Cotton potassium transporter protein ghkup3, gene encoding same and use thereof

Abstract

The application discloses a cotton potassium transporter protein GhKUP3, a coding gene thereof and application, and provides the protein, which is specifically obtained from a cotton variety "Lumianyan No.22", and is named as the GhKUP3 protein. The application also provides a gene coding the GhKUP3 protein, and is named as the GhKUP3 gene. The application also provides application of the GhKUP3 protein and the GhKUP3 gene. The application has great value for researching plant low potassium tolerance and exploring a signal regulation network of plants under adversity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering, and particularly relates to a cotton high-affinity potassium transporter protein GhKUP3, a coding gene thereof and application. BACKGROUND

[0002] Potassium is an essential mineral element for plant growth and development, accounting for about 10% of the dry weight of plants, and plays an important role in maintaining normal life activities of plants. Cotton, as an important economic crop, is a typical potassium-loving plant, and the absorption and utilization of potassium are closely related to the yield and quality of cotton. Potassium not only promotes the growth and development of the aboveground part and root system of cotton, but also increases dry matter accumulation. In recent years, with the gradual increase of cotton multiple cropping index and yield, and the popularization and application of transgenic insect-resistant cotton, the potassium deficiency in cotton fields in China is becoming more and more serious. In addition, due to unreasonable fertilizer management, the potassium deficiency in cotton production is further aggravated, which often leads to early decline and seriously affects the yield and quality.

[0003] The absorption and transport of potassium in higher plants mainly rely on low-affinity potassium ion channels and high-affinity potassium transporters. According to the structure and function, potassium ion channels are mainly composed of three gene families: Shaker, TPK (Tandem-pore K + ) and Kir (K + inward rectifier)-like gene family and CNGC (Cyclic Nucleotide-Gated Channels) family. Potassium transporters are mainly composed of KUP / HAK / KT, HKT and CPA three gene families. Among them, KUP / HAK / KT family genes are identified by homology with KUP / HAK in bacteria and fungi, and members thereof play a key role in mediating high-affinity absorption, transport and distribution of K + in plants. Since the traditional conventional breeding method to solve the problem of potassium deficiency in cotton often has problems such as high cost, large workload and low selection efficiency, the research on potassium transport system genes has become a new way to improve the potassium absorption characteristics of crops and breed potassium-efficient crop varieties.

[0004] The Arabidopsis KUP / HAK / KT family contains 13 members, and AtKUP1 has been confirmed to mediate the absorption of external K + , and is a dual-affinity potassium ion transporter. AtKUP2 can complement the growth of bacteria and yeast potassium-deficient strains under low potassium, and is a low-affinity potassium ion transporter, which is expressed at a high level under high potassium, and on the contrary, the expression of AtKUP3 is significantly induced by low potassium, which has been confirmed to mediate high-affinity potassium transport. However, there is no research report on the cloning and function of GhKUP3 gene in cotton. SUMMARY

[0005] The application aims to provide a cotton GhKUP3 protein, an encoding gene thereof and application thereof.

[0006] The application provides a protein obtained from cotton (Gossypium hirsutum), specifically from a cotton variety “Lumianyan No. 22”, named as a GhKUP3 protein, which is as follows (a) or (b):

[0007] (a) a protein consisting of an amino acid sequence shown in SEQ ID NO: 1;

[0008] (b) a protein derived from SEQ ID NO: 1, which has substitution, and / or deletion, and / or addition of one or more amino acid residues compared with the amino acid sequence shown in SEQ ID NO: 1 and is related to the low potassium stress tolerance and / or potassium ion absorption capacity of a plant.

[0009] The application also provides a gene encoding the GhKUP3 protein, named as a GhKUP3 gene.

[0010] In some embodiments, the nucleotide sequence of the GhKUP3 gene is shown in SEQ ID NO: 2 or a sequence with more than 90% homology with SEQ ID NO: 2.

[0011] The application also provides an expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacterium containing the GhKUP3 gene.

[0012] The application also provides application of any one of 1)-3) below in regulating the low potassium stress tolerance and / or potassium ion absorption capacity of a plant:

[0013] 1) the above-mentioned GhKUP3 protein;

[0014] 2) the above-mentioned GhKUP3 gene;

[0015] 3) the above-mentioned expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing the GhKUP3 gene.

[0016] In some embodiments, the expression amount and / or activity of the GhKUP3 protein or GhKUP3 gene in the plant is reduced, the low potassium stress tolerance and / or potassium ion absorption capacity of the plant is reduced.

[0017] In some embodiments, the expression amount and / or activity of the GhKUP3 protein or GhKUP3 gene in the plant is increased, the low potassium stress tolerance and / or potassium ion absorption capacity of the plant is increased.

[0018] The application also provides application of the substance for inhibiting expression of the GhKUP3 gene in cultivating a gene-silenced plant.

[0019] The application also provides a method for cultivating a gene-silenced plant, which comprises silencing the GhKUP3 gene in a target plant to obtain a silencing plant which has lower dry matter accumulation ability under low potassium stress than the target plant.

[0020] and / or, has lower chlorophyll content in leaves than the target plant under low potassium stress;

[0021] and / or, has lower potassium ion absorption ability than the target plant under low potassium stress;

[0022] and / or, has lower potassium content than the target plant under low potassium stress.

