A high potassium absorption utilization efficiency gene mutant and application thereof

Abstract

The application belongs to the field of agricultural biotechnology, and particularly relates to a gene mutant with high potassium absorption and utilization efficiency and application thereof. GhAKT1bD The gene mutant A is obtained by deleting the 111th-114th bases of the wild-type gene, and the gene mutant B is obtained by deleting the 112th-114th bases of the wild-type gene. GhAKT1bD The gene mutant A is obtained by deleting the 111th-114th bases of the wild-type gene, and the gene mutant B is obtained by deleting the 112th-114th bases of the wild-type gene. GhAKT1bD The gene mutant A is obtained by deleting the 111th-114th bases of the wild-type gene, and the gene mutant B is obtained by deleting the 112th-114th bases of the wild-type gene. The transgenic plant provided by the application has excellent low-potassium stress resistance compared with the wild-type plant. In addition, the transgenic plant provided by the embodiments of the application has growth advantages, higher stem potassium content and root potassium content compared with the wild-type plant under the conditions of sufficient potassium supply (2.5 mM) or severe potassium deficiency (0.03 mM).

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a gene mutant with high potassium absorption and utilization efficiency and its application. Background Technology

[0002] Potassium, an essential nutrient for plant growth and development, plays a vital role in maintaining normal physiological and biochemical processes in plant cells, such as osmotic regulation, ion homeostasis, and photosynthesis. Cotton is a potassium-loving economic crop, and potassium deficiency during both its vegetative and reproductive growth stages can lead to a decline in cotton yield and quality.

[0003] Plants primarily absorb potassium through their roots, and the absorption mechanism is complex, involving both high-affinity and low-affinity absorption systems. Under high potassium concentrations, plants mainly passively absorb potassium ions via low-affinity channel proteins; while under low potassium concentrations, high-affinity transport proteins play a dominant role, absorbing potassium ions through active transport. However, significant differences in potassium absorption efficiency exist among different plant species or even different varieties of the same plant. This is mainly controlled by genetic factors, including differences in the expression and function of potassium ion channel and transport protein genes.

[0004] In recent years, with the rapid development of molecular biology and genomics technologies, significant progress has been made in the study of the molecular mechanisms of potassium absorption and utilization in plants. Studies have shown that multiple gene families in plants are involved in the absorption, transport, and distribution of potassium ions, among which the HAK family genes play a crucial role in the high-affinity potassium absorption process in plants. For example, in rice, OsHAK1 , OsHAK5 and OsHAK16 Genes such as [specific genes] have been shown to be induced to express under low potassium conditions. Their loss of function leads to a significant decrease in the plant's potassium uptake capacity, while overexpression of these genes can significantly improve the plant's potassium accumulation and tolerance to low potassium.

[0005] Although some progress has been made in the study of the molecular mechanisms of potassium uptake and utilization in plants, existing plant materials still have shortcomings in potassium uptake efficiency, especially under low-potassium soil conditions, where crops have limited potassium acquisition capacity and cannot meet the demands of high yields. Therefore, creating plant materials with high potassium uptake and utilization efficiency through genetic engineering is of great significance for increasing crop yields, reducing potassium fertilizer input costs, and ensuring sustainable agricultural development. Summary of the Invention

[0006] To address the shortcomings of existing plant materials in terms of potassium absorption efficiency, especially under low-potassium soil conditions where crops have limited potassium uptake capacity and cannot meet the demands for high yields, this invention aims to develop a plant material with high potassium absorption and utilization efficiency, and to provide a gene mutant with high potassium absorption and utilization efficiency and its applications. To achieve the above objectives, this invention adopts the following technical solution.

[0007] This invention provides a gene mutant with high potassium absorption and utilization efficiency, wherein the gene mutant is a wild-type mutant. GhAKT1bD Gene mutant A and gene mutant B are obtained by mutation based on genes.

[0008] The gene mutant A is a wild-type mutant. GhAKT1bD The gene contains a deletion of bases from position 111 to position 114.

[0009] The gene mutant B is a wild-type mutant. GhAKT1bD The gene contains a deletion at positions 112 to 114.

[0010] The wild type GhAKT1bD The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] Preferably, the wild type GhAKT1bD The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4.

[0012] Preferably, the nucleotide sequence of the gene mutant A is shown in SEQ ID NO.2, and the nucleotide sequence of the gene mutant B is shown in SEQ ID NO.3.

[0013] Preferably, the amino acid sequence of the protein encoded by the gene mutant with high potassium absorption and utilization efficiency as shown in SEQ ID NO.2 is as shown in SEQ ID NO.5; and the amino acid sequence of the protein encoded by the gene mutant with high potassium absorption and utilization efficiency as shown in SEQ ID NO.3 is as shown in SEQ ID NO.6.

[0014] This invention also provides the application of the aforementioned high potassium absorption and utilization efficiency gene mutant in improving plant tolerance to low potassium stress.

[0015] Preferably, the wild type of plant is used. GhAKT1bD A gene mutant with high potassium absorption and utilization efficiency is mutated to obtain a transgenic plant that is resistant to low potassium abiotic stress. Transgenic plants resistant to low potassium abiotic stress are simply referred to as transgenic plants.

[0016] Understandably, the basic plant is the starting point for the transgenic plants provided in the embodiments of the present invention, namely, unmodified wild-type plants. As an example, the embodiments of the present invention use cotton as the basic plant and modify it using gene editing and other methods to obtain transgenic plants, thereby improving the plant's tolerance to low potassium stress.

[0017] Preferably, the wild type of plant is used. GhAKT1bD The steps for generating the gene mutant with the high potassium absorption and utilization efficiency are as follows:

[0018] A vector containing an sgRNA coding sequence was transferred into the plant, wherein the nucleotide sequence of the sgRNA coding sequence is shown in SEQ ID NO.7.

