observation are as Figure 2 shown, which shows the subcellular localization result map of the GhZAT10 protein in the examples of the present invention. Among them, GhZAT10 co-localizes with the nuclear localization marker, indicating that the GhZAT10 protein is a nuclear-localized protein.

[0121] Example 2 Low-potassium response pattern of the GhZAT10 gene in different tissues of "Jin668"

[0122] In this example, fluorescence real-time quantitative PCR was used to analyze the expression levels of the GhZAT10 gene in the roots, stems, leaves, etc. of cotton plants treated with normal potassium supply (cultured in a nutrient solution containing 2.5 mM K + all the time) and cotton plants treated with low potassium (cultured normally until the three-leaf stage and then placed in a nutrient solution containing 0.03 mM K + for 24 h) of the "Jin668" material.

[0123] (1) Samples of the roots, stems, and leaves of cotton plants treated with normal potassium supply and low-potassium stress were taken respectively with reference to step 1.3-(1). After extracting the total RNA, cDNA was obtained by reverse transcription.

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

[0125] The relative expression levels of the GhZAT10 gene in the roots, stems, and leaves of cotton at the three-leaf stage under normal potassium supply and low potassium treatment (setting the expression level of GhZAT10 in the roots under normal potassium supply as 1) are shown in Figure 3 . Among them, GhZAT10 was expressed in the roots, stems, and leaves of cotton plants, and its expression level increased after low potassium stress, indicating that GhZAT10 was induced by low potassium stress at the transcriptional level.

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

[0127] 2.1 Construction of recombinant plasmid

[0128] (1) In step 1.3-(1), total RNA of the roots of "Jin668" was extracted and reverse transcribed into cDNA.

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

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

[0131] R4: 5’-aacgtcgtatgggtaaggcctATTTAATTCTAAATCTTGTGCTAATGAAG-3’. (SEQ ID NO: 3)

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

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

[0134] (5) The digested product in step (3) was ligated with the vector backbone in step (4) to obtain the recombinant plasmid pCAMBIA2300::HA-GhZAT10.

[0135] According to the sequencing results, the structure of the recombinant plasmid pCAMBIA2300::HA-GhZAT10 is as follows: A double-stranded DNA molecule 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) The inflorescence of Arabidopsis thaliana grown in soil was decapitated one week after bolting and cultured until the multi-branched flowering stage.

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

[0139] (3) The correct positive clone was picked and inoculated into 5 mL of YEP liquid medium containing the corresponding antibiotics, and cultured overnight at 28 °C with shaking at 200 rpm.

[0140] (4) It was transferred to 25 mL of YEP liquid medium containing the corresponding antibiotics at a ratio of 1:50 and cultured at 28 °C with shaking at 200 rpm until OD 600 = 1.2 - 1.8.

[0141] (5) The bacterial liquid in (4) was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the bacteria were resuspended with the Arabidopsis infiltration transformation solution (1 L of the infiltration transformation solution contains 2.2 g of MS powder, 50 g of sucrose, 200 μL of Silwet L-77, 200 μL of 6-BA (5 mg·mL–1), pH = 5.60), and the OD 600 was adjusted to 0.8 - 1.0 to obtain the transformation solution.

[0142] (6) Pour the transformation solution into a 9-cm-diameter plastic round culture dish, and place the Arabidopsis plants to be transformed with the flowers, siliques, and white flower buds removed on their sides in the transformation solution and soak for 1 to 3 minutes. After the transformation is complete, place the Arabidopsis plants on their sides in a tray and cover them with a black plastic bag to protect them from light. After 16 to 24 hours, stand the flower pot upright, mark it to prevent cross-contamination with other materials, and culture it normally in the greenhouse. After about a month, harvest the seeds of the transformed materials, which are the T1 generation.

[0143] (7) After drying for more than 7 days, the T1 seeds were sterilized and sown on MS solid medium containing antibiotics. They were cultured in a light incubator. Seedlings with green leaves and normal growth were selected and transplanted into the soil. T2 seeds were collected from individual plants.