[0023] In some embodiments, the target plant is a monocotyledon or a dicotyledon. In some embodiments, the target plant is Arabidopsis thaliana or cotton.

[0024] Advantages and beneficial effects of the embodiments of the application:

[0025] The application discloses a new protein (GhKUP3 protein) and a new gene (GhKUP3 gene), and proves that after silencing the GhKUP3 gene in a plant, the plant is more sensitive to low potassium stress compared with a control group, has significantly reduced K + absorption ability, and further affects K + accumulation in the plant body, and shows more serious symptoms of potassium deficiency. The application has great value for studying plant tolerance to low potassium and exploring signal regulation networks of plants under stress. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 FIG. 1 is an evolutionary tree of cotton GhKUP3 and KUP family proteins of Arabidopsis thaliana, corn and rice in Embodiment 1 of the application.

[0027] Figure 2 FIG. 4 is a result of subcellular localization of cotton GhKUP3 in Embodiment 1 of the application.

[0028] Figure 3 FIG. 6 is an analysis result of tissue-specific expression of the cotton GhKUP3 gene in Embodiment 1 of the application.

[0029] Figure 4 FIG. 8 is a change in expression level of the cotton GhKUP3 gene under low potassium stress in Embodiment 1 of the application.

[0030] Figure 5 FIG. 11 is a result of phenotype identification of a transgenic microorganism in Embodiment 2 of the application.

[0031] Figure 6 Silencing efficiency of VIGS-GhKUP3 plants in Example 3 of the present application was detected.

[0032] Figure 7 Low potassium stress phenotype of VIGS-GhKUP3 plants in Example 3 of the present application.

[0033] Figure 8 Physiological indicators and K uptake rate of VIGS-GhKUP3 plants in Example 3 of the present application. + Absorption rate.

[0034] In the figure, * represents the result of difference significance analysis P<0.05; ** represents the result of difference significance analysis P<0.01; *** represents the result of difference significance analysis P<0.001; **** represents the result of difference significance analysis P<0.0001. DETAILED DESCRIPTION

[0035] The present application will be described in detail below in conjunction with specific embodiments. The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods, unless otherwise specified. In the following examples, the experimental materials used, unless otherwise specified, can be obtained from commercial channels. In the following examples, quantitative tests were set up with three repeated experiments, and the results were averaged. Based on the following description and these examples, those skilled in the art can determine the basic characteristics of the present application, and can make various changes and modifications to the present application without departing from the spirit and scope of the present application, so as to make it suitable for various uses and conditions.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application belongs.

[0037] The term "and / or" used in the present application includes any and all combinations of one or more related listed items.

[0038] In the embodiments of the present application:

[0039] pTRV GFP, pTRV RNA1 (pTRV1) is described in document 1.

[0040] pTRV2 (pYL156, pTRV RNA2), pTRV GhCLA1 are described in document 2.

[0041] Wild-type yeast R757, yeast R5421, p416-GPD vector are described in document 3.

[0042] Yeast R5421-GhHAK5 is described in patent CN108715609A.

[0043] Document 1: Mu, C., Zhou, L., Shan, L., Li, F., & Li, Z. (2019). Phosphatase GhDs PTP3 interacts with annexin protein Gh ANN 8b to reversely regulate salt tolerance in cotton (Gossypium spp.). New Phytologist, 223(4), 1856-1872.

[0044] Document 2: Yiru Wang, Ye Wang, Bo Li, Changming Xiong, A Egrinya Eneji, Mingcai Zhang, Fangjun Li, Xiaoli Tian, Zhaohu Li, The Cotton High Affinity K + Transporter, GhHAK5a, Is Essential for Shoot Regulation of K + Uptake in Root under Potassium Deficiency, Plant and Cell Physiology, Volume 60, Issue 4, April 2019, Pages 888-899, https: / / doi.org / 10.1093 / pcp / pcz003.

[0045] Document 3: Han, M., Wu, W., Wu, W. H., & Wang, Y. (2016). Potassium transporter KUP7 is involved in K+ acquisition and translocation in Arabidopsis root under K+-limited conditions. Molecular plant, 9(3), 437-446.

[0046] The present application provides a protein, which is obtained from cotton (Gossypium hirsutum), specifically from cotton variety "Lumianyan No. 22", and is named as GhKUP3 protein, and is as follows (a) or (b):

[0047] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0048] (b) a protein derived from SEQ ID NO: 1 having one or more substitutions and / or deletions and / or additions of amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 1 and being associated with the ability of a plant to tolerate low potassium stress and / or the ability to absorb potassium ions.