[0019] Preferably, the vector containing the sgRNA coding sequence is introduced into the callus tissue of the plant.

[0020] Preferably, the plant includes cotton.

[0021] Understandably, the basic plant is the starting point for the transgenic plants provided in the embodiments of the present invention, namely, unmodified wild-type plants. As an example, the embodiments of the present invention use cotton as the basic plant and modify it using gene editing and other methods to obtain transgenic plants, thereby improving the plant's tolerance to low potassium stress.

[0022] Preferably, the vector containing the sgRNA coding sequence is obtained by constructing the sgRNA coding sequence into a pTN vector.

[0023] Preferably, the method of introduction is Agrobacterium infection.

[0024] Preferably, the Agrobacterium is Agrobacterium strain GV3101.

[0025] In some embodiments, the sgRNA coding sequence is constructed into a pTN vector, and then the vector is co-cultured with cotton callus via infection with Agrobacterium tumefaciens strain GV3101, thereby transferring the vector containing the sgRNA coding sequence into the callus. The cotton callus can then grow into a mature plant through conventional differentiation, rooting, hardening-off transplanting, and subculture selection. GhAKT1bD Cotton with enhanced tolerance to low potassium due to gene suppression.

[0026] This application also provides a method for improving the tolerance of plants to low potassium stress, including the use of wild-type plants in the base plant. GhAKT1bD Gene mutations were performed to obtain transgenic plants that are resistant to low potassium stress.

[0027] Mutation refers to the wild type GhAKT1bD The gene undergoes at least one of the following mutations:

[0028] The TATC bases at positions 111 to 114 are missing.

[0029] The TCC bases at positions 112 to 114 are missing.

[0030] The mutated nucleotide sequences are shown in SEQ ID NO.2 and / or SEQ ID NO.3.

[0031] Or mutation refers to the wild type GhAKT1bD The protein expressed by the gene undergoes at least one of the following changes:

[0032] It encodes only 37 amino acids.

[0033] The 38th amino acid is deleted, and the arginine at position 39 is mutated to serine.

[0034] In some embodiments, the wild type GhAKT1bD The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0035] In some embodiments, the genetically modified plant is cotton.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. This invention provides a gene mutant with high potassium absorption and utilization efficiency, wherein the gene mutant with high potassium absorption and utilization efficiency is... GhAKT1bD Gene mutant, the gene mutant being wild-type GhAKT1bD Mutant A and mutant B were obtained by mutation based on the gene. Mutant A is a genetic variant derived from the wild-type gene. GhAKT1bD The gene contains a deletion at positions 111 through 114. Mutant B is derived from the wild-type gene. GhAKT1bD The gene contains a deletion at positions 112-114. This invention introduces... GhAKT1bD Following gene mutation, potassium supply capacity can be simultaneously enhanced, achieving "source-sink coordination" and supporting the realization of higher yield potential. For example, overexpression of this gene in crops such as cotton, rice, and corn... GhAKT1bD Gene mutants can significantly increase grain potassium content and yield. This invention utilizes wild-type... GhAKT1bD Introducing specific base deletion mutations into the gene resulted in a potassium ion channel mutant with enhanced function. This mutant exhibits the following effects:

[0038] (1) It significantly improved the potassium absorption rate and accumulation of plants under low potassium conditions.

[0039] (2) It enhances the plant’s adaptability to low-potassium soil and alleviates potassium stress.

[0040] (3) Reduce reliance on potassium fertilizer, improve fertilizer utilization, and promote green agriculture.

[0041] (4) Provide sufficient potassium nutrition support for high-yield crops to achieve synergy between high yield and high efficiency.

[0042] Therefore, this invention fundamentally improves the potassium uptake capacity of crops, solving the problem that existing plant materials still have insufficient potassium uptake efficiency in the prior art, especially under low potassium soil conditions, where the potassium uptake capacity of crops is limited and it is difficult to meet the demand for high yield. Thus, it provides core gene resources and innovative technical paths for solving the problem of high and stable crop yields under potassium-deficient arable land conditions in my country and around the world, and has significant scientific significance and application value.

[0043] Furthermore, the present invention, through the application of... GhAKT1bD Mutations in the gene cause premature termination of the encoded protein, resulting in proteins encoding only amino acid 37 (e.g., SEQ ID NO.5) and proteins with a deletion of amino acid 38 and a change in amino acid 39—arginine mutated to serine (e.g., SEQ ID NO.6). These proteins still exhibit growth advantage under low potassium stress, and their net potassium uptake rate by roots is significantly higher than that of the wild type. Therefore, they are beneficial to cotton. GhAKT1bD Genetic modification can improve cotton's tolerance to low potassium levels and enhance potassium utilization efficiency, thereby reducing the cost of potassium fertilizer application in conventional cotton production.

[0044] 2. This invention utilizes CRIPSR / Cas9 technology to regenerate wild-type plants. GhAKT1bD A gene mutation is induced, and then the mutation is transferred into a plant to obtain a transgenic plant that is resistant to low potassium stress. This transgenic plant has the following advantages over the wild-type plant:

[0045] Regardless of whether the potassium supply is sufficient at 2.5 mM or severely deficient at 0.03 mM, the transgenic plants provided in this embodiment of the invention have a growth advantage over the wild type (i.e., higher root, stem, and leaf dry weight).

[0046] Regardless of whether the potassium supply is sufficient at 2.5 mM or severely deficient at 0.03 mM, the transgenic plants provided in the embodiments of the present invention have higher stem potassium content and root potassium content compared to the wild type.

[0047] Regardless of whether the potassium supply is sufficient at 2.5 mM or severely deficient at 0.03 mM, the transgenic plants provided in this embodiment of the invention have higher root growth than the wild type, especially under severe potassium deficiency conditions.

