Application of GhMLDRLK Family Genes in Breeding Heat-tolerant Cotton

Abstract

The application belongs to the technical field of plant molecular biology, and relates to application of GhMLDRLK family genes in creating high-temperature-resistant cotton. Six genes are screened from GhMLDRLK family genes and are strongly associated with the high-temperature trait of cotton, and experiments prove that the six genes can all negatively regulate the reproductive fertility of cotton under high-temperature stress. The mutants of the above genes created through gene editing technology all show significantly enhanced heat resistance under high-temperature stress, and the application provides important gene resources and technical support for cotton high-temperature-resistant breeding and high yield and stable yield, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology and relates to the application of GhMLDRLK family genes in the creation of heat-resistant cotton. Background Technology

[0002] Against the backdrop of global warming, extreme heat events in summer are becoming more frequent and their duration significantly prolonged. Cotton is an important economic crop. Although it is a warm-loving crop, extreme heat stress can disrupt the plant's physiological metabolic homeostasis, hindering normal growth and development. This not only reduces fiber quality and yield but can also lead to plant death in severe cases. High temperatures inhibit both vegetative and reproductive growth in cotton. Compared to vegetative organs, reproductive organs are more sensitive to heat stress, with stamens exhibiting significantly lower heat tolerance than pistils. High temperatures easily cause decreased pollen viability, hindered anther dehiscence, and massive boll shedding, resulting in reduced seed setting rate and yield. Simultaneously, they inhibit normal cotton fiber development, leading to deterioration in fiber quality. Therefore, creating high-temperature-resistant cotton germplasm is of significant research value.

[0003] Introducing genes that positively regulate heat resistance into the cotton genome using transgenic technology is a major molecular breeding technique for cultivating heat-resistant cotton varieties. However, it has certain limitations in terms of biosafety. The development of gene editing technology has provided new ideas and technical support for heat-resistant cotton breeding. It can precisely target and knock out genes that negatively regulate heat resistance, creating heat-resistant mutant cotton materials, and then breeding stable new heat-resistant lines through multiple generations. Therefore, using gene editing technology to knock out or silence genes that negatively regulate heat resistance has become an important approach to improving cotton heat resistance and cultivating stress-resistant new lines. Summary of the Invention

[0004] In view of this, the present invention provides the application of GhMLDRLK family genes in the creation of heat-resistant cotton. The screened GhMLDRLK family genes can negatively regulate the reproductive fertility of cotton under high temperature conditions. Mutants created through gene editing technology all exhibit significantly enhanced heat resistance under high temperature stress. The purpose of this invention is to solve or at least partially solve the problems in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a gene associated with the heat resistance trait of cotton, said gene being selected from at least one of the following genes: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12.

[0007] Preferably, the nucleotide sequence of the GhMLDRLKA10 gene is shown in SEQ ID NO.1.

[0008] Preferably, the nucleotide sequence of the GhMLDRLKA13 gene is shown in SEQ ID NO.2.

[0009] Preferably, the nucleotide sequence of the GhMLDRLKA14 gene is shown in SEQ ID NO.3.

[0010] Preferably, the nucleotide sequence of the GhMLDRLKD10 gene is shown in SEQ ID NO.4.

[0011] Preferably, the nucleotide sequence of the GhMLDRLKD11 gene is shown in SEQ ID NO.5.

[0012] Preferably, the nucleotide sequence of the GhMLDRLKD12 gene is shown in SEQ ID NO.6.

[0013] Secondly, the present invention also provides the application of the above-mentioned gene in regulating the heat resistance of cotton.

[0014] Preferably, the gene is selected from at least one of the following genes: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12.

[0015] Preferably, the regulation is negative regulation.

[0016] More preferably, the regulation includes: inhibiting the expression of the gene to enhance the heat resistance of the cotton plant.

[0017] More preferably, the reagent used to inhibit the expression of the gene is selected from one of the following (A)-(C):

[0018] (A) An sgRNA for knocking down the gene, wherein the target site of the sgRNA is selected from at least one of the nucleotide sequences shown in SEQ ID NO. 7-12;

[0019] (B) An editing vector containing the sgRNA described in (A);

[0020] (C) Recombinant microorganisms containing the editing vector described in (B).

[0021] More preferably, inhibiting the expression of the gene includes the following steps:

[0022] A CRISPR / Cas9 recombinant vector was constructed, transformed into wild-type cotton, and transgenic cotton plants with reduced gene expression were obtained.