[0049] MGPESGISIPSPPRNPSSLSWVNLSRNLILAYQSLGVVYGDLSTSPLYVYSSTFIGNLQDHQ

[0050] NEDAIFGAFSLIFWTLTLIPLLKYVFIMLGADDSGEGGTFALYSLLCRHAKFSLLPNQQAA

[0051] DEELSAYKYGPSMQVATGSPLKRFLEKHKTLRTALLVVVLFGASMVIGDGVLTPAISVLSS

[0052] VSGLQVTEKKLASGEIVVLACVILVGLFALQHFGTRRVAFMFAPIVIIWLVSIFSIGLYNIIF

[0053] WNPKIVRAISPYYIIKFFRETGKDGWFSLGGILLSVTGTEAMFADLGHFTALSIRLAFAFVV

[0054] YPCLVVQYMGQAAFLSRNPQKVLNSFYDSIPGPVFWPIFVIATLATIVGSQAIITATFSLIKQ

[0055] CHALGCFPRVKVVHTSKHIRGQIYIPEINWILMILTLAITIGFRSTTLIGNAYGLACITVMFIT

[0056] TFLMALVVTFVWQKSVLLAAMFLVFFWFSEGVYLSSALMKVPQGGWVPLVLSVIFMLV

[0057] MYIWHYGTRKKYNFDLHNKVSLKWLLGLGPSLGIVRVPGIGLIYSELATGVPAIFSHFVT

[0058] NLPAFHKVLVFVCVKSVPVPYVSPEERFLIGRVCPRPYRMYRCIVRYGYKDIQRDDGDFE

[0059] EQLIQSIAEFIQMEAGDAQFCSSESSSYDGRLAVISSRTIQSSSSLIVTEREDCDGIESIQSSKS

[0060] LMLQSLRSAYPDDENPGVRRRHVRFEVAASPGMDTGVREELMDLIEAKEAGVAYIMGHSYVKARRSSSYLKKLVIDMGYSFLRKNCRGPSVALNIPHINLIEVGMIYYV (SEQ ID NO: 1)

[0061] The present application also provides a gene encoding the GhKUP3 protein, which is named as GhKUP3 gene.

[0062] In some embodiments, the nucleotide sequence of the GhKUP3 gene is as shown in SEQ ID NO: 2 or a sequence having more than 90% homology with SEQ ID NO: 2.

[0063] ATGGGGCCGGAATCAGGAATTTCGATTCCTTCTCCTCCGCGAAATCCTTCCTCGCTTTCATGGGTCAATCTCTCCAGGAACCTAATATTAGCATACCAGAGCCTCGGGGTGGTGTATGGAGACCTGAGCACTTCACCTCTTTATGTCTACTCAAGCACGTTCATAGGGAACTTGCAGGATCATCAGAATGAAGATGCAATATTTGGAGCATTTTCCTTGATTTTTTGGACCCTTACATTGATACCCCTTCTTAAATATGTGTTTATCATGCTGGGTGCAGATGATAGCGGTGAAGGTGGCACTTTTGCTCTTTACTCGCTGCTTTGTAGGCATGCAAAATTTAGCTTGCTT