[0048] Compared to the wild type, the transgenic plants provided in this embodiment of the invention have a stronger potassium uptake capacity. Attached Figure Description

[0049] Figure 1 The wild-type HM and mutant CRI in Example 1 of this invention #GhAKT1bD -11 and mutant CRI # GhAKT1bD Phenotypic characteristics of -84 cells cultured for 7 days under severe potassium deficiency and adequate potassium conditions: Severe potassium deficiency condition: 0.03 mM K + Sufficient potassium supply conditions: 2.5 mM K + ;in:

[0050] A represents wild-type HM and mutant CRI# under normal potassium supply conditions. GhAKT1bD -11 and mutant CRI# GhAKT1bD -84 cotyledon potassium deficiency phenotype; where normal potassium supply conditions are equivalent to adequate potassium supply conditions;

[0051] B represents the wild-type HM and mutant CRI under low potassium stress conditions. #GhAKT1bD -11 and mutant CRI # GhAKT1bD -84 cotyledon potassium deficiency phenotype; among which, low potassium stress condition is severe potassium deficiency condition;

[0052] Wild-type HM-1 is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI #GhAKT1bD -84 is abbreviated as CRI #GhAKT1bD -84.

[0053] Figure 2 The wild-type HM and mutant CRI in Example 1 of this invention #GhAKT1bD -11 and mutant CRI # GhAKT1bD Dry matter weight of roots, stems, and cotyledons after 7 days of cultivation under severe potassium deficiency and adequate potassium supply conditions: Severe potassium deficiency condition: 0.03 mM K + Sufficient potassium supply conditions: 2.5 mM K + ;in:

[0054] A represents the wild-type HM and the mutant CRI under normal potassium supply conditions and low potassium stress conditions. #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84 cotyledon dry matter weight;

[0055] B represents the wild-type HM and the mutant CRI under normal potassium supply conditions and low potassium stress conditions. #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84 stem mass weight;

[0056] C represents the conditions of normal potassium supply and low potassium stress, and the wild-type HM and mutant CRI. #GhAKT1bD -11 and mutant CRI #GhAKT1bD The root dry matter weight was -84;

[0057] Normal potassium supply conditions refer to adequate potassium supply conditions, while low potassium stress conditions refer to severe potassium deficiency conditions; wild-type HM is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI #GhAKT1bD -84 is abbreviated as CRI # GhAKT1bD -84.

[0058] Figure 3 The wild-type HM and mutant CRI in Example 1 of this invention #GhAKT1bD -11 and mutant CRI # GhAKT1bD Potassium content of roots, stems, and cotyledons after 7 days of cultivation under severe potassium deficiency and adequate potassium supply conditions: Severe potassium deficiency condition: 0.03 mM K + Sufficient potassium supply conditions: 2.5 mM K + ;in:

[0059] A represents the wild-type HM and the mutant CRI under normal potassium supply conditions and low potassium stress conditions. #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84% potassium content in leaves;

[0060] B represents the wild-type HM and the mutant CRI under normal potassium supply conditions and low potassium stress conditions. #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84% stem potassium content;

[0061] C represents the conditions of normal potassium supply and low potassium stress, and the wild-type HM and mutant CRI. #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84% potassium content in the roots;

[0062] Normal potassium supply conditions refer to adequate potassium supply conditions, while low potassium stress conditions refer to severe potassium deficiency conditions; wild-type HM is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI #GhAKT1bD -84 is abbreviated as CRI # GhAKT1bD -84.

[0063] Figure 4 The wild-type HM and mutant CRI in Example 2 of this invention #GhAKT1bD -11 and mutant CRI # GhAKT1bD -84 Root phenotypic characteristics after 21 days of cultivation under severe potassium deficiency and adequate potassium supply conditions; Severe potassium deficiency condition: 0.03 mM K + Sufficient potassium supply conditions: 2.5 mM K + ;in:

[0064] A represents the wild-type HM and mutant CRI under normal potassium supply conditions. #GhAKT1bD -11 and mutant CRI # GhAKT1bD -84 root phenotype; among which, normal potassium supply conditions are equivalent to sufficient potassium supply conditions;

[0065] B represents the wild-type HM and mutant CRI under low potassium stress conditions. #GhAKT1bD -11 and mutant CRI # GhAKT1bD The root phenotype was -84; among which, low potassium stress conditions are severe potassium deficiency conditions.

[0066] Wild-type HM is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI #GhAKT1bD -84 is abbreviated as CRI #GhAKT1bD -84.

[0067] Figure 5 The wild-type HM and mutant CRI in Example 3 of this invention #GhAKT1bD -11 and mutant CRI # GhAKT1bD The exhaustion test results were -84.

[0068] Figure 6 The wild-type HM and mutant CRI in Example 4 of this invention #GhAKT1bD -11 and mutant CRI # GhAKT1bD Results of the net potassium ion absorption rate of the -84 root system. Detailed Implementation

[0069] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0070] This invention is based on the inventor's following understanding:

[0071] Potassium, an essential nutrient for plant growth and development, plays a vital role in maintaining normal physiological and biochemical processes in plant cells, such as osmotic regulation, ion homeostasis, and photosynthesis. Cotton is a potassium-loving economic crop, but current cultivated soils are generally deficient in potassium, and my country's overall potassium fertilizer resources are scarce, severely impacting cotton yield and quality. Therefore, developing cotton materials with high potassium absorption and utilization efficiency is of paramount importance for cotton growth and development and for reducing potassium fertilizer input costs.

[0072] The plant root system contains both parents and K + The absorption mechanism involves different pathways. When the external potassium ion concentration is high (>0.3 mM), the low-affinity potassium uptake system (LATS) is mainly mediated by potassium ion channels. When the external potassium ion concentration is low (<0.2 mM), the high-affinity potassium transport and uptake system (HATS) is mainly mediated by potassium transporters. Based on protein characteristics, plant potassium ion channels can be classified into three main families: Shaker, TPK, and Kir-like potassium channel families. AKT1, as an important member of the Shaker potassium channel family, has ambidextrous properties and is an important channel mediating the absorption of potassium ions from the soil by plant roots.

