A method of reducing cotton sensitivity to defoliant

Abstract

This invention discloses a method for reducing the sensitivity of cotton to defoliants. The method includes the following steps: reducing the content and / or activity of GhBRI1 protein in cotton. This invention reduces the sensitivity of cotton to defoliants. GhBRI1 Gene knockout, to construct GhBRI1 Gene knockout cotton, through research GhBRI1 The sensitivity of gene knockout cotton to the defoliant TDZ was discovered. GhBRI1 Gene knockout reduces or weakens the sensitivity of cotton to the defoliant TDZ. This invention is of great significance for addressing the differences in sensitivity to TDZ among different cotton varieties, thereby improving the stability and adaptability of cotton defoliation effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for reducing the sensitivity of cotton to defoliants. Background Technology

[0002] Cotton is an important economic crop in my country, widely used in multiple core industries such as textiles and light industry. Its planting efficiency and harvest quality directly affect the upgrading of the agricultural industry and the economic benefits for cotton farmers. With the rapid advancement of modern agricultural mechanization, mechanized cotton harvesting has gradually replaced traditional manual harvesting methods, becoming the mainstream development trend for large-scale, industrialized cotton planting. Chemical defoliation, as a key supporting technology for machine-harvested cotton, involves spraying defoliants before harvesting to induce the natural formation of the abscission layer on the cotton plant petiole and promote orderly leaf shedding. This effectively reduces the impurity content of machine-harvested seed cotton, avoids leaf debris contaminating the cotton fiber, and significantly improves cotton harvesting efficiency and finished product quality. It is a core technical link ensuring high-quality and high-yield machine-harvested cotton and is indispensable in modern large-scale cotton planting.

[0003] Currently, the mainstream cotton defoliants used in agricultural production are mainly plant growth regulators such as thidiazuron and diuron. Their mechanism of action is to regulate hormone signaling pathways such as ethylene and cytokinin in cotton plants, activate the differentiation of abscission layer cells in the petiole, thereby achieving leaf shedding. They are characterized by high defoliation efficiency and wide applicability, and have been widely promoted and applied. However, for some cotton varieties that are sensitive to defoliants, spraying conventional doses of defoliants not only induces the shedding of ineffective old and diseased leaves, but also easily causes premature shedding of functional leaves and new tender leaves. At the same time, it can easily cause endogenous hormone disorders in cotton plants and damage the photosynthetic system, seriously affecting the normal boll opening and nutrient accumulation in the later stages of cotton boll production. This problem has become a key bottleneck restricting the high-quality and stable yield of machine-harvested cotton.

[0004] Current industry-standard control measures primarily focus on external interventions such as optimizing defoliant formulations, adjusting spraying time and dosage, and improving the application environment. These approaches only fine-tune the defoliation effect at the pesticide application level and cannot improve the cotton's inherent sensitivity at the intrinsic level. In actual field applications, even with precise control of parameters such as pesticide dosage, temperature, and timing, highly sensitive cotton varieties are still prone to problems such as excessive defoliation, premature plant senescence, and stunted boll development, directly leading to decreased yield, reduced lint percentage, and deterioration of fiber quality. Furthermore, to avoid the risk of excessive defoliation, farmers are often forced to reduce the defoliant dosage and advance or delay application time, resulting in incomplete defoliation, residual dead leaves, and inconsistent defoliation cycles. This significantly increases the cost of cleaning impurities during machine harvesting and severely reduces the efficiency of machine harvesting and the commercial grade of cotton. Therefore, developing a method that can effectively reduce cotton's sensitivity to defoliants, balance defoliation effects with cotton plant growth and development, and improve field adaptability is of great significance for cotton harvesting and production. Summary of the Invention

[0005] One object of the present invention is to provide a method for reducing the sensitivity of cotton to defoliants.

[0006] The method for reducing the sensitivity of cotton to defoliants provided by the present invention includes the following steps: reducing the content and / or activity of GhBRI1 protein in cotton.