[0064] CCCAATCAGCAAGCAGCTGATGAAGAACTCTCCGCCTACAAATATGGGCCCTCGATGC

[0065] AGGTGGCCACTGGTTCTCCATTGAAGCGATTTTTGGAGAAGCATAAAACGTTAAGGAC

[0066] AGCCCTTCTTGTTGTGGTACTCTTTGGTGCTTCTATGGTCATAGGTGATGGTGTGCTCA

[0067] CTCCAGCAATATCAGTTCTGTCATCAGTTTCAGGACTTCAAGTTACTGAAAAGAAATT

[0068] AGCTAGTGGTGAAATTGTCGTGCTTGCTTGTGTCATATTGGTGGGCCTATTTGCCCTGC

[0069] AGCATTTTGGAACACGCAGGGTAGCTTTTATGTTTGCACCAATTGTGATCATCTGGCTG

[0070] GTATCAATTTTTTCCATTGGCTTGTACAACATAATATTTTGGAACCCAAAGATTGTTCGT

[0071] GCAATTTCCCCATATTATATTATCAAGTTCTTTAGAGAGACAGGTAAAGATGGCTGGTT

[0072] TTCGCTAGGAGGGATCCTTCTTTCAGTAACTGGCACCGAAGCTATGTTTGCAGACCTT

[0073] GGTCATTTCACTGCCTTATCAATTAGGCTTGCGTTTGCATTTGTAGTATACCCATGTCTG

[0074] GTTGTCCAATACATGGGTCAGGCTGCGTTCTTGTCAAGAAACCCACAAAAAGTTCTTA

[0075] ACAGTTTTTATGACTCAATTCCTGGTCCGGTTTTTTGGCCTATCTTTGTGATTGCCACCC

[0076] TTGCAACTATTGTTGGGAGTCAGGCTATTATAACTGCCACTTTTTCTTTAATTAAACAAT

[0077] GCCATGCCCTTGGATGCTTCCCTCGAGTCAAAGTAGTTCACACCTCAAAACATATACG

[0078] TGGCCAGATCTACATCCCAGAAATAAACTGGATACTCATGATACTTACTCTTGCAATAA

[0079] CTATTGGATTTCGAAGTACTACTTTAATTGGAAATGCTTATGGACTTGCATGCATTACGG

[0080] TGATGTTTATTACAACATTTCTCATGGCTCTTGTGGTAACCTTTGTTTGGCAGAAAAGT

[0081] GTTTTACTTGCTGCTATGTTCCTTGTGTTCTTCTGGTTTAGTGAAGGGGTCTACTTATCA

[0082] TCAGCACTCATGAAAGTGCCTCAAGGAGGATGGGTGCCTCTTGTGCTGTCTGTCATCT

[0083] TTATGTTGGTTATGTACATATGGCACTATGGGACTCGCAAGAAGTACAACTTTGACCTG

[0084] CACAACAAAGTATCACTGAAATGGTTGCTTGGCTTGGGTCCTAGCCTCGGTATTGTGC

[0085] GTGTGCCTGGGATAGGCCTCATTTACTCCGAACTGGCAACAGGAGTCCCTGCAATATT

[0086] TTCCCACTTTGTCACAAACCTGCCAGCGTTCCATAAGGTGCTGGTTTTTGTTTGTGTGA

[0087] AATCAGTTCCAGTCCCATATGTTTCACCAGAAGAACGCTTTCTTATTGGTCGGGTATGC

[0088] CCCAGGCCATATCGTATGTATAGGTGCATTGTCAGGTATGGGTACAAAGATATCCAGCG

[0089] AGATGATGGGGATTTTGAGGAGCAGCTTATACAAAGCATAGCAGAGTTCATCCAGATG

[0090] GAAGCTGGAGATGCACAATTTTGTAGTTCTGAGAGTTCATCATATGATGGGAGGCTAG

[0091] CTGTTATAAGTAGCAGGACCATCCAATCAAGCTCAAGTTTAATAGTTACTGAGAGAGA

[0092] GGATTGTGATGGTATCGAGTCCATACAGAGCAGCAAATCCCTCATGTTACAGAGCTTG

[0093] CGAAGTGCTTATCCCGATGATGAGAATCCAGGAGTGAGGAGGAGGCATGTGAGGTTC

[0094] GAAGTAGCAGCAAGTCCAGGTATGGATACAGGTGTTAGGGAAGAGCTGATGGATTTG

[0095] ATAGAGGCAAAGGAAGCAGGCGTTGCATATATAATGGGACATTCATATGTAAAGGCCA

[0096] GGAGAAGTTCATCATATTTGAAAAAGCTAGTAATAGACATGGGGTATTCATTTCTTAGA

[0097] AAGAACTGCAGGGGGCCTTCAGTTGCACTAAACATTCCTCACATCAATCTCATTGAAG

[0098] TTGGAATGATATACTATGTCTAG(SEQ ID NO:2)

[0099] The application also provides an expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacterium containing the GhKUP3 gene.

[0100] The application also provides the use of any one of 1)-3) below in regulating the low potassium stress tolerance and / or potassium ion absorption capacity of a plant.

[0101] 1) the GhKUP3 protein described above;

[0102] 2) the above-mentioned GhKUP3 gene;

[0103] 3) the above-mentioned expression cassette containing the GhKUP3 gene, recombinant vector, transgenic cell line or recombinant bacteria.

[0104] In some embodiments, the expression amount and / or activity of the GhKUP3 protein or GhKUP3 gene in a plant is reduced, the low potassium stress tolerance of the plant is reduced, and / or the potassium ion absorption capacity of the plant is reduced.

[0105] In some embodiments, the expression amount and / or activity of the GhKUP3 protein or GhKUP3 gene in a plant is increased, the low potassium stress tolerance of the plant is increased, and / or the potassium ion absorption capacity of the plant is increased.

[0106] The embodiments of the present application also provide use of a substance that inhibits expression of a GhKUP3 gene in breeding of a gene-silenced plant.

[0107] The embodiments of the present application also provide a method for breeding a gene-silenced plant, which comprises silencing a GhKUP3 gene in a target plant to obtain a silenced plant that has a lower dry matter accumulation capacity than the target plant under low potassium stress.

[0108] and / or, a lower chlorophyll content in leaves than the target plant under low potassium stress;

[0109] and / or, a lower potassium ion absorption capacity than the target plant under low potassium stress;

[0110] and / or, a lower potassium content than the target plant under low potassium stress.

[0111] In some embodiments, the target plant is a monocotyledonous plant or a dicotyledonous plant. In some embodiments, the target plant is Arabidopsis thaliana or cotton.

[0112] In some embodiments, the substance that inhibits expression of the GhKUP3 gene comprises an interference vector. The interference vector can be a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown in SEQ ID NO: 3 into the Kpn I and Sma I enzyme cutting sites of a pTRV2 vector. The GhKUP3 gene in the target plant is silenced by introducing the interference vector into the target plant.

[0113] GTGGTGTATGGAGACCTGAGCACTTCACCTCTTTATGTCTACTCAAGCACGTTCATAGGGAACTTGCAGGATCATCAGAATGAAGATGCAATATTTGGAGCATTTTCCTTGATTTTTTGGACCCTTACATTGATACCCCTTCTTAAATATGTGTTTATCATGCTGGGTGCAGATGATAGCGGTGAAGGTGGCACTTTTGCTCTTTACTCGCTGCTTTGTAGGCATGCAAAATTTAGCTTGCTTCCCAATCAGCAAGCAGCTGATGAAGAACTCTCCGCCTACAAATATGGGCCCTCGATGCAGGTGGCCACTGGTTCTCCATTGAAGCGATTTTTGGAGAAGCATAAAACGTTAAGGACAGCCCTTCTTGTTGTGGTACTCTTTGGTGCTTCTATGGTCATAGGTGATGGTGTGCTCACTCCAGCAATATCAGTTCTGTCATCAGTTTCAGGACTTCAAGTTACTGAAAAGAAATTAGCTAGTGGTGAAATTGTCGTGCTT (SEQ ID NO: 3)

[0114] In some embodiments, the substance for inhibiting the expression of the GhKUP3 gene further comprises pTRV RNA1 and Agrobacterium GV3101.