[0073] The inventors of this invention previously cloned from the potassium-efficient genotype Liaomian 17. GhAKT1 Potassium channel genes were identified, and their subcellular localization, tissue-specific expression, and potassium uptake function were determined. Details of this research can be found in Chinese patent document CN103396477B. GhAKT1 It is wild type GhAKT1bD Gene, abbreviated as GhAKT1bD Gene.

[0074] Subsequently, the research group of the inventors of this invention obtained overexpression via Agrobacterium transformation. GhAKT1 The homozygous cotton lines were evaluated for their tolerance to low potassium during the indoor seedling stage and in severely potassium-deficient field soils. The results showed that they exhibited certain yield and growth advantages compared to the recipient variety R15. The inventors of this invention further utilized CRISPR / Cas9 technology to… GhAKT1bD Gene editing revealed that the cotton recipient variety HM-1... GhAKT1bD After the gene is mutated into a nucleotide mutant as shown in SEQ ID NO.1 or SEQ ID NO.2, the resulting transgenic plants have the same, or even better, tolerance to low potassium.

[0075] Among them, overexpression GhAKT1 The homozygous cotton lines were published in the literature "Luo Bixue." GhAKT1 Identification of potassium-efficient cotton lines [D]. China Agricultural University, 2018.

[0076] The receptor variety R15 is disclosed in the literature "Luo Bixue. Translated GhAKT1 Identification of potassium-efficient cotton lines [D]. China Agricultural University, 2018.

[0077] The cotton recipient variety HM-1 was provided by Wuhan Tianwen Company.

[0078] The first aspect of this invention is to provide a gene mutant with high potassium absorption and utilization efficiency, compared to the wild type. GhAKT1bD The gene has at least one of the following mutations:

[0079] Bases 111 through 114 are missing.

[0080] Bases 112 to 114 are missing.

[0081] Alternatively, the protein encoded by the gene mutant with high potassium uptake and utilization efficiency undergoes at least one of the following changes:

[0082] It encodes only 37 amino acids.

[0083] The 38th amino acid is deleted, and the arginine at position 39 is mutated to serine.

[0084] wild type GhAKT1bD The gene sequence information is Gh_D07G0028.1, and its nucleotide sequence is shown in SEQ ID NO.1:

[0085]

[0086] Wild type GhAKT1bD The sequence information of the gene is Gh_D07G0028.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 4:

[0087] MFRGSVLCGQEEIEHLSRESSHYSLTTGIIPSLGARSIRRVKLRSYIISPHDRRYRVWETFLVLLVIYTAWVSPFEFGFLGKPDTPLAVTDNVVNGFFALDIILTFFVAYLDKAAYLLIDDPKKIAWKYGTSWLAFDVISTIPSELARRISPKPLRSYGLFNMLRLWRLRRVSALFSRLEKDKNFNYFWVRCAKLICVTVFAVHCAGCFYYRIAARYRDPGRTWIGASMGDNFHEQSLSIRYVTSMYWSITTLTTVGYGDLHPVNTPEMIFDIFYMFFNLGLTAYLIGNMTNLVVHGTSRTRQFRDTIQAASSFAQRNQLPPRLLDQMVAHLSLKYRTDSEGLQQQETLDSLPKAIRSSISHYLFYSLVDNVYLFHGVSNDLLFQLVSEMKAEYFPPKEDVILQNEAPTDFYILVTGAVDLLVRKNGVEQVVGEASAGDLCGEIGVLCYRPQLFTVRTKRLCQLLRLNRTTFLNIIQANVGDGTIIMNNLLQHLKDMDDPIMEGVLIETQNMLARGSMDLPLNLCFAAVKGDDSLLNKLLEKGHNANESDDNGRTPLHIAASKGSENCVLILLDHNADPNIKDSEGSVPLWEAILGGHDNIAKLLKDNGANINVGDVGHFACIAAEQNDLNLLKEIVRYGGNVTCPRYNGYTALHVAVCEGNFEIVKYLLEQGSDIDKRDIHGWTPRDLAAQQGHEDIEMIFKSLTEKKKTQPIMTIPEKHEARFLGRFTSEPVISTATSGDGLDGSSRGKSRRPRRKSSNFNNSLFGIMSQNVEKDLLLSVHQPKGMKDPLVKSSRVIISCPEKGDTIGKLVLVPGSMEELVEIGANKFGIFGGKVMNKGGGEIDDIEVIRDGDHLVFVSDGYMQQEINDTQNP。

[0088] The nucleotide sequences of gene mutants with high potassium absorption and utilization efficiency are shown in SEQ ID NO.2 or SEQ ID NO.3.

[0089] The gene mutant with high potassium absorption and utilization efficiency, as shown in SEQ ID NO.2, is the mutated GhAKT1bD gene 1, and its nucleotide sequence is as follows:

[0090]

[0091] The gene mutant with high potassium absorption and utilization efficiency, as shown in SEQ ID NO.3, is the mutated GhAKT1bD gene 2, and its nucleotide sequence is as follows:

[0092]

[0093] The amino acid sequence of the protein encoded by the gene mutant with high potassium absorption and utilization efficiency shown in SEQ ID NO.2 is shown in SEQ ID NO.5;

[0094] MFRGSVLCGQEEIEHLSRESSHYSLTTGIIPSLGARSAGLSSGAILFLLTIVVTGYGRLFWSY.

[0095] The amino acid sequence of the protein encoded by the gene mutant with high potassium absorption and utilization efficiency, as shown in SEQ ID NO.3, is shown in SEQ ID NO.6:

[0096] .