[0023] More preferably, the wild-type cotton is upland cotton J668.

[0024] More preferably, the CRISPR / Cas9 recombinant vector includes an sgRNA for knocking down the gene, wherein the target site of the sgRNA is selected from at least one of the nucleotide sequences shown in SEQ ID NO. 7-12.

[0025] More preferably, the conversion of wild-type cotton includes the following steps:

[0026] The CRISPR / Cas9 recombinant vector was transferred into wild-type cotton using Agrobacterium-mediated transformation.

[0027] Thirdly, the present invention also provides a method for cultivating heat-resistant cotton, comprising the following steps:

[0028] A CRISPR / Cas9 recombinant vector was constructed and transformed into wild-type cotton to obtain transgenic cotton plants with reduced gene expression levels associated with cotton's heat tolerance trait.

[0029] Preferably, the gene associated with the heat resistance trait of cotton is selected from at least one of the following genes: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12.

[0030] More preferably, the nucleotide sequences of the GhMLDRLKA10 gene, GhMLDRLKA13 gene, GhMLDRLKA14 gene, GhMLDRLKD10 gene, GhMLDRLKD11 gene and GhMLDRLKD12 gene are shown in SEQ ID NO. 1-6, respectively.

[0031] Preferably, the CRISPR / Cas9 recombinant vector includes an sgRNA for knocking down the gene, wherein the target site of the sgRNA is selected from at least one of the nucleotide sequences shown in SEQ ID NO. 7-12.

[0032] Preferably, the conversion of wild-type cotton includes the following steps:

[0033] The CRISPR / Cas9 recombinant vector was transferred into wild-type cotton using Agrobacterium-mediated transformation.

[0034] Preferably, the wild-type cotton is upland cotton J668.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0036] This invention screened six genes in the GhMLDRLK family that are strongly associated with high-temperature traits in cotton, revealing the core genetic targets regulating cotton's high-temperature resistance. Based on these genes, the gene-edited mutants created in this invention exhibit excellent high-temperature resistance: pollen viability is unaffected by high temperatures, yield traits are superior to the wild type under high temperatures, and the traits are stable under high temperatures. This provides excellent and highly resistant genetic material and germplasm resources for cotton high-temperature resistance genetic breeding. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 Figure 1 shows the gene editing results of mutant cotton plants; Figure 2 shows the specific gene editing of mutant Ghmldrlka10-1; Figure 3 shows the specific gene editing of mutant Ghmldrlka13-1; Figure 4 shows the specific gene editing of mutant Ghmldrlka14-1; Figure 5 shows the specific gene editing of mutant Ghmldrlkd10-1; Figure 6 shows the specific gene editing of mutant Ghmldrlkd11-1; Figure 7 shows the specific gene editing of mutant Ghmldrlkd12-1.

[0039] Figure 2 The images show the TTC staining results of mutant pollen at 34℃ and 37℃.

[0040] Figure 3 Phenotypic diagram of seed cotton yield in mutant plants under high temperature. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this invention, the term "comprising" means "including but not limited to". Various embodiments of the invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0043] The GhMLDRLK family of genes is a class of receptor-like kinase genes containing a Malectin-like domain. Typical characteristics of GhMLDRLK family genes in cotton include the presence of a Malectin domain, a transmembrane domain, and a kinase domain. The Malectin domain is a conserved carbohydrate recognition domain, primarily located in the plant cell membrane, and plays a central role in plant signal sensing and stress response. Kinases containing the Malectin domain can recognize cell wall polysaccharides, glycosylated proteins, and stress-induced extracellular signaling molecules through this domain, thereby activating their own kinase phosphorylation activity and initiating downstream cascade signaling pathways. These kinases are widely involved in the regulation of plant temperature, oxidative stress, and growth and development. They can sense changes in the extracellular environment, transmit stress signals through protein phosphorylation, and regulate the expression of downstream stress-related genes. Simultaneously, they can also participate in maintaining cellular redox homeostasis, regulate the transcriptional levels of functional genes such as peroxidases and phosphatases, and mediate the development of plant heat tolerance and stress adaptation. The Malectin domain, relying on its carbohydrate recognition function, assists kinases in signal sensing and transduction. It is a key functional module connecting extracellular signals and intracellular phosphorylation regulatory networks, and has important research value in the molecular regulation of crop stress resistance.