[0115] In some embodiments, the silencing of the GhKUP3 gene in the target plant is achieved by means of a VIGS system.

[0116] In some embodiments, the silencing of the GhKUP3 gene in the target plant is achieved by injecting the target plant with an infection solution. The infection solution is a bacterial solution of two recombinant Agrobacterium, one obtained by introducing the interference vector into Agrobacterium GV3101 and the other obtained by introducing pTRV-RNA1 into Agrobacterium GV3101.

[0117] In this context, 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 normal growth and development; it can be a potassium ion concentration of ≤0.1 mM or less, specifically a potassium ion concentration of ≤0.05 mM or less, and more specifically a potassium ion concentration of ≤0.03 mM, but not zero.

[0118] In this context, being more sensitive to low potassium stress can be manifested in a decrease in the biomass of the plant (root dry weight, stem dry weight, leaf dry weight, etc.), a decrease in the chlorophyll content of the leaves, etc.

[0119] Cloning and mapping of GhKUP3 protein and its encoding gene

[0120] I. Cloning of GhKUP3 gene

[0121] A gene which was significantly up-regulated after low potassium stress was screened from the low potassium stress transcriptome, and was aligned to the protein sequence of Gh_D04G0700 using the cotton database. The protein homologous gene sequence of Arabidopsis thaliana was searched, and an evolutionary tree was generated according to the multiple sequence alignment results of the protein using the Neighbor Joining method. Figure 1 The evolutionary tree results show that Gh_D04G0700 has the highest homology with Arabidopsis AtKUP3 / AtKT4 (AT3G02050), and therefore the gene is named GhKUP3. A new protein is obtained from the cotton variety "Lumianyan No. 22", which is named GhKUP3.

[0122] According to the sequence of GhKUP3 gene obtained from the cotton database, two specific primers were designed, F1: ATGGGGCCGGAATCAG (SEQ ID NO: 4) and R1: GACATAGTATATCATTCCAACTTCAAT (SEQ ID NO: 5). The total RNA of polysaccharide polyphenol plant was extracted from cotton leaves by using a polysaccharide polyphenol plant total RNA extraction kit (Beijing Genesil Biological Technology Co., Ltd.), and the first strand cDNA was synthesized by using an M-MLV reverse transcription kit (Baori Medical Biotechnology (Beijing) Co., Ltd.). The full-length of GhKUP3 gene was amplified by using a high-fidelity DNA polymerase kit (Nanjing Novozyme Biological Technology Co., Ltd.). The 50 μL PCR reaction system included: 25 μL of 2x DNA polymerase buffer, 1 μL of 10 mM nucleotide premix, 1 μL of cDNA template, 1 μL of DNA polymerase, 2 μL of upstream primer, 2 μL of downstream primer, and 18 μL of ddH2O. The PCR amplification program was as follows: 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 58 °C for 15 s, and 72 °C for 2 min; and finally 72 °C for 5 min. The PCR product was electrophoresed on a 1.5% agarose gel. After electrophoresis, the target band was cut under a UV lamp, purified by using an agarose gel DNA recovery kit (Jiangsu Kangwei Century Biological Technology Co., Ltd.), and the operation steps were referred to the instruction manual of the kit. The recovered fragment end needed to be added with A, and the 10 μL reaction system was as follows: 1 μL of 10x Buffer, 1 μL of dATP, 0.5 μL of Taq enzyme, and 7.5 μL of recovered fragment, and 72 °C reaction for 30 min. The recovered fragment after adding A was connected with PMD18-T vector (purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.), and the operation was performed according to the instruction manual. The PCR tube was sequentially added with 4.5 μL of the recovered fragment after adding A, 0.5 μL of PMD18-T, and 5 μL of Solution I, with a total volume of 10 μL; and connected at 16 °C overnight. 5 μL of the connection product was taken, transformed into E. coli DH5α by using the heat shock method (referred to J. Sambrook, et al., Huang Peitang, et al., Molecular Cloning Laboratory Guide (3rd Edition), Science Press, 2002), and the positive clones were screened on LB solid plates containing 50 mg / L of ampicillin. Five clones were picked for sequencing (the sequencing work was performed by Beijing Genesil Biological Technology Co., Ltd.), and the required full-length gene cDNA was obtained. The sequencing results showed that the sequence of the full-length gene was 2367 bp, and the complete ORF reading frame encoded 788 amino acids.

[0123] The amino acid sequence of GhKUP3 protein is shown as SEQ ID NO: 1 (788 aa). The gene encoding GhKUP3 protein is named as GhKUP3 gene, and the sequence is shown as SEQ ID NO: 2 (2367 bp).