[0097] The following embodiments are used to further illustrate the advantages and features of this method, and are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0098] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0099] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0100] Example 1

[0101] (1) Constructing a vector pTN-sgRNA containing the sgRNA coding sequence: The sgRNA coding sequence is constructed into the pTN vector.

[0102] Among them, the vector pTN-sgRNA containing the sgRNA coding sequence is simply referred to as the vector containing the sgRNA coding sequence or the pTN-sgRNA vector.

[0103] The nucleotide sequence encoding the sgRNA is shown in SEQ ID NO.7:

[0104] TGGTGCCAGAAGTATCCGCAGGG.

[0105] The pTN carrier was provided by Wuhan Tianwen Company.

[0106] (2) Disinfect the seeds of the cotton recipient variety HM-1. After the seeds germinate, cut the hypocotyl of the sterile seedling and cut the hypocotyl into 5mm stem segments to obtain cotton callus tissue, i.e. explant.

[0107] The cotton recipient variety HM-1 was provided by Wuhan Tianwen Company.

[0108] (3) Soak the explants with pTN-sgRNA vector for 8 minutes by infecting them with Agrobacterium tumefaciens strain GV3101 to transfer the pTN vector into the cotton callus. Then, use sterile filter paper to absorb the excess bacterial solution. Finally, place the cotton callus on a co-culture medium covered with filter paper and co-culture in the dark for 2 days.

[0109] The co-culture medium is MSB medium, which is made from materials with the following final concentrations:

[0110] The co-culture medium consisted of 1×MS inorganic salts, 100 mg / L inositol, 2.0 mg / L glycine, 0.5 mg / L nicotinic acid, 0.5 mg / L pyridoxine hydrochloride, 0.1 mg / L thiamine hydrochloride, 0.1 mg / L 4-dichlorophenoxyacetic acid, 0.1 mg / L 6-furfurylaminopurine, 0.3 g / L MgCl₂·6H₂O, 3% D-glucose, and 0.25% plant gel, in water as the solvent; the pH of the co-culture medium was 5.85.

[0111] (4) Transfer the cotton callus tissue cultured in (3) to a resistant callus induction medium and add 400 mg•L -1 Cef cephalosporin inhibited the growth of Agrobacterium and was cultured for 4 months at 28℃±2℃ for 16 / 8 days / night to induce the growth of resistant callus. Subculture was performed every 30 days.

[0112] The resistant callus induction medium was prepared by adding MSB medium to a final concentration of 400 mg / L. -1 Cef cephalosporin.

[0113] (5) Differentiation induction of the resistant callus cultured in (4) is performed by cutting off the loose pale yellow or white callus at both ends. When the callus grows to the size of a fingernail, it is transferred to a differentiation medium without any hormones and antibiotics to continue proliferation induction until embryogenic callus is produced.

[0114] The differentiation medium, free of any hormones and antibiotics, was prepared from materials at the following final concentrations:

[0115] The medium consisted of 1× MSB medium powder (NH4NO3 removed), 0.5 mg / L indole-3-butyric acid, 0.15 mg / L 6-furfurylaminopurine, 30 g / L L-glucose, 1.0 g / L glutamine, 0.5 g / L asparagine, and 2.5 g / L plant gel, in water as the solvent; the pH of the differentiation medium was 5.90, with all hormones and antibiotics removed.

[0116] (6) Embryogenic callus tissue was induced into embryoids and plant seedlings. When the seedlings sprouted true leaves and were about 3 cm tall, they were transferred to seedling culture medium for further culture. When the seedlings grew to 6 cm, the regenerated seedlings were in a semi-lignified state and were grafted in a greenhouse until they survived. PCR molecular detection was performed and positive plants were retained until the T1 generation seeds of the transgenic line were harvested.

[0117] The seedling culture medium is made from materials with the following final concentrations:

[0118] The medium consisted of 1 / 2 MSB medium powder, 0.5 g / L glutamine, 0.25 g / L asparagine, 15 g / L D-glucose, and 2.5 g / L plant gel, in water as the solvent; the pH of the seedling medium was 5.90.

[0119] The steps for PCR molecular detection are as follows:

[0120] 1) Primer design:

[0121] Based on the target gene GhAKT1 nucleotide sequence and screening gene NptII The nucleotide sequence was obtained, and primers were designed using Primer3 and DNAMAN. The primer amplification fragments were approximately 390 bp and 720 bp, respectively. These primers were designed and synthesized by BGI Genomics. Primer information is as follows:

[0122] CRISPR-AKT1-R: 5'-TCCAAAACAAAGGCTCACGA-3', as shown in SEQ ID NO.8.

[0123] CRISPR-AKT1-F: 5'-AGAGGGTGTTTGATGCTTGT-3', as shown in SEQ ID NO.9.

[0124] NptII-F: 5'-TCCGGCCGCTTGGGTGGAGAG-3', as shown in SEQ ID NO.10.

[0125] NptII-R: 5'-CTGGCGCGAGCCCCTGATGCT-3', as shown in SEQ ID NO.11.

[0126] 2) The reaction system and the volume of each component are shown in Table 2.

[0127] Table 2. Components and volumes of the PCR reaction system

[0128]

[0129] The 2× high-fidelity enzyme premix was purchased from Kangwei Century Company, catalog number: CW3405S. Forward primers are short nucleotide chains complementary to the sense strand of the template DNA, providing the initiation binding site for DNA polymerase, including CRISPR-AKT1-F and NptII-F; reverse primers are short nucleotide chains complementary to the antisense strand of the template DNA, providing the initiation binding site for DNA polymerase, including CRISPR-AKT1-R and NptII-R. Template DNA refers to the DNA extracted using the CTAB cleavage method, i.e., the target gene. GhAKT1 .

[0130] 3) PCR amplification conditions:

[0131] Pre-denaturation at 94℃ for 5 minutes.

[0132] Denaturation at 94℃ for 30 seconds.