[0044] In view of this, the present invention uses gene editing technology to knock down the GhMLDRLK family gene that negatively regulates high-temperature resistance in cotton, creating mutants of the GhMLDRLK family gene, providing a reference for creating high-temperature resistant cotton lines with excellent pollen viability and yield traits under high temperatures.

[0045] The following specific embodiments further illustrate the application of the GhMLDRLK family genes of the present invention in the creation of heat-resistant cotton. This section further illustrates the content of the present invention in conjunction with specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, such as the conditions described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0046] In the following embodiments, the cotton is upland cotton J668, which is derived from the cotton genetic improvement team of the National Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University, as detailed in the reference ([1] Li J, Wang M, Li Y, et al. Multi‐omics analyses reveal epigenomics basis for cotton somaticembryogenesis through successive regeneration acclimation process[J]. Plantbiotechnology journal, 2019, 17(2): 435-450.).

[0047] In this embodiment of the invention, the term "normal temperature" means 34 ℃ and below, and the term "high temperature" means 37 ℃ and above.

[0048] This invention identified six GhMLDRLK family genes associated with high-temperature male sterility in cotton: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12. The nucleotide sequences of these genes are shown in SEQ ID NO. 1-6, respectively.

[0049] GhMLDRLKA10:

[0050]

[0051] GhMLDRLKA13:

[0052]

[0053] GhMLDRLKA14:

[0054]

[0055] GhMLDRLKD10:

[0056]

[0057] GhMLDRLKD11:

[0058]

[0059] GhMLDRLKD12:

[0060]

[0061] To reduce or suppress gene expression, the present invention preferably employs a gene knockdown method. In a preferred embodiment, a recombinant CRISPR / Cas9 vector is used to knock down at least one of the GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12 genes. The recombinant CRISPR / Cas9 vector includes sgRNAs that target the above six genes respectively, and their nucleotide sequences are shown in SEQ ID NO. 7-12 respectively.

[0062] This invention does not specifically limit the steps of the transgenic method used; conventional transgenic steps in the art are acceptable. In a preferred embodiment, the transgenic method is Agrobacterium-mediated transformation.

[0063] The technical solution of the present invention and the technical effects achieved will be described in detail below through more specific embodiments.

[0064] 1. Design and synthesis of sgRNA

[0065] Target sgRNAs were designed using the coding regions of the GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12 genes. The nucleotide sequences and PAM sites (Protospacer Adjacent Motifs) of each gene's sgRNA are shown in Table 1.

[0066] Table 1: sgRNA sequences and PAM sites targeting 6 genes

[0067]

[0068] 2. Construction of CRISPR / Cas9 recombination vectors

[0069] The above sgRNA sequence was ligated into the vector pRGEB32-GhU6.7-NPTⅡ, which was cut by BsaI enzyme (New England Biolabs, Inc., catalog number: R3733), via polycistronic tRNA-gRNA to obtain the CRISPR / Cas9 recombinant vector. The polycistronic tRNA-gRNA and the vector pRGEB32-GhU6.7-NPTⅡ were prepared and obtained according to the method described in the literature ([2] Wang F, Liang S, Wang G, et al. CRISPR–Cas9-mediated construction of a cotton CDPKmutant library for identification of insect-resistance genes[J]. Plant Communications, 2024, 5(11).).

[0070] 3. Using the CRISPR-Cas9 system to convert cotton

[0071] All CRISPR / Cas9 recombinant vectors constructed in step 2 were transformed into cotton using Agrobacterium-mediated transformation. The specific transformation steps are as follows:

[0072] (1) Select plump and healthy cotton seeds, peel off the seed coat, soak them in 0.1% HgCl2 solution for 10 min, wash them 3 times with sterile water, inoculate the sterilized cotton seeds onto sterile seedling culture medium, and culture them in the dark at 28 ℃ for 6-7 days.

[0073] (2) Take a sterile hypocotyl, cut a 0.6-0.7 cm segment and inoculate it into Agrobacterium (Beijing Qingke Biotechnology Co., Ltd., product number: TSC-A01) suspension in Agrobacterium activation medium with an OD value of 0.5. After 10 min of infection, use sterile filter paper to blot dry the bacterial solution on the surface of the hypocotyl.

[0074] (3) The hypocotyl was inoculated into co-culture medium and co-cultured at 21 °C for 48 h; then transferred to selective medium and subcultured once a month until embryogenic callus was obtained; the embryogenic callus was transferred into differentiation medium to obtain embryoids;

[0075] (4) After the embryoids were transformed into transgenic cotton seedlings, they were transferred into rooting medium and labeled as Ghmldrlka10-1, Ghmldrlka13-1, Ghmldrlka14-1, Ghmldrlkd10-1, Ghmldrlkd11-1 and Ghmldrlkd12-1 mutants, which correspond to transgenic cotton plants with the GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11 and GhMLDRLKD12 genes knocked down, respectively.

