Method for improving pigment gland traits and gossypol traits of cotton seeds and use thereof

Abstract

The present invention relates to a method for improving pigment gland traits and gossypol traits of cotton seeds and a use thereof. For the key gene Gl2 / Gl3 which determines the development of pigment glands in cotton, a cotton seed-specific promoter is utilized to specifically inhibit the expression of the gene or the activity of an encoding protein of the gene in a seed development process, so that novel cotton of which the seeds have no pigment glands and the plants have normal pigment glands can be obtained, wherein the gossypol content in the seeds of the cotton is zero, and the gossypol content in other tissues is normal.

Description

Method for improving cotton seed pigment gland traits and gossypol traits and use thereof Technical Field

[0001] The present invention belongs to the field of plant bioengineering, and specifically relates to a method for improving cotton seed pigment gland traits and gossypol traits by genetic engineering means and its use. Background Art

[0002] Cotton is not only an important natural fiber crop worldwide, but also a significant oilseed and high-protein food crop. China cultivates approximately 50 million mu (approximately 1.5 million hectares) of cotton annually, producing over 10 million tons of seeds, containing approximately 2 million tons of oil and 2 million tons of protein. However, the presence of high levels of gossypol in cotton seeds significantly limits their use as a food and feed ingredient.

[0003] Currently, both upland cotton plants and cotton seeds, which are widely cultivated around the world, contain large amounts of gossypol. Gossypol is a sesquiterpenoid secondary metabolite that is synthesized during the development of cotton pigment glands (referred to as "glands") and accumulates in the cotton glands. Gossypol is essential for cotton plants to resist insect pests. However, secondary metabolites such as gossypol contained in cotton seeds are harmful to humans and monogastric animals, seriously affecting their safety as food ingredients, bringing complexity to processing and detoxification, and consumer concerns. Therefore, maintaining the gossypol content of plants unchanged, eradicating toxic substances in cotton seeds in a targeted manner, and making cotton an integrated "grain, cotton, and oil" crop are scientific problems that need to be solved urgently internationally.

[0004] Traditional breeding primarily involves introducing low-phenol traits into other superior lines using phenol-free strains as parents. Currently selected low-phenol varieties include Xiangmian 18, Zhongmian 22, Lumian 12, Zhemian 10, Jinmian 22, Wanmian 18, Giza 77, and Giza 81, with seed gossypol contents of 0.0304%, 0.0051%, 0.008%, 0.006%, 0.002%, 0.00037%, 0.023%, and 0.021%, respectively. With the exception of Wanmian 18, the seed gossypol contents of most of these varieties, while below the World Health Organization standard of ≤600 mg / kg, are higher than the 50 mg / kg standard for first-grade finished cottonseed oil in my country's latest national standard, GB / T1537-2019, posing a potential safety risk. More importantly, since the glandless varieties have no gossypol or have very low gossypol content, they are easily harmed by rats, rabbits, leaf-feeding pests, etc., and have not yet been widely promoted and applied.

[0005] Using bioengineering methods, a large number of genetic improvement practices have been carried out at home and abroad, but no substantial progress has been made so far. For example, with the help of RNAi, American scientists use seed-specific promoters to drive the dsRNA of the CAD gene, specifically silence seed glandular genes and cotton phenol synthesis genes, and create new cotton germplasms with normal seed low phenol plants (Sunilkumar G, Campbell LAM, Puckhaber L, et al. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proceedings of the National Academy of Sciences, 2006, 103 (48): 18054-18059). The cotton breeding team of Zhejiang University silenced the key gene Gl2 / Gl3 of cotton gland synthesis by RNAi method, and also created similar cotton germplasm (Gao W, Zhu X, Ding L, et al. Development of the engineered "glanded plant and glandless seed" cotton. Food Chemistry: Molecular Sciences, 2022, 5: 100130). However, RNAi can only reduce but not completely block the translation of target genes, so a small amount of glands and low levels of gossypol still exist in the seeds, and other secondary metabolites in the glands still pose a great threat to the health of humans and monogastric animals.

[0006] Therefore, there is an urgent need to develop a more effective method to reduce the gossypol content in cotton seeds, so as to achieve zero gossypol in cotton seeds while leaving the gossypol content in other tissues of the cotton plant unaffected.