[0007] Another object of the present invention is to provide a method for breeding transgenic cotton with reduced sensitivity to defoliants.

[0008] The method for cultivating transgenic cotton with reduced sensitivity to defoliants provided by the present invention includes the following steps: reducing the content and / or activity of GhBRI1 protein in recipient cotton to obtain transgenic cotton; wherein the transgenic cotton is less sensitive to defoliants than the recipient cotton.

[0009] In the above method, the GhBRI1 protein is any one of the following: A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares 75% or more identity with A1) and has the same function.

[0010] In the protein described in A2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0011] In the protein described in A3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0012] In the protein described in A4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the identity of a pair of amino acid sequences, the identity value (%) can be obtained. The identity includes amino acid sequences that have 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequence shown in Sequence 2 of this invention.

[0013] In the above method, the genetically modified cotton's lower sensitivity to defoliants compared to the recipient cotton is manifested in any of the following ways: 1) Under defoliant treatment (e.g., 100 mg / L TDZ treatment), the defoliation rate of the transgenic cotton is lower than that of the recipient cotton; 2) Under defoliant treatment (e.g., 100 mg / L TDZ treatment), the CAT activity of the transgenic cotton was higher than that of the recipient cotton; 3) Under defoliant treatment (e.g., 100 mg / L TDZ treatment), the H2O2 content of the transgenic cotton is lower than that of the recipient cotton; 4) Under defoliant treatment (e.g., 100 mg / L TDZ treatment), the MDA content of the transgenic cotton is lower than that of the recipient cotton; 5) Under defoliant treatment (e.g., 100 mg / L TDZ treatment), the POD activity of the transgenic cotton is higher than that of the recipient cotton; 6) Under defoliant treatment (e.g., 100 mg / L TDZ treatment), the SOD activity of the transgenic cotton is higher than that of the recipient cotton.

[0014] In the above method, the method for reducing the content and / or activity of GhBRI1 protein in cotton is to knock out the GhBRI1 protein encoding gene in cotton.

[0015] Furthermore, the method for knocking out the GhBRI1 protein-coding gene in cotton involves introducing a substance that knocks out the GhBRI1 protein-coding gene into recipient cotton.

[0016] The substance used to knock out the GhBRI1 protein-coding gene in cotton is a CRISPR / Cas9 gene editing vector that knocks out the GhBRI1 protein-coding gene.

[0017] The methods of introduction include, but are not limited to: transfecting cotton cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and then cultivating the transfected cotton cells or tissues into plants.

[0018] Furthermore, the CRISPR / Cas9 gene editing vector expresses sgRNA and Cas9 protein targeting the GhBRI1 protein-coding gene.

[0019] In some implementations, the target sequence of the sgRNA is shown as positions 154-172 and 662-681 of Sequence 1.

[0020] Another object of the present invention is to provide new uses for substances that reduce the content and / or activity of GhBRI1 protein.

[0021] This invention provides the use of substances that reduce the content and / or activity of GhBRI1 protein in any of the following: a1) Reduce the sensitivity of cotton to defoliants; a2) Develop transgenic cotton with reduced sensitivity to defoliants; a3) Cotton breeding or cotton variety improvement.

[0022] The substance that reduces GhBRI1 protein content may be a substance that inhibits GhBRI1 protein synthesis, promotes GhBRI1 protein degradation, inhibits or interferes with the expression of the GhBRI1 protein-encoding gene, or knocks out the GhBRI1 protein-encoding gene.

[0023] The substance that reduces the activity of GhBRI1 protein can be a protein, polypeptide, or small molecule compound that inhibits the function of GhBRI1 protein.

[0024] Furthermore, the substance that reduces the content and / or activity of GhBRI1 protein is any one of the following: D1) Inhibit or interfere with the expression of the GhBRI1 protein-coding gene or knock out the nucleic acid molecules of the GhBRI1 protein-coding gene; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3).