[0076] The components and preparation methods of the culture medium for the above transformation operation are as follows:

[0077] (1) Sterile seedling culture medium: 1 / 2 MS medium (Merck KGaA, catalog number: M3900-50ML), 15g / L glucose, 2.5g / L Phytagel (Merck KGaA, catalog number: P8169-100G), pH value 6.1~6.2;

[0078] (2) Agrobacterium activation medium: tryptone 5 g / L, NaCl 5 g / L, MgSO4•7H2O 0.1 g / L, KH2PO4 0.25 g / L, mannitol 5 g / L, glycine 1.0 g / L, pH 5.85~5.95;

[0079] (3) Co-culture medium: MS medium (Merck KGaA, catalog number: M3900-50ML), ammonium nitrate 1.65 g / L, 2,4-D (2,4-dichlorophenoxyacetic acid, CAS: 94-75-7) 0.1 mg / L, KT (kinetin, CAS: 525-79-1) 0.1 mg / L, 3% glucose, 0.25% Phytagel, pH 5.8;

[0080] (4) Selective culture medium: MS medium, ammonium nitrate 1.65 g / L, 2,4-D 0.1 mg / L, KT 0.1 mg / L, 3% glucose, 0.3% Phytagel, kanamycin 50 mg / L, cephalosporin 400 mg / L, pH 5.85~5.95;

[0081] (5) Differentiation medium: MS medium, KNO3 1.9 g / L, glutamine 1.0 g / L, asparagine 0.5 g / L, IBA (indolebutyric acid, Merck KGaA, catalog number: I5386) 0.5 mg / L, KT 0.15 mg / L, 3% glucose, 0.25% Phytagel, pH 6.1~6.2;

[0082] (6) Rooting medium: 1 / 2 MS medium, 15 g / L glucose, 2.5 g / L Phytagel, pH 5.90~5.95.

[0083] CRISPR-Cas9 targets the GhMLDRLKA10, GhMLDRLKA13, and GhMLDRLKA14 genes. Editing effects of GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12 genes

[0084] Gene editing was detected in the regenerated plants obtained by next-generation sequencing (Hi-TOM), specifically following the method described in the literature ([3] Liu Q, Wang C, Jiao X, et al. Hi-TOM: a platform for high-throughputtracking of mutations induced by CRISPR / Cas systems[J]. Science China LifeSciences, 2019, 62(1): 1-7.).

[0085] Figure 1 The results of gene editing in mutant cotton plants are shown in the diagrams. Figure A shows the specific gene editing of mutant Ghmldrlka10-1; Figure B shows the specific gene editing of mutant Ghmldrlka13-1; Figure C shows the specific gene editing of mutant Ghmldrlka14-1; Figure D shows the specific gene editing of mutant Ghmldrlkd10-1; Figure E shows the specific gene editing of mutant Ghmldrlkd11-1; and Figure F shows the specific gene editing of mutant Ghmldrlkd12-1. In the figures, "-" indicates base deletion, and red boxes indicate base insertion. The results show that the construction of each mutant was effective, with the main editing types being base deletion and base insertion.

[0086] Mutants Ghmldrlka10-1, Ghmldrlka13-1, Ghmldrlka14-1, Ghmldrlkd10-1, Phenotypic verification of Ghmldrlkd11-1 and Ghmldrlkd12-1

[0087] Pollen from wild-type cotton (WT group), Ghmldrlka10-1, Ghmldrlka13-1, Ghmldrlka14-1, Ghmldrlkd10-1, Ghmldrlkd11-1 and Ghmldrlkd12-1 mutant plants were collected under normal temperature (34℃) and high temperature (37℃) conditions, respectively, and their activity was detected by TTC staining. After TTC staining, pollen with good activity was stained red, while aborted pollen was stained yellow or gray. Samples were taken 10 days after treatment.