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

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

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

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

[0148] The results of plant growth phenotype identification are shown in Figure 4 . Figure 4 The results of the effect of the GhZAT10 gene of the present invention on plant growth phenotype under different potassium ion conditions are shown, wherein Figure 4 Part A shows the growth phenotype results of Arabidopsis wild type (Col-0), Arabidopsis AtZAT10 T-DNA insertion mutant (atzat10) and GhZAT10 overexpressing Arabidopsis strains (OE#9, OE#14) cultured on low potassium and low ammonium medium without exogenous K+, with 0.015 mM K+ and with 5 mM K+ for 10 days. The root length photos show that the root length of Arabidopsis thaliana was significantly higher when no exogenous K+ was added. + and added 0.015 mM K +On the culture medium, OE#9 and OE#14 were significantly greater than Col-0, indicating that overexpression of GhZAT10 is beneficial to the growth of Arabidopsis thaliana under low potassium and low ammonium culture conditions and is more conducive to Arabidopsis thaliana's resistance to low potassium stress.

[0149] Figure 4 Part B shows the schematic diagram of the comparison results of the primary root lengths of Col-0, atzat10, OE#9, and OE#14 cultured on a low potassium and low ammonium culture medium for 10 days under the conditions of no exogenous addition of K+, addition of 0.015 mM K+, and addition of 5 mM K+, respectively. Among them, the root length statistical results showed that without exogenous addition of K + and addition of 0.015 mM K + On the culture medium, the primary root lengths of OE#9 and OE#14 were significantly greater than Col-0, indicating that overexpression of GhZAT10 is beneficial to the growth of Arabidopsis thaliana under low potassium and low ammonium culture conditions and is more conducive to Arabidopsis thaliana's resistance to low potassium stress.

[0150] Example 3 Identification of the growth phenotypes of VIGS-GhZAT10-silenced plants

[0151] 3.1 Construction of the GhZAT10 recombinant plasmid

[0152] (1) Extract the total RNA from the leaves of the cotton variety "Jin668" and reverse transcribe it into cDNA.

[0153] (2) Using the cDNA obtained in step (1) as a template, perform PCR amplification with the primer pair composed 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) Double-digest the PCR amplification product obtained in step (2) with the restriction endonucleases EcoRI and KpnI, and recover the digested product.

[0157] (4) Double-digest the pTRV2 vector with the restriction endonucleases EcoRI and KpnI, and recover the vector backbone.

[0158] (5) Connect the digested product in step (3) and the vector backbone in step (4) to obtain the recombinant plasmid pTRV2-GhZAT10.

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

[0160] SEQ ID NO: 5: GCCTTTCACCAACAAATACGATGACGTGGACAACAATTACGTCGAGACATGGAAGAAAGGCAAGCGTTCGAAGCGCCAACGTGGCGACTCTCCTGCTGCTGTTGAACTTCAACCCACCACCGAAGAAGAGTACCTCGCTCTTTGTCTCATCATGCTCGCTCGCGGCTCTTCCGGTGCTGATCGTGATGTTATTCGTCGGTCTTCCTCTTCGTCGTCACCGCCTCCGCCGCCGCCTGCTTTGAAGTTGTCTTACAAGTGTAGTGTTTGTGACAAGGCGTTCCCTTCTTATCAAGCTTTGGGCGGTCATAAAGCCAGCCACCGCAAACCCCTTTCCGCCGACGCCGCTACCACCACCGCCGCCGTCAACGTCGATAACCCATCAACAACCAGCACCGCCACCACCATCACCAGCAGCGGTAGGCTTCACGAGTGTTCCAT.

[0161] 3.2 Obtaining VIGS-GhZAT10 Silenced Plants

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

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

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

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

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

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

[0168] (6) The mixtures were injected in groups, and the specific grouping is as follows:

[0169] Experimental group: Operations were performed on Xinshi 17 plants at the cotyledon stage, that is, the mixture A was injected into the lower surface of the cotyledons (both cotyledons of each plant were injected until the cotyledons were filled).