[0025] In the above D1), the nucleic acid molecule that inhibits or interferes with the expression of the GhBRI1 protein-coding gene or knocks out the GhBRI1 protein-coding gene can be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0026] In some embodiments, the nucleotide sequence of the GhBRI1 protein-coding gene is shown in Sequence 1.

[0027] In some embodiments, the nucleic acid molecule that knocks out the GhBRI1 protein-coding gene is any one of the following: F1) The DNA molecule shown in sequence 3; DNA molecules that have 75% or more identity with the nucleotide sequences defined by F2 and F1, and have the same function.

[0028] In D2) above, the expression cassette may include a promoter, the nucleic acid molecule described in D1) above, and a terminator. Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0029] In D3) above, the vector refers to a vector capable of delivering the nucleic acid molecule described in D1) into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). The recombinant vector refers to a recombinant DNA molecule constructed by ligating the nucleic acid molecule described in D1) to the vector in vitro. Existing plant expression vectors can be used to construct recombinant vectors containing the nucleic acid molecule described in D1).

[0030] In some embodiments, the substance for knocking out the GhBRI1 protein-coding gene is a CRISPR / Cas9 gene-editing vector that knocks out the GhBRI1 protein-coding gene. The CRISPR / Cas9 gene-editing vector expresses sgRNA and Cas9 protein targeting the GhBRI1 protein-coding gene. Preferably, the target sequence of the sgRNA is shown as positions 154-172 and 662-681 of Sequence 1.

[0031] In some specific embodiments, the CRISPR / Cas9 gene editing vector for knocking out the GhBRI1 protein-coding gene is the recombinant plasmid pWMV016-GhBRI1. The recombinant plasmid pWMV016-GhBRI1 is formed by inserting the gRNA fragment shown in sequence 3 into the pWMV016 vector. Bsa The vector obtained after I restriction site digestion.

[0032] In D4) above, the microorganism can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The recombinant microorganism refers to a recombinant microorganism whose function has been altered by manipulating and modifying the genes of the target microorganism. For example, the recombinant microorganism obtained after introducing the aforementioned recombinant vector into the target microorganism. The recombinant microorganism can be understood not only as a specific recombinant microorganism, but also as the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0033] Another objective of this invention is to provide a method for cotton breeding or cotton variety improvement.

[0034] The method for cotton breeding or cotton variety improvement provided by the present invention includes the following steps: using the transgenic cotton prepared according to the above method as a parent for breeding.

[0035] The final objective of this invention is to provide a method for preparing cotton with reduced sensitivity to defoliants.

[0036] The method for preparing cotton with reduced sensitivity to defoliants provided by this invention includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] GhBRI1 Deleting (or removing) positions 159-163 and 665-667 of the gene yields cotton with reduced sensitivity to defoliants; GhBRI1 The nucleotide sequence of the gene is shown in Sequence 1.

[0037] The defoliant mentioned above is thidiazuron.

[0038] The indicators for cotton breeding mentioned above include sensitivity to defoliants (such as sensitivity to the defoliant thiabendazole).

[0039] The purpose of any of the above-mentioned cotton breeding programs includes breeding cotton with reduced sensitivity to defoliants (such as breeding cotton with reduced sensitivity to the defoliant thiabendazole).

[0040] The cotton mentioned above includes any cotton germplasm resource, variety, strain or single plant, preferably cotton varieties sensitive to the defoliant thiabendazole (such as BL34, ZM49).