[0007] Summary of the Invention

[0008] The present invention provides a new method for improving the pigment gland traits and gossypol traits of cotton seeds. This method can achieve the goal of producing cotton seeds without pigment glands and with zero gossypol content, while maintaining the pigment glands and gossypol content of the cotton plant. Cotton plants created using this method are able to normally resist pests and diseases, and the resulting seeds, because they do not contain gossypol, are significantly superior to "low-phenol cotton" in terms of food safety. The new cotton has the advantages of both pigment gland-free and pigment gland-containing cotton, without compromising the plant's ability to resist pests and diseases. It also completely eliminates the dephenolization process during cotton seed utilization, providing a large amount of high-quality protein and oil resources for humans and animals, greatly improving the economic benefits of cotton cultivation.

[0009] Accordingly, in a first aspect, the present invention provides a method for inhibiting the formation of pigment glands in cotton seeds, the method comprising causing the cotton seeds to express a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

[0010] At the same time, the present invention also provides a method for reducing the gossypol content in cotton seeds, which comprises expressing a Gl2 / Gl3 protein mutant in the cotton seeds, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has a reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

[0011] In some embodiments, the transcriptional activation function of the mutant tetramer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the wild-type tetramer formed by wild-type Gl2 / Gl3 protein alone.

[0012] In some embodiments, the method comprises the step of introducing into the cotton a nucleotide sequence encoding a G12 / G13 protein mutant. In some embodiments, the method comprises integrating the nucleotide sequence encoding a G12 / G13 protein mutant into the genome of the cotton.

[0013] In some embodiments, the Gl2 / Gl3 protein mutants of the present invention have one or more amino acid substitutions, insertions, and / or deletions at positions selected from amino acids 1-282 and 374-475 compared to wild-type Gl2 / Gl3 proteins. In some embodiments, the Gl2 / Gl3 protein mutants have one or more amino acid substitutions, insertions, and / or deletions at positions 20-202 or 300-313. In some preferred embodiments, the Gl2 / Gl3 protein mutants have one or more mutations selected from the following: amino acid A to K (A43K) at position 43, amino acid mutations 21-24 to alanine, amino acid mutations 54-59 to alanine, deletions of amino acids 43-52, deletions of amino acids 72-79, deletions of amino acids 94-99, deletions of amino acids 121-126, and deletions of amino acids 300-313. In some more preferred embodiments, the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:3-10.

[0014] In some embodiments, compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant of the present invention is a truncated version of the N-terminus and / or C-terminus of the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant does not contain a functional transcriptional activation domain, and the Gl2 / Gl3 protein mutant contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with amino acids 283-373 of the wild-type Gl2 / Gl3 protein. In some embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 283-373 or amino acids 141-475 of the wild-type Gl2 / Gl3 protein. In some preferred embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12.

[0015] In some embodiments, the nucleotide sequence encoding the G12 / G13 protein mutant is operably linked to a cotton seed-specific promoter.

[0016] The seed-specific promoter suitable for the method of the present invention has the following characteristics:

[0017] (1) initiating expression of the Gl2 / Gl3 protein mutant at a time no later than that of the Gl2 / Gl3 protein during seed development in wild-type cotton;

[0018] (2) ensuring that the expression level of the Gl2 / Gl3 protein mutant is no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and

[0019] (3) The expression of the Gl2 / Gl3 protein mutant is initiated only during cotton seed development, and does not or substantially does not initiate after mature seed germination and other stages of cotton plant development.

[0020] In some embodiments, the seed-specific promoter is a promoter of the OLE1_D gene, the OLE1_A gene, or the ALB2S gene. In some preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 13-15. In some more preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14.

[0021] In some embodiments, the methods of the present invention comprise integrating into the genome of cotton a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 or 23.

[0022] In some embodiments, the methods of the present invention comprise the following steps:

[0023] (1) transforming a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0024] (2) contacting cotton cells, seeds, tissues, organs or plants with the Agrobacterium carrying the nucleotide sequence in step (1), thereby introducing the nucleotide sequence into the cotton cells, seeds, tissues, organs or plants.