[0124] II. Subcellular localization of GhKUP3 protein

[0125] The total RNA of the root system of "LuMianyan 22" was extracted and reverse-transcribed into cDNA, and PCR amplification was performed using F2 and R2 as primers. The obtained PCR amplification product was double-digested with restriction endonucleases BamHI and StuI, and the pHBT-GFP vector was also double-digested with restriction endonucleases BamHI and StuI. The recovered products were subjected to homologous recombination, and the cDNA sequence of GhKUP3 was constructed on the pHBT-GFP expression vector and transiently transformed in cotton leaf protoplasts. Microscopic observation results showed that GhKUP3 and the membrane localization marker appeared to be co-localized (Fig. 2), indicating that the GhKUP3 protein is a membrane-localized protein. Figure 2

[0126] F2: CTCCCCTTGCTCCGTGGATCCATGGGGCCGGAATCAG (SEQ ID NO: 6)

[0127] R2: CTCGCCCTTGCTCACAGGCCTGACATAGTATATCATTCCAACTTCAAT (SEQ ID NO: 7).

[0128] III. Tissue-specific expression of GhKUP3 gene

[0129] The expression site of GhKUP3 gene in "LuMianyan 22" material was analyzed by fluorescent real-time quantitative PCR. The instrument used for fluorescent real-time quantitative PCR was ABI 7500 Fast (Applied Biosystem), and the primer pair used was F3: AACCTGCCAGCGTTCCATAA (SEQ ID NO: 8) and R3: AGCTGCTCCTCAAAATCCCC (SEQ ID NO: 9). The sample for detecting tissue-specific expression was the cDNA obtained by reverse transcription of the total RNA of each part of the three-leaf cotton under normal nutritional level, and the PCR program was denaturation at 94℃ for 30 s, denaturation at 94℃ for 5 s, annealing at 60℃ for 35 s, 40 cycles, and the relative expression amount was calculated by 2 -ΔΔCt Method, and cotton Actin9 gene was used as a control (the primer pair for identifying cotton Actin9 gene was 5'-GCCTTGGACTATGAGCAGGA-3' (SEQ ID NO: 10) and 5'-AAGAGATGGCTGGAAGAGGA-3' (SEQ ID NO: 11). The relative expression amount of GhKUP3 gene in each part of the three-leaf cotton under normal nutritional level (the expression amount of GhKUP3 in pistil was set as 1) is shown in Table 1. Figure 3 ​GhKUP3 is constitutively expressed in cotton plants, and is expressed in roots, stems, leaves, boll shells and fibers.

[0130] The cotton variety Luyi 22 was used as the test material, and 0.03 mM K + was used to treat the seedlings at the three-leaf stage. RT-qPCR was used to detect the expression level of GhKUP3 in the leaves and roots. The results, as shown in Figure 4 , showed that the transcription level of GhKUP3 in the roots was rapidly up-regulated by low potassium stress, and reached a significant level at 3 h of stress, and reached a peak at 12 h of stress Figure 4 . The expression level of GhKUP3 in the leaves was induced to a certain extent, and reached a peak at 12 h of stress Figure 4 . This indicates that GhKUP3 also plays a certain role in the leaves.

[0131] Example 2, Obtaining and Phenotypic Identification of Transgenic Microorganisms

[0132] Yeast R5421 is a potassium absorption-deficient yeast strain obtained by silencing the trk1 gene and the trk2 gene of wild-type yeast R757. The p416-GPD vector is a yeast transformation vector.

[0133] I. Construction of Recombinant Plasmid

[0134] 1. Total RNA was extracted from the roots of Luyi 22 and reverse transcribed into cDNA.

[0135] 2. The cDNA obtained in step 1 was used as the template for PCR amplification with the primer pair F4 and R4 to obtain the PCR amplification product.

[0136] F4: 5'-CTTAGTTTCGACGGATTCTAGAATGGGGCCGGAATCAG-3'; (SEQ ID NO: 12)

[0137] R4: 5'-ATATCGAATTCCTGCAGCCCGGGGACATAGTATATCATTCCAACTTCAAT-3'. (SEQ ID NO: 13)

[0138] 3. The PCR amplification product obtained in step 2 was double-digested with restriction enzymes XbaI and SmaI, and the digested product was recovered.

[0139] 4. The p416-GPD vector was double-digested with restriction enzymes XbaI and SmaI, and the vector backbone was recovered.

[0140] 5. The digested product of step 3 and the vector backbone of step 4 were ligated to obtain the recombinant plasmid p416-GPD-GhKUP3.

[0141] According to the sequencing result, the recombinant plasmid p416-GPD-GhKUP3 is described as follows: the double-stranded DNA molecule shown in SEQ ID NO: 2 is inserted between the XbaI and Smal enzyme cutting sites of the p416-GPD vector.

[0142] II. Obtaining of the recombinant yeast

[0143] The recombinant plasmid p416-GPD-GhKUP3 is introduced into the yeast R5421 to obtain the recombinant yeast, which is named as the yeast R5421-GhKUP3. The p416-GPD vector is introduced into the yeast R5421 to obtain the recombinant yeast, which is named as the yeast R5421-EV.

[0144] III. Phenotype identification

[0145] The yeasts to be tested are: the yeast R757, the yeast R5421, the yeast R5421-GhHAK5, the yeast R5421-GhKUP3 and the yeast R5421-EV.

[0146] 1. The yeasts to be tested are inoculated into YPDA medium and cultured at 30°C with 200 rpm shaking until OD 600nm = 0.8.