[0041] This invention, by studying the effects of TDZ on the defoliation mechanism of cotton varieties with different sensitivities, discovered that in the BR signal transduction pathway... GhBRI1 Genes may influence the sensitivity of cotton to the defoliant TDZ, and further research can be conducted by constructing... GhBRI1 Gene-edited cotton was tested and its sensitivity to the defoliant TDZ was verified, confirming the effectiveness of knockout. GhBRI1 Genes can reduce the sensitivity of cotton to the defoliant TDZ. This invention is of great significance for solving the problem of different cotton varieties' varying sensitivity to the defoliant TDZ, thereby improving the stability and adaptability of cotton defoliation effects. Attached Figure Description

[0042] Figure 1 This study presents the dynamic changes in leaf drop rate and reactive oxygen species (ROS) metabolism response of BL34 under different concentrations of TDZ treatment. AD represents the phenotypic changes of the sensitive material BL34 at 0, 36, 72, and 108 h after treatment with 0, 50, 100, and 200 mg / L TDZ, respectively. EI represents the results of the determination of physiological indicators H2O2 content, MDA content, CAT activity, SOD activity, and POD activity. J represents the statistical results of BL34 leaf drop rate. All values ​​are the mean ± standard deviation of three independent biological replicates (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, comparisons between treatment concentrations.

[0043] Figure 2The dynamic changes in defoliation rate and reactive oxygen species (ROS) metabolism of LMY28 under different concentrations of TDZ treatment are shown. AD represents the phenotypic changes of the insensitive material LMY28 at 0, 36, 72, and 108 h after treatment with 0, 50, 100, and 200 mg / L TDZ, respectively. EI represents the results of the determination of physiological indicators H2O2 content, MDA content, CAT activity, SOD activity, and POD activity. J represents the statistical results of LMY28 defoliation rate. All values ​​are the mean ± standard deviation of three independent biological replicates (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, comparisons between treatment concentrations.

[0044] Figure 3 This study uses combined transcriptomic and metabolomic analysis to reveal the temporal modules of the TDZ response. Figure A shows co-expression analysis, revealing three clusters of BL34 and LMY28 exhibiting similar metabolite accumulation and gene expression after TDZ treatment. The left-hand figure illustrates the co-expression patterns of gene expression and metabolite accumulation in these three clusters. The middle figure presents a heatmap of differentially accumulated metabolites and differentially expressed genes in each cluster. The right-hand figure summarizes the types and correlations of enriched differentially expressed metabolites and genes in each cluster. Figures B and C, respectively, show the distribution and differential changes of key genes in the brassinolide signaling pathway in BL34 and LMY28 after TDZ treatment.

[0045] Figure 4 Treatment with 100 mg / L TDZ GhBRI1 Dynamic changes in defoliation rate and reactive oxygen species metabolic response in gene knockout cotton and wild-type cotton. A is... GhBRI1 Phenotypic changes in gene knockout cotton at 0, 36, 72, and 108 h after treatment with 100 mg / L TDZ. B shows phenotypic changes in wild-type cotton at 0, 36, 72, and 108 h after treatment with 100 mg / L TDZ. C shows... GhBRI1 CAT activity in gene knockout cotton and wild-type cotton after treatment with 100 mg / L TDZ for 0, 36, and 72 h. D represents... GhBRI1 H2O2 content of gene knockout cotton and wild-type cotton after treatment with 100 mg / L TDZ for 0, 36, and 72 h. E represents... GhBRI1 MDA content of gene knockout cotton and wild-type cotton after treatment with 100 mg / L TDZ for 0, 36, and 72 h. F represents... GhBRI1 POD activity of gene knockout cotton and wild-type cotton after treatment with 100 mg / L TDZ for 0, 36, and 72 h. G represents... GhBRI1 SOD activity of gene knockout cotton and wild-type cotton after treatment with 100 mg / L TDZ for 0, 36, and 72 h. H represents... GhBRI1Defoliation rates of gene knockout cotton and wild-type cotton after treatment with 100 mg / L TDZ for 0, 36, 72, and 108 h. WT-N represents the wild-type cotton control group without TDZ treatment, WT-T represents the wild-type cotton group treated with TDZ, and BRI1-N represents the wild-type cotton control group without TDZ treatment. GhBRI1 Gene knockout cotton, BRI1-T represents the TDZ treatment group GhBRI1 Gene knockout cotton. All values ​​are mean ± standard deviation of three independent biological replicates (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, comparisons between treatment concentrations. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] 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 this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0048] The cotton variety BL34 used in the following examples is described in the literature “Xu B, Liu L, Zhao R, et al.Establishment of a high-throughput field defoliation data survey strategy combined with genome-wide association studies to reveal the genetic basis of defoliation in cotton[J]. Plant Phenomics, 2025: 100109.”, which is available to the public from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and may not be used for other purposes.