[0025] In some embodiments, the methods of the present invention result in the abortion of pigment glands in cotton seeds compared to pigment glands in wild-type cotton seeds, and the development of pigment glands in cotton plants is not affected.

[0026] In some embodiments, the methods of the invention reduce the gossypol content in cotton seeds by 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% compared to the gossypol content of wild-type cotton seeds.

[0027] In some embodiments, the methods of the present invention result in gossypol levels in tissues of a cotton plant that are no different or substantially no different than the gossypol levels in tissues of a wild-type cotton plant.

[0028] In a second aspect, the present invention provides a cotton cell, seed, tissue, organ or plant obtained by the method of the first aspect of the present invention.

[0029] In a third aspect, the present invention provides cotton cells, seeds, tissues, organs or plants comprising a nucleotide sequence encoding a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has reduced or eliminated transcriptional activation function compared to the wild-type tetramer formed by only the wild-type Gl2 / Gl3 protein.

[0030] In some embodiments, the transcriptional activation function of the mutant tetramer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared to the wild-type tetramer formed by wild-type Gl2 / Gl3 protein alone.

[0031] In some embodiments, the nucleotide sequence encoding the G12 / G13 protein mutant is integrated into the genome of a cotton cell, seed, tissue, organ, or plant.

[0032] In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions, and / or deletions at positions selected from amino acids 1-282 and 374-475 compared to wild-type Gl2 / Gl3. In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions, and / or deletions at positions 20-202 or 300-313. In some preferred embodiments, the Gl2 / Gl3 protein mutant has one or more mutations selected from the following: amino acid A at position 43 is mutated to K (A43K), amino acids 21-24 are mutated to alanine, amino acids 54-59 are mutated to alanine, amino acid deletions at positions 43-52, amino acid deletions at positions 72-79, amino acid deletions at positions 94-99, amino acid deletions at positions 121-126, and amino acid deletions at positions 300-313. In some more preferred embodiments, the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:3-10.

[0033] In some embodiments, the Gl2 / Gl3 protein mutant is a truncated version of the wild-type Gl2 / Gl3 protein at the N-terminus and / or C-terminus, the Gl2 / Gl3 protein mutant does not comprise a functional transcriptional activation domain, and the Gl2 / Gl3 protein mutant comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 283-373 of the wild-type Gl2 / Gl3 protein. In some embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 283-373 or amino acids 141-475 of the wild-type Gl2 / Gl3 protein. In some preferred embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:11 or SEQ ID NO:12.

[0034] In some embodiments, the nucleotide sequence encoding the G12 / G13 protein mutant in the cotton cell, seed, tissue, organ or plant of the present invention is operably linked to a cotton seed-specific promoter.

[0035] The seed-specific promoter in the cotton cell, seed, tissue, organ or plant of the present invention has the following characteristics:

[0036] (1) Initiating expression of the Gl2 / Gl3 protein mutant at a time no later than that of the wild-type Gl2 / Gl3 protein during seed development;

[0037] (2) ensuring that the expression level of the Gl2 / Gl3 protein mutant is no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and

[0038] (3) The expression of the Gl2 / Gl3 protein mutant is initiated only during cotton seed development, and does not or substantially does not initiate after mature seed germination and other stages of cotton plant development.

[0039] In some embodiments, the seed-specific promoter is a promoter of the OLE1_D gene, the OLE1_A gene, or the ALB2S gene. In some preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 13-15. In some more preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14.

[0040] In some embodiments, the cotton cells, seeds, tissues, organs or plants of the present invention are obtained by integrating a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 21 or 23 into the genome of cotton.

[0041] In some embodiments, the cotton cell, seed, tissue, organ or plant of the present invention is obtained by the following steps:

[0042] (1) transforming a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0043] (2) contacting cotton cells, seeds, tissues, organs or plants with the Agrobacterium carrying the nucleotide sequence in step (1), thereby obtaining the cotton cells, seeds, tissues, organs or plants.

[0044] In a fourth aspect, the present invention provides a nucleic acid construct comprising, from 5' to 3', a cotton seed-specific promoter and a gene encoding a Gl2 / Gl3 protein mutant.

[0045] In some embodiments, the seed-specific promoter in the nucleic acid construct of the present invention is the promoter of the OLE1_D gene, the OLE1_A gene, or the ALB2S gene. In some preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 13-15. In some more preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14.