[0133] 56℃ refolding time: 30s.

[0134] Extended time at 72℃: 2 minutes.

[0135] Extended time at 72℃: 2 minutes.

[0136] 12℃ insulation: ∞.

[0137] Steps 2 through 4 are repeated 35 times.

[0138] 4) Condition assessment:

[0139] Take 5 μL of PCR product for agarose gel electrophoresis to detect the target gene. GhAKT1 and gene screening NPT II Primers.

[0140] (7) DNA was extracted from the leaves of the transgenic plant lines using the CTAB method, and PCR detection was performed by screening for marker gene-specific primers to determine whether the transgenic plant lines were successfully obtained.

[0141] The method for screening specific primers for marker genes for PCR detection is the same as the PCR molecular detection step in step (6).

[0142] 2. Identification of successful gene editing:

[0143] PCR detection results showed that the positive rate of the plants reached 87.5%, and the sequencing peak of the 369bp gene fragment near the target gene was found to be a single peak, indicating that the method of the present invention can efficiently obtain transgenic plant lines with enhanced low potassium tolerance.

[0144] Among them, transgenic plant lines with enhanced tolerance to low potassium are known as transgenic plants or CRIs. #GhAKTbD Mutant, CRI #GhAKTbD Mutants include mutant CRI #GhAKT1bD -11 and mutant CR I#GhAKT1bD -84.

[0145] Example 2

[0146] Wild-type HM and the mutant CRI obtained above were taken respectively. #GhAKT1bD -11 and mutant CR I#GhAKT1bD For the -84 strain, two treatment groups were designed for the three types of strains mentioned above: a severe potassium deficiency group and a sufficient potassium supply group.

[0147] Among them, the severe potassium deficiency group is the severe potassium deficiency treatment, in which potassium nutrient solution 1 is used to treat the above three types of strains respectively.

[0148] The potassium-sufficient group, also known as the potassium-sufficient treatment, uses potassium nutrient solution 2 to treat the three types of strains mentioned above.

[0149] Wild-type HM refers to the cotton receptor variety HM-1.

[0150] Potassium nutrient solution 1 is made from materials with the following final concentrations:

[0151] 0.03mM KNO3, Ca(NO3)2, 1mM MgSO4, 0.5mM (NH4)H2PO4, 0.1mM FeNaEDTA, 2×10 -4 mMCuSO4, 1×10 -3 mM ZnSO4, 2×10 -2 mM H3BO3, 5×10 -6 mM (NH4)6Mo7O 24 and 1×10 -3 mM MnSO4.

[0152] The treatment method for the fully potassium-supplyed group is the same except for the composition of the potassium nutrient solution. The potassium nutrient solution for the fully potassium-supplyed group is potassium nutrient solution 2, which is made from materials with the following final concentrations:

[0153] 2.5mM KNO3, Ca(NO3)2, 1mM MgSO4, 0.5mM (NH4)H2PO4, 0.1mM FeNaEDTA, 2×10 -4 mMCuSO4, 1×10 -3 mM ZnSO4, 2×10 -2 mM H3BO3, 5×10 -6 mM (NH4)6Mo7O 24 and 1×10 -3 mM MnSO4.

[0154] The seeds of the three types of strains were disinfected with 9% hydrogen peroxide. After 15 minutes, they were rinsed several times with clean water and soaked in deionized water for 12 hours. Seeds with white sprouts were selected and planted in a clean and dry sand bed. Water was sprayed on the seed surface to keep it moist. After covering with non-woven fabric, a thin layer of sand was placed on top. After culturing in the dark for 2 days, the seed coat was removed. After 1 day of light exposure, the seedlings were transferred directly to potassium nutrient solution 1 after the cotyledons unfolded.

[0155] Each treatment group had three replicates. Seedlings with uniform growth were selected, and the cotyledons and hypocotyls were wrapped in sponges and transferred to perforated trays. They were then placed in nutrient solutions containing different potassium levels. Phenotypic photos were taken on day 7, and the dry matter weight and potassium content of each line were recorded.

[0156] The seedling cultivation conditions were as follows: light / dark duration of 14h / 10h, day / night temperature of 30±2℃ / 22±2℃, relative humidity of 75%±5%, and light intensity of 600 μmol / cm². -2 s -1 .

[0157] See the results of the photograph. Figure 1 .according to Figure 1 As can be seen, at a potassium supply level of 0.03 mM, CRI was found to be... #GhAKTbD The mutant exhibited the low potassium phenotype later, and the proportion of potassium-deficient spots in the cotyledons was less than that of the wild-type HM. Among these, CRI... #GhAKTbD Mutants include mutant CRI #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84. Wild-type HM is abbreviated as HM; mutant CRI # GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI #GhAKT1bD -84 is abbreviated as CRI #GhAKT1bD -84.

[0158] Method for detecting dry matter weight: Divide the seedlings into three parts: roots, stems, and cotyledons. Place them at 105℃ for 30 minutes to blanch, then dry at 80℃ for 48 hours. Weigh and record the results. Figure 2 As shown. According to Figure 2 As can be seen, culturing at a potassium supply level of 0.03 mM revealed that CRI #GhAKTbD The mutant's cotyledon dry weight, stem dry weight, and root dry weight were all significantly higher than those of the wild-type HM, and under a normal potassium supply level of 2.5 mM, the CRI (Cellular Intake) was significantly lower. #GhAKTbD The mutants also maintain their growth advantage.