[0049] The cotton variety LMY28 used in the following examples is described in the literature “Xu B, Liu L, Zhao R, et al. Establishment of a high-throughput field defoliation data survey strategy combined with genome-wide association studies to reveal the genetic basis of defoliation in cotton[J]. Plant Phenomics, 2025: 100109.”, which is available to the public from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and may not be used for other purposes.

[0050] The cotton variety ZM49 in the following examples is described in the literature “Liu L, Liu Z, Fan L, et al. Natural variation in the GhTTL promoter modulates cotton fiber elongation through the GhTALE–GhTTL–GhBIN2 module[J]. Plant Communications, 2025, 6(10).”, which is available to the public from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and shall not be used for other purposes.

[0051] The pWMV016 vector in the following examples is described in the literature “Liu L, Liu Z, Fan L, et al. Natural variation in the GhTTL promoter modulates cotton fiber elongation through the GhTALE–GhTTL–GhBIN2 module[J]. Plant Communications, 2025, 6(10).” and is publicly available from the applicant. This biological material is only for repeating the relevant experiments of the present invention and shall not be used for other purposes.

[0052] The TDZ (thiafenuron) used in the following examples is a product of Sichuan Guoguang, with product number PD20101581, purity 98%, CAS number 51707-55-2, and molecular formula C9H8N4OS.

[0053] Example 1: Investigation into the optimal application concentration of TDZ (thiamethoxam). Test materials: TDZ-sensitive cotton variety BL34 and TDZ-insensitive cotton variety LMY28.

[0054] Experimental methods: The test materials were planted according to conventional methods. At the 7-leaf stage of cotton plant growth, different concentrations (50 mg / L, 100 mg / L, 200 mg / L) of TDZ solution (solvent is DMSO) were sprayed to completely wet the leaves (about 10-15 mL / plant). The spraying was performed once. At the same time, an equal amount of distilled water was sprayed as a control.

[0055] Phenotypic images of plants in each treatment group were taken before TDZ treatment (0 h) and at 36 h, 72 h, and 108 h after treatment. Leaf color changes, wilting degree, and leaf drop were recorded for subsequent leaf drop rate statistics and phenotypic analysis. Simultaneously, to investigate the effects of TDZ treatment on the physiological state of cotton leaves, this study measured the activities of five key physiological and biochemical indicators related to oxidative stress and cell damage: peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), as well as the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2). All measurements were performed using kits manufactured by Suzhou Grease Biotechnology Co., Ltd.

[0056] The results are as follows Figure 1 and Figure 2 As shown, the results indicate that the defoliation response of the TDZ-sensitive cotton variety BL34 was significantly faster than that of the TDZ-insensitive cotton variety LMY28, and the increase in oxidative stress index was greater. The optimal defoliation concentration of TDZ was 100 mg / L for both TDZ-sensitive and TDZ-insensitive varieties, LMY28.

[0057] Example 2: Identification of GhBRI1, a gene related to BR signal transduction. To investigate the genetic basis of differences in cotton leaf defoliation sensitivity, whole transcriptome and metabolome sequencing were performed on BL34 and LMY28 plants treated with 200 mg / LTDZ, followed by combined analysis. After background gene knockout and cluster analysis, transcriptome and metabolome cluster analysis divided differentially expressed genes and metabolites into three clusters each, showing a clear correspondence, indicating a close synergistic effect between transcriptional regulation and metabolomics in cotton leaves under TDZ treatment. Functional enrichment results showed that the co-enriched pathways mainly focused on plant hormone signal transduction, glutathione metabolism, phenylalanine metabolism, and flavonoid biosynthesis. The brassinolide signaling pathway was significantly enriched in the cluster, and analysis of differentially expressed genes and metabolites showed significant changes in GhBRI1, suggesting a potential role for both in cotton leaf defoliation sensitivity. Figure 3 ).