[0046] In some more preferred embodiments, the nucleic acid construct comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:21 or 23.

[0047] In a fifth aspect, the present invention provides the nucleic acid construct according to the fourth aspect of the present invention, which is used to inhibit the formation of pigment glands in cotton seeds.

[0048] In a sixth aspect, the present invention provides the nucleic acid construct according to the fourth aspect of the present invention, which is used to reduce the gossypol content in cotton seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 shows the development of cotton pigment glands. Figure 1a shows the pseudo-chronological process of cotton immature embryo gland development. Figure 1b shows the process of gland morphogenesis in the cotyledon of upland cotton immature embryo. Figure 1c shows the development of cotton immature embryo cotyledons.

[0050] FIG2 shows the results of measuring gossypol content in cotton immature embryos at different developmental stages.

[0051] Figure 3 shows the number of glands and gossypol content in plants with different Gl2 / Gl3 genotypes. Figure 3a shows the true leaf phenotypes of plants with different Gl2 / Gl3 genotypes. Figure 3b shows the gossypol content in different tissues of a Gl2 / Gl3 loss-of-function mutant (gl2gl2gl3gl3). Figure 3c shows the statistical results of the number of glands in the second true leaf after germination of plants with different Gl2 / Gl3 genotypes. Figure 3d shows the results of the measurement of gossypol content in the second true leaf after germination of plants with different Gl2 / Gl3 genotypes.

[0052] Figure 4 shows that secondary metabolites such as gossypol are synthesized and accumulated in situ in pigment glands. Figure 4a is a schematic diagram of non-glandular cell sampling. Figure 4b shows the results of gossypol content determination in immature embryos and non-glandular cells. Figure 4c shows the transcriptome expression profiles of gossypol synthesis pathway genes in glandular cells in TM-1 immature embryos, leaves, and other parts, as well as glandular mutant strains. Figure 4d shows the GUS staining results of glands in immature embryos, leaves, and roots of transgenic strains after the promoter of CDNC, a key gene in the gossypol synthesis pathway, was fused to the GUS gene and introduced into the plant.

[0053] FIG5 shows the expression levels of Gl2 / Gl3 genes in glandular and non-glandular tissues.

[0054] Figure 6 shows that the Gl2 / Gl3 proteins regulate glandular development and growth by forming homotetramers. Figure 6a shows a schematic diagram of the predicted structure of the Gl2 protein. Figure 6b shows the results of a Gl2 / Gl3 bimolecular fluorescence complementation experiment, demonstrating that Gl2 / Gl3 protein molecules can interact with themselves. Figure 6c shows the results of a size exclusion chromatography analysis of Gl2 protein multimers. Figure 6d shows a structural model of the Gl2 protein homotetramer.

[0055] Figure 7 shows the use of ChIP-seq to discover Gl2 binding sites. Figure 7a is a Venn diagram of two ChIP-seq results for Gl2 protein, showing the overlap of the two ChIP-seq results. Figure 7b shows the distribution of Gl2 protein binding regions across the genome. Figure 7c shows the distribution of Gl2 binding regions near gene start codons, near stop codons, and on genes. Figure 7d shows the DNA motifs predicted by MEME-ChIP to which Gl2 protein binds. Figure 7e is a Venn diagram of Gl2-bound genes and transcriptome differentially expressed gene analysis (DEGs).

[0056] Figure 8 demonstrates the ability of the artificial mutant protein mute9 to form tetramers and bind to DNA. Figure 8 (b) shows the results of a bimolecular fluorescence complementation assay detecting the interaction between Gl2 protein and the mute9 mutant protein. Figure 8 (b) shows the ability of Gl2 protein or the mute9 mutant protein to bind to target DNA as detected by EMSA, confirming that the mutant proteins can still bind to the target DNA molecule (E-box).

[0057] FIG9 shows the transcriptional activation ability of wild-type G12 protein and artificial mutant proteins.

[0058] FIG10 shows the expression levels of candidate promoter genes in different tissues of TM-1.

[0059] FIG. 11 shows the gene expression levels of seed-specifically expressed genes Ghα-globulin, GhOLE1_D, GhALB2S, and Gl2+Gl3 genes during seed development.