[0170] Positive control group: Operations were performed on Xinshi 17 plants at the cotyledon stage, that is, the mixtures B and C were injected into the lower surface of the cotyledons (both cotyledons of each plant were injected until the cotyledons were filled).

[0171] Negative control group: Operations were performed on Xinshi 17 plants at the cotyledon stage, that is, the mixture D was injected into the lower surface of the cotyledons (both cotyledons of each plant were injected until the cotyledons were filled).

[0172] Among them, each group of plants was hydroponically cultured in Hoagland's nutrient solution (2.5 mM KNO 3 , 2.5 mM Ca(NO 3 ) 2 , 1 mM MgSO 4, 0.5 mM (NH 4 )H 2 PO 4 , 0.1 mM FeNa-EDTA, and trace elements (2×10 -4 mM CuSO 4 , 1×10 -3 mM ZnSO 4 , 2×10 -2 mM H 3 BO 3 , 5×10 -6 mM (NH 4 ) 6 Mo 7 O 24 and 1×10 -3 mM MnSO 4 ), and the nutrient solution was replaced with a new one every week.

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

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

[0175] Cotton plants with the GhZAT10 gene silenced were obtained according to the method in 3.2. After 7 days of injection, the control plants injected with the pTRV2-GhCLA1 bacterial solution showed an albino phenotype. The VIGS-GhZAT10 silenced plants and the control VIGS-Ctrl were cultured in normal nutrient solution (CK, 2.5 mM K + ) and low-potassium nutrient solution (LK, 0.03 mM K + ) for 21 days.

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

[0177] (2) Observe the phenotypes. Among them, the detection method for dry weight is as follows: Dry the plants at 80 °C until constant weight and weigh them; detect the dry weight of leaves / stems / roots. Samples of 12 plants in each group were taken under each treatment, and the results were averaged.

[0178] Figure 5 The detection results of the silencing efficiency of the GhZAT10 gene in the examples of the present invention are shown, where VIGS-GhZAT10 is the plant with the GhZAT10 gene silenced, and VIGS-Ctrl is the control group. It can be seen that the GhZAT10 gene was successfully silenced.

[0179] Figure 6 The growth phenotypes of the GhZAT10 gene-silenced plants in the examples of the present invention are shown, Figure 6 Part A of shows the growth phenotypes of the whole plants of the GhZAT10 gene-silenced plants VIGS-GhZAT10 and the control group VIGS-Ctrl under normal and low-potassium stress conditions; Figure 6 Part B of shows the leaf phenotypes of VIGS-GhZAT10 and VIGS-Ctrl under normal and low-potassium stress conditions. Under low-potassium conditions, the leaves of the experimental group plants were more severely yellowed compared with those of the negative control group plants.

[0180] Figure 7Shows the potassium accumulation in different parts of the GhZAT10 gene-silenced plants VIGS-GhZAT10 and the control group VIGS-Ctrl in the embodiments of the present invention under normal and low potassium stress conditions. Among them, under the low potassium treatment condition, compared with the plants in the negative control group, the K + content in different parts (roots, stems, leaves) of the test group plants is lower. Under the condition of normal potassium supply, there is no significant difference in the K + content in different parts (roots, stems, leaves) of the test group plants compared with the control plants.

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

[0182] 3.4 Determination of the potassium ion absorption rate of the roots of VIGS-GhZAT10-silenced plants

[0183] (1) After obtaining the VIGS-GhZAT10-silenced plants according to the method in 3.2, grow them to the three-leaf stage under the culture condition of sufficient potassium supply.

[0184] (2) Divide the VIGS-GhZAT10-silenced plants into 2 groups. One group is cultured under normal potassium supply (CK, 2.5 mM K + ), and the other group is cultured under the condition of low potassium stress (LK, 0.03 mM K + ) for 8 days.