[0159] Potassium content detection method: Dry roots, stems, and cotyledons of the plant were ground into powder. 0.2g of each powder was accurately weighed, and three replicates were made. Each powder was placed in a 10mL centrifuge tube, and 5mL of mol / L hydrochloric acid was added for extraction. The mixture was incubated overnight at 28℃ using a shaker. The extract was filtered through 9cm qualitative filter paper, diluted 100 times, and the potassium content of each plant part was determined using an atomic absorption spectrophotometer. The results are shown below. Figure 3 As shown. According to Figure 3 As can be seen, after one week of culture under normal potassium supply and 0.03 mM potassium supply levels, CRI was found to be... #GhAKTbDThe potassium content of the cotyledons, stems, and roots of the mutant was higher than that of the wild-type HM, and in some cases, the difference was statistically significant, except for the mutant CR. I#GhAKT1bD -84 on the outside of the blade.

[0160] In this context, "overnight" refers to a duration of 12 hours or less.

[0161] Example 3

[0162] Wild-type HM and the mutant CRI obtained above were taken respectively. #GhAKT1bD -11 and mutant CR I#GhAKT1bD For the -84 strain, two treatment groups were designed for the three types of strains mentioned above: a severe potassium deficiency group and a sufficient potassium supply group.

[0163] Among them, the severe potassium deficiency group is the severe potassium deficiency treatment, in which potassium nutrient solution 1 is used to treat the above three types of strains respectively.

[0164] The potassium-sufficient group, also known as the potassium-sufficient treatment, uses potassium nutrient solution 2 to treat the three types of strains mentioned above.

[0165] Wild-type HM refers to the cotton receptor variety HM-1.

[0166] The formula for potassium nutrient solution 1 is the same as that in Example 2.

[0167] The treatment method for the fully potassium-supply group is the same except for the composition of the potassium nutrient solution. The potassium nutrient solution for the fully potassium-supply group is potassium nutrient solution 2, and its formula is the same as in Example 2.

[0168] The seeds of the three types of strains were disinfected with 9% hydrogen peroxide. After 15 minutes, they were rinsed several times with clean water and soaked in deionized water for 12 hours. Seeds with white sprouts were selected and planted in a clean and dry sand bed. Water was sprayed on the seed surface to keep it moist. After covering with non-woven fabric, a thin layer of sand was placed on top. After culturing in the dark for 2 days, the seed coat was removed. After 1 day of light exposure, the seedlings were transferred directly to potassium nutrient solution 1 after the cotyledons unfolded.

[0169] Each treatment group was configured with three replicates. The culture medium was changed every 5 days in each treatment group. On day 21, the roots of the plants in each group were collected and scanned using an EPSON root scanner. The results are as follows: Figure 4 As shown.

[0170] The seedling cultivation conditions are the same as in Example 2.

[0171] according to Figure 4 As can be seen, after three weeks of culture under normal potassium supply and 0.03 mM potassium supply levels, CR I#GhAKTbD The root growth of the mutant was significantly higher than that of the wild-type HM.

[0172] Among them, CRI #GhAKTbD Mutants include mutant CRI #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84. Wild-type HM is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI # GhAKT1bD -84 is abbreviated as CRI #GhAKT1bD -84.

[0173] Example 4

[0174] Wild-type HM and the mutant CRI obtained above were taken respectively. #GhAKT1bD -11 and mutant CR I#GhAKT1bD For the -84 strain, two treatment groups were designed for the three types of strains mentioned above: a severe potassium deficiency group and a sufficient potassium supply group.

[0175] Among them, the severe potassium deficiency group is the severe potassium deficiency treatment, in which potassium nutrient solution 1 is used to treat the above three types of strains respectively.

[0176] The potassium-sufficient group, also known as the potassium-sufficient treatment, uses potassium nutrient solution 2 to treat the three types of strains mentioned above.

[0177] Wild-type HM refers to the cotton receptor variety HM-1.

[0178] The formula for potassium nutrient solution 1 is the same as that in Example 2.

[0179] The treatment method for the fully potassium-supply group is the same except for the composition of the potassium nutrient solution. The potassium nutrient solution for the fully potassium-supply group is potassium nutrient solution 2, and its formula is the same as in Example 2.

[0180] The seeds of the three types of strains were disinfected with 9% hydrogen peroxide. After 15 minutes, they were rinsed several times with clean water and soaked in deionized water for 12 hours. Seeds with white sprouts were selected and planted in a clean and dry sand bed. Water was sprayed on the seed surface to keep it moist. After covering with non-woven fabric, a thin layer of sand was placed on top. After culturing in the dark for 2 days, the seed coat was removed. After 1 day of light exposure, the seedlings were transferred directly to potassium nutrient solution 1 after the cotyledons unfolded.

[0181] Each treatment group was configured with three replicates. After the cotyledons of the seedlings had fully expanded, three representative and uniformly growing seedlings were selected from each group. These seedlings were placed in 50 mL centrifuge tubes containing 40 mL of starvation solution for 48 hours, with the starvation solution replaced every 24 hours. The cotton seedlings were then removed and placed in a depletion solution containing 0.068 mM potassium. 1 mL of the depletion solution was collected at 0 h, 1 h, 3 h, 5 h, and 8 h. After sampling, the fresh weight of the roots was weighed, and the potassium content in the depletion solution was determined using an atomic absorption spectrophotometer. The results are shown below. Figure 5 As shown.

[0182] The seedling cultivation conditions are the same as in Example 2.

[0183] The starvation fluid is made from materials with the following final concentrations:

[0184] 5 mM 2-morpholinoethanesulfonic acid and 0.2 mM Ca2SO4, with water as the solvent.

[0185] The depletion solution is made from materials with the following final concentrations:

[0186] The pH of the depleted solution was adjusted to 6.8 using tris(hydroxymethyl)aminomethane, with 5 mM 2-morpholine ethanesulfonic acid, 0.2 mM Ca2SO4, and 0.068 mM KNO3.

[0187] The plants in each group are the same as in Example 1.

[0188] according to Figure 5 As can be seen, in the depletion experiment conducted at an actual potassium supply level of 0.054 mM, the CRI decreased with increasing culture time. #GhAKTbD The potassium content in the culture medium of the mutant was much lower than that of the wild-type HM, indicating that CRI #GhAKTbD The mutant has a strong ability to absorb potassium.