[0058] Example 3 GhBRI1 Construction of gene knockout cotton and analysis of its defoliation effect To further verify GhBRI1 It has a potential role in cotton defoliant sensitivity and has been developed. GhBRI1 Gene knockout cotton was analyzed. GhBRI1 Changes in the sensitivity of gene knockout cotton to the defoliant TDZ (thiabendazole). The specific steps are as follows: one, GhBRI1 Construction of gene knockout cotton 1. Design using CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) GhBRI1 The gene editing target was ultimately selected, and the gRNA target sequence is shown in positions 154-172 and 662-681 of Sequence 1.

[0059]

[0060] 3. Use the pWMV016 vector Bsa The vector was obtained by digestion with an enzyme I endonuclease.

[0061] 4. Using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novizan), the gRNA fragment obtained in step 2 and the linearized vector obtained in step 3 were ligated to obtain the ligation product and sequenced. The plasmid with correct sequencing was named pWMV016-GhBRI1.

[0062] The recombinant plasmid pWMV016-GhBRI1 is formed by ligating the gRNA fragment shown in sequence 3 into the pWMV016 vector. Bsa The vector obtained after I restriction site digestion. The recombinant plasmid pWMV016-GhBRI1 expresses two target genes. GhBRI1 The gRNA of the gene (target sequences are positions 154-172 and 662-681 of sequence 1, respectively) and the Cas9 protein.

[0063] 5. The recombinant plasmid pWMV016-GhBRI1 was transformed into Agrobacterium EHA105. After identification, the recombinant bacteria EHA105 / pWMV016-GhBRI1 containing the recombinant plasmid pWMV016-GhBRI was obtained.

[0064] 6. Using cotton variety ZM49 as the recipient, the recombinant plasmid pWMV016-GhBRI1 was introduced into the recipient material via shoot tip transformation using the recombinant strain EHA105 / pWMV016-GhBRI1. The specific method was as described in the literature "Ge X, Xu J, Yang Z, et al. Efficient genotype‐independent cotton genetic transformation and genome editing[J]. Journal of Integrative Plant Biology, 2023, 65(4): 907-917." After multiple generations of screening, the stably inherited T3 generation was obtained. GhBRI1 Homozygous gene-edited lines were confirmed as generation T3 by next-generation sequencing after planting. GhBRI1 In homozygous gene-edited lines GhBRI1 Stability of gene editing.

[0065] Compared with the genome sequence of cotton variety ZM49, the T3 generation GhBRI1 homozygous gene-edited lines in GhBRI1A 5bp segment deletion occurred at positions 159-163 of the gene sequence (Sequence 1), and... GhBRI1 A 3bp segment was deleted at positions 665-667 of the gene sequence (Sequence 1), resulting in... GhBRI1 When a gene undergoes a frameshift mutation, its function is completely lost.

[0066] two, GhBRI1 Analysis of the defoliation effect of gene knockout cotton Test materials: Wild-type cotton variety ZM49 (abbreviated as WT), T3 generation GhBRI1 The homozygous gene-edited line (BRI1).

[0067] Experimental methods: The test materials were planted according to conventional methods. When the cotton plants were at the 7-leaf stage, they were sprayed with 100 mg / L TDZ solution (solvent is DMSO) to completely wet the leaves (about 10-15 mL / plant). The spraying was done once. At the same time, an equal amount of distilled water was sprayed as a control group.