[0147] 2. After step 1 is completed, the whole system is diluted by 10 times gradient using 0.9% NaCl as solvent, and then 10 μL of the bacterial liquid of each dilution is spotted on the AP medium with different potassium concentrations, the flat plate is blown dry on the clean bench, and is cultured in the 30°C incubator for 2 days in the dark and then is photographed.

[0148] Preparation method of the AP medium (adjusting pH value to 6.5 by L-arginine): 50× salt solution 20 mL, H3PO4 546 μL, L-arginine 1.742 g, 1000× vitamin 1 mL, 1000× trace element 1 mL, supplementing ingredients without uracil 0.77 g, 100× Ura 10 mL, glucose 20 g, agar powder 15 g, KCl (determined by the required K + concentration), and water is added to 1 L.

[0149] 50× salt solution: 100 mM MgSO4, 10 mM CaCl2, and the rest is water.

[0150] Preparation method of 1000× vitamin: biotin 0.011 g, nicotinic acid 0.04 g, myo-inositol 0.2 g, pantothenic acid 0.04 g, and water is added to 100 mL.

[0151] 1000x micro-preparation method: KI 0.01 g, H3BO3 0.05 g, MnSO4-H2O 0.0447 g, ZnSO4-7H2O 0.071 g, CuSO4-5H2O 0.0078 g, Na2MnO4-2H2O 0.0235 g, and water to 100 mL.

[0152] Uracil powder 0.2 g, and water to 100 mL.

[0153] Uracil powder 0.2 g, and water to 100 mL.

[0154] Results are shown in Table 1. Figure 5 . Figure 5 In the present application, 10 -1 In the present application, 10 -2 In the present application, 10 -3 In the present application, 10 + Yeast R5421 and yeast R5421-EV could not grow normally on AP medium with K + Yeast R5421-GhHAK5 and yeast R5421-GhKUP3 could grow on AP medium with K

[0155] Example 3, VIGS silencing plant phenotype

[0156] I. Construction of VIGS-GhKUP3 silencing vector

[0157] 1. Extract total RNA from leaves of cotton variety "Lumianyan No. 22" and reverse transcribe into cDNA.

[0158] 2. Use the cDNA obtained in step 1 as a template, and use the primer pair composed of F5 and R5 to perform PCR amplification to obtain PCR amplification products.

[0159] F5: GTGAGTAAGGTTACCGAATTCGTGGTGTATGGAGACCTGAGCA (SEQ ID NO: 14) R5: GAGACGCGTGAGCTCGGTACCAAGCACGACAATTTCACCACTA (SEQ ID NO: 15)

[0160] 3. Double enzyme digestion of the PCR product obtained in step 2 with restriction enzymes EcoRI and Kpnl, and recover the digested product.

[0161] 4. Double enzyme digestion of the pTRV2 vector with restriction enzymes EcoRI and Kpnl, and recover the vector backbone.

[0162] 5. Ligation of the digested product of step 3 and the vector backbone of step 4 to obtain the recombinant plasmid pTRV2-GhKUP3.

[0163] According to the sequencing results, the recombinant plasmid pTRV2-GhKUP3 is described as follows: the double-stranded DNA molecule shown in SEQ ID NO: 3 is inserted between the Xbal and Smal enzyme digestion sites of the pTRV2 vector.

[0164] II. Obtaining of VIGS-GhKUP3 silenced plants

[0165] 1. Introduce the recombinant plasmid pTRV2-GhKUP3 into Agrobacterium GV3101 to obtain a recombinant Agrobacterium. Then, suspend the recombinant Agrobacterium in a VIGS solution (50 μg / mL kanamycin, 50 μg / mL gentamicin, 10 mM 2-(N-morpholino)ethanesulfonic acid, and 20 μΜ acetyl-syringone in 500 mL YEP liquid medium) to obtain a bacterial solution with OD 600nm = 1.5.

[0166] 2. Introduce the pTRV1 vector into Agrobacterium GV3101 to obtain a recombinant Agrobacterium. Then, suspend the recombinant Agrobacterium in a VIGS solution to obtain a bacterial solution with OD 600nm = 1.5.

[0167] 3. Mix the bacterial solution obtained in step 1 and the bacterial solution obtained in step 2 in equal volumes to obtain a mixed solution A.

[0168] 4. Introduce the pTRV-GhCLA1, pTRV2-GhKUP3, and pTRV-GFP vectors into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Then, suspend the recombinant Agrobacterium in a VIGS solution to obtain bacterial solutions with OD 600nm = 1.5.

[0169] 5. Mix the pTRV-GhCLA1, pTRV2-GhKUP3, and pTRV-GFP bacterial solutions obtained in step 4 and the bacterial solution obtained in step 2 in equal volumes to obtain mixed solutions B, C, and D, respectively.

[0170] 6. Group injection

[0171] Test group: operation was performed on cotyledon stage Xinshiyi plants: cotyledon lower surface injection of mixed solution A (each plant was injected into two cotyledons, and the injection was stopped until the cotyledon was full).

[0172] Positive control group: operation was performed on cotyledon stage Xinshiyi plants: cotyledon lower surface injection of mixed solution B and C (each plant was injected into two cotyledons, and the injection was stopped until the cotyledon was full).