[0060] FIG. 12 shows a schematic diagram of a vector containing an expression cassette of the proGhOLE1_D-mute9 mutant gene.

[0061] FIG13 shows the glandular phenotype and gossypol content of novel cotton seeds.

[0062] FIG. 14 shows the plant gland phenotype of the novel cotton seeds.

[0063] FIG. 15 shows the novel cotton material fiber phenotype.

[0064] FIG16 shows the gossypol content in various tissues of the novel cotton.

[0065] FIG. 17 shows a schematic diagram of a vector containing an expression cassette for the proGhALB2S-mute4 mutant gene.

[0066] FIG18 shows a comparison of amino acid sequences of wild-type Gl2 / Gl3 proteins from different cotton origins. DETAILED DESCRIPTION

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0068] In order to make the present disclosure more easily understood, certain terms are first defined below. The following terms and additional definitions of other terms are set forth throughout the specification.

[0069] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0070] As used herein, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including, but not limited to, ").

[0071] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items. For example, a composition comprising A and / or B can be interpreted as a composition comprising A, a composition comprising B, or a composition comprising A and B.

[0072] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and are optionally varied (+) or (-) in increments of 1.0 or 0.1, or by variations of + / - 15%, 10%, 5%, or 2%, as appropriate. It should be understood that all numerical designations are preceded by the term "about." It should also be understood that the agents described herein are exemplary only and that equivalents thereof are known in the art. As used herein, the term "about" when referring to a measurable value such as an amount or concentration, etc., is intended to include variations within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0073] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or double-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting of, or consisting essentially of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0074] The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably and in their broadest sense refer to a polymeric form of two or more amino acid subunits, amino acid analogs, or peptidomimetics. "Protein," "peptide," "polypeptide," and "amino acid sequence" contain at least two amino acids, and there is no limitation on the maximum number of amino acids. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0075] The term "encoding" when applied to a nucleotide sequence refers to a nucleotide sequence that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide that "encodes" RNA or "encodes" a polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0076] As used herein, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, amino acid sequence, or protein. In some cases, "expression" as used herein also includes splicing, modification, processing, etc. of mRNA molecules.

[0077] "Carrier" refers to a delivery vehicle or element of genetic material. In this article, the term "carrier" refers to one or a series of macromolecules comprising polynucleotides that promote the delivery of polynucleotides to target cells, target tissues or target organs in vitro or in vivo. The polynucleotides to be delivered are sometimes referred to as "transgenes", including but not limited to the coding sequence of certain proteins or polypeptides that can enhance, suppress, weaken, protect, trigger or prevent certain biological functions and physiological functions (such as the nucleotide sequence of the Gl2 / Gl3 protein mutant of the present invention), the coding sequence of RNAi components (for example, shRNA, siRNA, microRNA, antisense oligonucleotides), or optional markers. In the carrier, the coding sequence of protein, polypeptide, RNAi components etc. is usually operably connected to an expression control element, such as a promoter. In some embodiments, the carrier can be a linear DNA molecule. In some embodiments, the carrier can be a circular DNA molecule.

[0078] As used herein, the term "operably linked" refers to the functional spatial arrangement of two or more nucleic acid fragments or nucleic acid sequences. For example, if a promoter is placed at a specific position relative to a nucleic acid sequence encoding a protein of interest such that transcription of the nucleic acid sequence encoding the protein of interest is regulated by the promoter, the promoter is said to be "operably linked" to the nucleic acid sequence encoding the protein of interest.

[0079] As used herein, the term "specific promoter" refers to a promoter that is capable of directing the expression of a gene of interest at a specific time and / or specific space (e.g., a specific cell, tissue, or organ). In this article, a "seed-specific promoter" refers to a promoter that is capable of directing the specific expression of its downstream gene in the seeds of cotton (cottonseed). Generally, if the mRNA level in a specific cell, tissue, or organ is at least 5 times higher, preferably at least 10 times higher, more preferably at least 100 times higher, and most preferably at least 1000 times higher than in other cells, tissues, or organs, the expression of the gene of interest is considered to be cell-, tissue-, or organ-specific, and the promoter of the gene of interest is referred to as the specific cell-, tissue-, or organ-specific promoter.