[0185] (3) Select plants with similar sizes for the determination of the K + absorption rate, that is, after subjecting the VIGS plants to K + starvation treatment for 2 days, replace them with the depletion solution with an initial K + concentration of 0.08 mM, and measure the K + content in the depletion solution after 10 hours of depletion, and convert it to the K + absorption rate per unit fresh weight per unit time.

[0186] Figure 8 Shows the net K+ absorption rate of the GhZAT10 gene-silenced plants VIGS-GhZAT10 and the control group VIGS-Ctrl in the embodiments of the present invention. Among them, for the cotton seedlings with GhZAT10 silenced, their K + absorption ability is significantly reduced, which in turn affects the accumulation of K + in the plants, showing more severe potassium deficiency symptoms.

[0187] The above results indicate that the test group plants (i.e., GhZAT10 gene-silenced plants) have a significantly reduced K + absorption ability.

[0188] In addition, 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 quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0189] In the present invention, unless otherwise clearly specified and defined, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely means that the horizontal height of the first feature is higher than that of the second feature. The first feature being "under", "below" and "beneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely means that the horizontal height of the first feature is lower than that of the second feature.

[0190] In the present invention, the terms "an embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

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

Claims

1. Application of the GhZAT10 gene in promoting the ability of plants to tolerate low potassium stress, characterized in that: include: The ability of the plant to promote tolerance to low potassium stress and / or the ability of the plant to absorb potassium ions is improved by overexpressing the GhZAT10 in the plant, wherein the nucleotide sequence of the GhZAT10 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The overexpression of GhZAT10 in the plant comprises: Inserting the DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector; and The recombinant vector is introduced into the plant to overexpress the GhZAT10. Optionally, the RNA of the plant is reverse transcribed to obtain a template DNA, and PCR amplification is performed based on the template DNA using a primer pair as shown in SEQ ID NO: 2 and SEQ ID NO: 3, thereby obtaining a DNA fragment of the GhZAT10 gene, Optionally, the step of inserting the DNA fragment of the GhZAT10 gene into a vector to obtain a recombinant vector comprises: The pCAMBIA2300::HA vector was double-digested with restriction endonucleases BamHI and StuI to obtain the vector framework; The DNA fragment of the GhZAT10 gene is connected to the vector frame to obtain the recombinant vector pCAMBIA2300::HA-GhZAT10.

3. The use according to claim 1, characterized in that: The plant is a monocot or a dicot; preferably, the plant is Arabidopsis thaliana or cotton.

4. The use according to claim 1, characterized in that: The GhZAT10 gene encodes a GhZAT10 protein having an amino acid sequence as shown in SEQ ID NO:

4.

5. A recombinant vector, characterized in that: comprising the nucleotide sequence shown in SEQ ID NO: 1, Preferably, the recombinant vector is pCAMBIA2300::HA-GhZAT10.

6. A recombinant microorganism, characterized in that Comprising the recombinant vector according to claim 7.

7. Use of any one of the following a)-d) in promoting the ability of plants to tolerate low potassium stress; a) a GhZAT10 protein having an amino acid sequence as shown in SEQ ID NO: 4 or a variant having a sequence with a homology of more than 90% with SEQ ID NO: 4; b) the recombinant vector according to claim 5; c) the recombinant microorganism according to claim 6; d) An expression cassette and / or a transgenic cell line comprising the nucleotide sequence of the GhZAT10 gene as shown in SEQ ID NO: 1 or a sequence having a homology of more than 90% to SEQ ID NO:

1.

8. A method for improving plant tolerance to low potassium stress, characterized in that: Include at least one of the following: overexpressing the GhZAT10 gene in the plant; increasing the activity of the GhZAT10 protein in the plant; and The content of GhZAT10 protein in the plant is increased.

9. A plant breeding method, characterized in that: include: 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, and the plant is a monocotyledonous plant or a dicotyledonous plant, preferably, the plant is Arabidopsis thaliana or cotton.

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