[0173] Negative control group: operation was performed on cotyledon stage Xinshiyi plants: cotyledon lower surface injection of mixed solution D (each plant was injected into two cotyledons, and the injection was stopped until the cotyledon was full).

[0174] Each group of plants was watered with Hoagland's nutrient solution (2.5mM KNO3, 2.5mM Ca(NO3)2, 1mM MgSO 4, 0.5mM (NH4)H2PO4, 0.1mM FeNa-EDTA, and trace elements (2×10 -4 mM CuSO4, 1×10 -3 mM ZnSO4, 2×10 -2 mM H3BO3, 5×10 -6 mM (NH4)6Mo7O 24 and 1×10 -3 mM MnSO4), and the nutrient solution was replaced every week.

[0175] III. Phenotype of VIGS-GhKUP3 silenced plants

[0176] After the GhKUP3 silenced plants were obtained by the above method, the control plants injected with pTRV2-GhCLA1 bacterial solution showed albinism phenotype on the 7th day after injection, and the gene silencing efficiency of the silenced plants was detected. Figure 6 After the VIGS-GhKUP3 silenced plants and the control VIGS-GFP were cultured in normal nutrient solution (CK, 2.5mM K + ) and low potassium nutrient solution (LK, 0.03mM K + ) for 24 days, the phenotype was observed.

[0177] The relative chlorophyll content was determined using a SPAD chlorophyll meter to determine the relative chlorophyll value of the functional leaves (the third leaf from the bottom) of the cotton plants. Under each treatment, 12 plants were sampled from each group of plants, and the results were averaged.

[0178] The detection method of dry weight: 80℃ (drying to constant weight), weighing. The dry weight of leaves (Leaf) / stem (Stem) / root (Root) was detected. Under each treatment, 12 plants were sampled from each group of plants, and the results were averaged.

[0179] Figure 7 Image A shows a phenotypic photograph of the entire plant. Figure 7 Image B shows a phenotypic photograph of the plant's leaves from bottom to top. Under low potassium conditions, the experimental group showed more severe leaf yellowing compared to the negative control group.

[0180] The results for chlorophyll content and dry weight are shown in [link to results]. Figure 8 .

[0181] Under low potassium conditions, the experimental group plants had lower chlorophyll content in their leaves compared to the negative control group plants. Figure 8 K in A) and various parts (root, stem, and leaves) + Lower content ( Figure 8 (D) Lower dry weight in all parts (roots, stems, leaves) Figure 8 (F). Under normal potassium supply conditions, compared with the control plants, the K content of various parts (roots, stems, and leaves) of the experimental group plants was significantly higher. + content( Figure 8 C), dry weight of each part (root, stem, leaf) Figure 8 There were no significant differences between E and E.

[0182] The above results indicate that the experimental group plants (i.e., GhKUP3 gene-silenced plants) are more sensitive to low potassium.

[0183] IV. Potassium ion uptake rate in the roots of VIGS-GhKUP3-silenced plants

[0184] After obtaining VIGS-GhKUP3 silent plants, they were grown to the three-leaf stage under conditions with sufficient potassium supply. A portion of these plants were then allowed to continue normal potassium supply (CK, 2.5 mM K). + One part was cultured, and the other part was subjected to low potassium stress (LK, 0.03 mM K). + Cultured under these conditions for 8 days. Plants of similar size were selected for K... + Determination of absorption rate ( Figure 8 (B) VIGS plants were all treated with 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.

[0185] Silent cotton seedlings of GhKUP3, their K + 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.

[0186] Prospects for the application of this invention

[0187] Cotton is an important economic crop in my country, holding a vital position in the national economy. However, with the widespread adoption of Bt gene-modified insect-resistant cotton in the Yellow River and Yangtze River cotton-growing areas, coupled with irrational fertilizer and water management, potassium deficiency in cotton is becoming increasingly severe. Cotton is a potassium-loving crop, and potassium deficiency leads to premature aging, seriously affecting yield and quality. Solving the potassium deficiency problem in cotton is crucial for increasing yield and improving quality. With the continuous development of molecular biology, it has become possible to cultivate low-potassium-tolerant cotton varieties using biotechnology. While cotton gene cloning has achieved some results, it lags far behind that of grain crops such as rice, corn, and wheat. Virus-induced gene silencing (VIGS), as an effective reverse genetics technique, is 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 study discovered and cloned the cotton GhKUP3 gene, silenced GhKUP3 in cotton using VIGS technology, and explored the biological function of the GhKUP3 gene. Our current work mainly focuses on the function of the cotton potassium transporter GhKUP3 in cotton potassium absorption, elucidating the regulatory pathway of GhKUP3 in cotton's response to low potassium stress, studying gene expression and regulation under low potassium stress at the molecular level, improving the signal transduction and gene expression regulation network under stress conditions, and further investigating the plant's response mechanism to low potassium stress signals, laying a good molecular foundation for effectively improving plant tolerance to low potassium stress.

[0188] 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.

[0189] 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. Use of any one of 1) to 3) below in regulating the low potassium stress tolerance of a plant; 1) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; 2) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; 3) an expression cassette, a recombinant vector, or a recombinant bacterium containing a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, wherein the plant is cotton, and in the use, the expression amount of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or the encoding gene thereof in the plant is increased, and the low potassium stress tolerance of the plant is increased.

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