[0088] The TTC staining procedure is as follows: Dissolve 8 g of TTC (2,3,5-triphenyltetrazolium chloride, CAS: 298-96-4) in 1 L of phosphate buffer (containing 26.6 g of dipotassium hydrogen phosphate trihydrate and 10.2 g of dipotassium hydrogen phosphate per liter) to obtain the TTC staining stock solution; dilute the TTC staining stock solution to prepare a 2% (v / v) TTC staining working solution, and store it at 4 ℃ in the dark; when needed, aliquot the solution into 1.5 mL centrifuge tubes, add the anthers, react at 28 ℃ for 2 h, and then observe under a microscope.

[0089] Figure 2 The results of TTC staining of the mutant are shown; red indicates active pollen, and yellow or gray indicates aborted pollen. The results show that knockout of the GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12 genes all maintained high pollen activity at 34℃ and 37℃, while the pollen activity of wild-type plants at 37℃ was significantly lower than that at 34℃.

[0090] Figure 3 The phenotypic diagram of seed cotton yield in mutant plants after high temperature treatment is shown; the scale bar in the figure represents 10 cm. The results show that the seed cotton yield of the mutants under high temperature conditions is better than that of the WT group. In addition, the number of bolls in the mutant plants after high temperature treatment is also greater than that of the wild type.

[0091] In conclusion, by knocking out gene fragments of GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12 in cotton, it is possible to create cotton plants resistant to high temperatures.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gene associated with the heat resistance trait of cotton, characterized in that, The gene is selected from at least one of the following genes: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12. The nucleotide sequence of the GhMLDRLKA10 gene is shown in SEQ ID NO.1; The nucleotide sequence of the GhMLDRLKA13 gene is shown in SEQ ID NO.2; The nucleotide sequence of the GhMLDRLKA14 gene is shown in SEQ ID NO.3; The nucleotide sequence of the GhMLDRLKD10 gene is shown in SEQ ID NO.4; The nucleotide sequence of the GhMLDRLKD11 gene is shown in SEQ ID NO.5; The nucleotide sequence of the GhMLDRLKD12 gene is shown in SEQ ID NO.

6.

2. The application of the gene as described in claim 1 in regulating the heat resistance of cotton.

3. The application as described in claim 2, characterized in that, The gene is selected from at least one of the following genes: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12.

4. The application as described in claim 2 or claim 3, characterized in that, The regulation mentioned is a negative regulation; The regulation includes: inhibiting the expression of the gene to enhance the heat resistance of cotton plants.

5. The application as described in claim 4, characterized in that, The reagent used to inhibit the expression of the gene is selected from one of the following (A)-(C): (A) sgRNA for knocking down the gene, wherein the target site of the sgRNA is selected from at least one of the nucleotide sequences shown in SEQ ID NO.7-12; (B) An editing vector containing the sgRNA described in (A); (C) Recombinant microorganisms containing the editing vector described in (B).

6. The application as described in claim 4, characterized in that, Suppressing the expression of the gene includes the following steps: A CRISPR / Cas9 recombinant vector was constructed, transformed into wild-type cotton, and transgenic cotton plants with reduced gene expression were obtained. The wild-type cotton is upland cotton J668.

7. The application as described in claim 6, characterized in that, The CRISPR / Cas9 recombinant vector includes an sgRNA for knocking down the gene, wherein the target site of the sgRNA is selected from at least one of the nucleotide sequences shown in SEQ ID NO. 7-12.

8. The application as described in claim 6, characterized in that, The conversion of wild-type cotton includes the following steps: The CRISPR / Cas9 recombinant vector was transferred into wild-type cotton using Agrobacterium-mediated transformation.

9. A method for cultivating heat-resistant cotton, characterized in that, Includes the following steps: A CRISPR / Cas9 recombinant vector was constructed, transformed into wild-type cotton, and transgenic cotton plants with reduced gene expression levels associated with cotton heat tolerance were obtained. The gene associated with the heat resistance trait of cotton is selected from at least one of the following genes: GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11, and GhMLDRLKD12. The nucleotide sequences of the GhMLDRLKA10, GhMLDRLKA13, GhMLDRLKA14, GhMLDRLKD10, GhMLDRLKD11 and GhMLDRLKD12 genes are shown in SEQ ID NO. 1-6, respectively. The CRISPR / Cas9 recombinant vector includes an sgRNA for knocking down the gene, wherein the target of the sgRNA is selected from at least one of the nucleotide sequences shown in SEQ ID NO.7-12; The conversion of wild-type cotton includes the following steps: The CRISPR / Cas9 recombinant vector was transferred into wild-type cotton using Agrobacterium-mediated transformation.

10. The method as described in claim 9, characterized in that, The wild-type cotton is upland cotton J668.

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