[0068] Phenotypic images of plants in each treatment group were taken before TDZ treatment (0 h) and at 36 h, 72 h, and 108 h after treatment. Leaf color changes, wilting degree, and leaf drop were recorded for subsequent leaf drop rate statistics and phenotypic analysis. This data was also used to explore... GhBRI1 To investigate the effects of TDZ treatment on the physiological state of cotton leaves, this study measured five key physiological and biochemical indicators related to oxidative stress and cell damage: the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), as well as the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2). All measurements were performed using kits manufactured by Suzhou Greens Biotechnology Co., Ltd.

[0069] The results are as follows Figure 4 As shown. The results showed that, under the condition of spraying with 100 mg / L TDZ solution, compared with wild-type cotton ZM49, GhBRI1 The defoliation rate of gene knockout cotton was significantly reduced, indicating that the knockout... GhBRI1 The gene reduced or weakened the cotton’s sensitivity to the defoliant TDZ, and the results of the determination of the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and hydrogen peroxide (H2O2) also support this conclusion.

[0070] In practical applications, for cotton varieties sensitive to TDZ treatment, knockout can be used. GhBRI1 Genes can be used to reduce the plant's sensitivity to the defoliant TDZ.

[0071] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for reducing the sensitivity of cotton to defoliants, comprising the steps of: reducing the content and / or activity of GhBRI1 protein in cotton; wherein the GhBRI1 protein is any one of the following: A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares 75% or more identity with A1) and has the same function.

2. A method for cultivating transgenic cotton with reduced sensitivity to defoliants, comprising the following steps: reducing the content and / or activity of GhBRI1 protein in recipient cotton to obtain transgenic cotton; wherein the transgenic cotton is less sensitive to defoliants than the recipient cotton; wherein the GhBRI1 protein is any one of the following: A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares 75% or more identity with A1) and has the same function.

3. The method according to claim 1 or 2, characterized in that: The method for reducing the content and / or activity of GhBRI1 protein in cotton is to knock out the GhBRI1 protein encoding gene in cotton.

4. The method according to claim 3, characterized in that: The method for knocking out the GhBRI1 protein-coding gene in cotton involves introducing a substance that knocks out the GhBRI1 protein-coding gene into recipient cotton. Alternatively, the substance used to knock out the GhBRI1 protein-coding gene in cotton may be a CRISPR / Cas9 gene editing vector that knocks out the GhBRI1 protein-coding gene.

5. The method according to claim 4, characterized in that: The CRISPR / Cas9 gene editing vector expresses sgRNA and Cas9 protein targeting the GhBRI1 protein-coding gene; Alternatively, the target sequence of the sgRNA is shown as positions 154-172 and 662-681 of Sequence 1.

6. The use of substances that reduce the content and / or activity of GhBRI1 protein in any of the following: a1) Reduce the sensitivity of cotton to defoliants; a2) Develop transgenic cotton with reduced sensitivity to defoliants; a3) Cotton breeding or cotton variety improvement; The GhBRI1 protein is any one of the following: A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares 75% or more identity with A1) and has the same function.

7. The application according to claim 6, characterized in that: The substance that reduces the content and / or activity of GhBRI1 protein is any one of the following: D1) Inhibit or interfere with the expression of the GhBRI1 protein-coding gene or knock out the nucleic acid molecules of the GhBRI1 protein-coding gene; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3).

8. A method for cotton breeding or cotton variety improvement, comprising the following steps: using transgenic cotton prepared according to any one of claims 2-5 as a parent for breeding.

9. A method for preparing cotton with reduced sensitivity to defoliants, comprising the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] GhBRI1 Deleting positions 159-163 and 665-667 of the gene yields cotton with reduced sensitivity to defoliants; GhBRI1 The nucleotide sequence of the gene is shown in Sequence 1.

10. The method according to any one of claims 1-5, or the application according to claim 6 or 7, or the method according to claim 8 or 9, characterized in that: The defoliant is thiamethoxam.

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