[0189] Among them, CRI #GhAKTbD Mutants include mutant CRI #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84. Wild-type HM is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI # GhAKT1bD -84 is abbreviated as CRI #GhAKT1bD -84.

[0190] Example 5

[0191] Wild-type HM and the mutant CRI obtained above were taken respectively. #GhAKT1bD -11 and mutant CR I#GhAKT1bDFor the -84 strain, two treatment groups were designed for the three types of strains mentioned above: a severe potassium deficiency group and a sufficient potassium supply group.

[0192] Among them, the severe potassium deficiency group is the severe potassium deficiency treatment, in which potassium nutrient solution 1 is used to treat the above three types of strains respectively.

[0193] The potassium-sufficient group, also known as the potassium-sufficient treatment, uses potassium nutrient solution 2 to treat the three types of strains mentioned above.

[0194] Wild-type HM refers to the cotton receptor variety HM-1.

[0195] The formula for potassium nutrient solution 1 is the same as that in Example 2.

[0196] The treatment method for the fully potassium-supply group is the same except for the composition of the potassium nutrient solution. The potassium nutrient solution for the fully potassium-supply group is potassium nutrient solution 2, and its formula is the same as in Example 2.

[0197] Seeds of the three types of strains were disinfected using 9% hydrogen peroxide. After rinsing the seeds several times with deionized water, they were rolled in germination paper, placed in deionized water, and cultured in a 28℃ incubator for 4 days. The seedling roots were then rinsed with distilled water and soaked for 30 minutes, then transferred to 20 mL of test solution and soaked for 10 minutes. After transferring to a fresh 20 mL test solution and testing for 10 minutes, the net potassium ion uptake rate of the roots in each group was detected using a non-destructive ion meter. The measurement site was the principal root 300 μm from the root tip. Each treatment was replicated with 3 plants. The results are as follows: Figure 6 As shown.

[0198] The test solution formulation consists of 0.1 mM KCl, 0.1 mM CaCl2, and 0.3 mM MES, with a pH of 6.0.

[0199] according to Figure 6 As can be seen, CRI #GhAKTbD The net potassium uptake rate of the mutant roots was significantly higher than that of the wild-type HM, indicating that CRI #GhAKT1bD The mutant's root potassium uptake capacity was significantly higher than that of the wild-type HM.

[0200] Among them, CRI #GhAKTbD Mutants include mutant CRI #GhAKT1bD -11 and mutant CRI #GhAKT1bD -84. Wild-type HM is abbreviated as HM; mutant CRI #GhAKT1bD -11 is abbreviated as CRI #GhAKT1bD -11; mutant CRI # GhAKT1bD -84 is abbreviated as CRI #GhAKT1bD -84.

[0201] The experimental results above show that the CRI provided by this invention... #GhAKTbD The mutant exhibits a strong growth advantage under low potassium stress, with smaller potassium-deficient patches on cotyledons compared to the wild-type HM. Furthermore, the potassium content in the roots is significantly increased, and the net potassium uptake capacity of the roots is significantly enhanced. These findings indicate that CRI #GhAKTbD By enhancing the potassium absorption capacity of the root system, the tolerance to low potassium levels is increased.

[0202] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0203] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A gene mutant with high potassium absorption and utilization efficiency, characterized in that, The gene mutant is in the wild type. GhAKT1bD Mutant A or mutant B obtained by mutation based on a gene: The gene mutant A is a wild-type mutant. GhAKT1bD The gene contains a deletion of bases from position 111 to position 114. The gene mutant B is a wild-type mutant. GhAKT1bD The gene contains a deletion at positions 112 to 114. The wild type GhAKT1bD The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The gene mutant with high potassium absorption and utilization efficiency according to claim 1, characterized in that, The nucleotide sequence of mutant A is shown in SEQ ID NO.2, and the nucleotide sequence of mutant B is shown in SEQ ID NO.

3.

3. The application of the gene mutant with high potassium absorption and utilization efficiency as described in any one of claims 1 to 2 in improving plant tolerance to low potassium abiotic stress, characterized in that, The plant in question is cotton.

4. The application of the high potassium absorption and utilization efficiency gene mutant according to claim 3 in improving plant tolerance to low potassium stress, characterized in that, The wild type of plant GhAKT1bD Gene mutations were performed to create a mutant with high potassium absorption and utilization efficiency, resulting in transgenic plants that are resistant to low potassium abiotic stress.

5. The application of the high potassium absorption and utilization efficiency gene mutant according to claim 4 in improving plant tolerance to low potassium stress, characterized in that, The wild type of plant GhAKT1bD The steps for generating the gene mutant with the high potassium absorption and utilization efficiency are as follows: A vector containing an sgRNA coding sequence was transferred into the plant, wherein the nucleotide sequence of the sgRNA coding sequence is shown in SEQ ID NO.

7.

6. The application of the gene mutant with high potassium absorption and utilization efficiency according to claim 5 in improving plant tolerance to low potassium abiotic stress, characterized in that, The vector containing the sgRNA coding sequence is transferred into the callus tissue of the plant.

7. The application of the high potassium absorption and utilization efficiency gene mutant according to claim 6 in improving plant tolerance to low potassium stress, characterized in that, The vector containing the sgRNA coding sequence is obtained by constructing the sgRNA coding sequence into a pTN vector.

8. The application of the gene mutant with high potassium absorption and utilization efficiency according to claim 6 in improving plant tolerance to low potassium stress, characterized in that, The method of introduction was Agrobacterium infection.

9. The application of the gene mutant with high potassium absorption and utilization efficiency according to claim 8 in improving plant tolerance to low potassium stress, characterized in that, The Agrobacterium species in question is Agrobacterium strain GV3101.

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