[0080] The term "sequence identity" refers to the sequence similarity between two polypeptides or between two nucleic acid sequences. The percent identity can be determined by comparing positions in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences depends on the number of shared matching positions. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity with one of the sequences of the present invention. The alignment and percent sequence identity of the nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / tools-bin / clustalw / ) and using ClustalW.

[0081] The terms "equivalent" or "functional variant" are used interchangeably when referring to specific molecules, biological materials or cellular substances and refer to those that have minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference polypeptide (e.g., a Gl2 / Gl3 protein mutant described herein); or polypeptides encoded by polynucleotides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference polynucleotide (e.g., a polynucleotide encoding a Gl2 / Gl3 protein mutant described herein).

[0082] Equivalents having one or more amino acid modifications compared to the reference polypeptide of the present invention (e.g., the Gl2 / Gl3 protein mutant of the present invention) are also encompassed within the scope of the present invention, provided that the one or more amino acid modifications do not affect or substantially do not affect the function of the reference polypeptide. As used herein, amino acid modifications may be amino acid substitutions, amino acid deletions, or amino acid insertions. Amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions (also known as conservative mutations, conservative substitutions, or conservative variations) are amino acid substitutions in proteins that change a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, "conservative substitutions" refer to replacement of an amino acid residue by another biologically similar residue. Examples of conservative substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another; or substitution of one charged or polar residue for another, such as substitution of arginine for lysine, substitution of glutamic acid for aspartic acid, substitution of glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and the like.

[0083] Cotton pigment glands are a trait unique to the genus Gossypium and its closely related species, also known as gossypol glands. Glands are visible to the naked eye as small dark brown dots distributed on the plants and seeds of cotton plants. Glands are only observed in tissues such as anthers, seed coats, and fibers. Under a microscope, the glands are multicellular cavities, with the central cavity formed by the lysis of multiple cells and the exterior composed of multiple layers of flat cells. Glands in cotyledons or leaves occur under the palisade tissue, glands in new stems occur in the hypodermis, and glands in roots are distributed in the parenchyma tissue near the cambium. The central cavity of mature glands contains a large amount of terpene secondary metabolites such as gossypol, as well as components of residual cell walls. The gossypol content can reach 35-50% of the total weight of the pigment glands.

[0084] Upland cotton (Gossypium hirsutum) is one of the most widely cultivated cotton species in the world, possessing an allotetraploid AD genome. The synthesis and accumulation of gossypol involves the regulation of multiple genes in cotton. Among these, Gl2 / Gl3 are the most important genes controlling glandularization, encoding the Gl2 / Gl3 proteins, respectively, which control glandularization in various tissues of cotton. Other genes only affect glandularization in limited tissues or alter the number and morphology of glands. The Gl2 gene is located on the A subgenome of upland cotton, while the Gl3 gene is an allele located on the D subgenome. The Gl2 / Gl3 proteins encoded by these two genes differ by only two amino acids.

[0085] As used herein, the terms "Gl2 / Gl3" and "Gl2 and / or Gl3" are used interchangeably to refer to either or both of the Gl2 and Gl3 proteins or the Gl2 and Gl3 genes. It should be understood that due to the high similarity in amino acid sequence and function between the Gl2 and Gl3 proteins, the mutational approaches designed for the Gl2 protein in the present invention are also applicable to the Gl3 protein. It should also be understood that the technical solutions related to Gl2 protein mutants disclosed herein are also applicable to Gl3 protein mutants, and those skilled in the art will appreciate that similar technical effects can be achieved by implementing the technical solutions disclosed herein using Gl2 and / or Gl3 protein mutants.

[0086] The term "dominant negative effect" refers to the ability of certain proteins, after mutation, to inhibit or block the function or effect of the wild-type protein within the same cell. Accordingly, the resulting mutant protein is called a "dominant negative mutation" or "dominant negative mutant." For example, certain proteins function within cells as multimers. When a dominant negative mutation is present within the cell, the mutant protein can form multimers with the wild-type protein, thereby affecting the normal function of the multimer.

[0087] When referring to a Gl2 / Gl3 protein mutant, "does not contain a functional transcriptional activation domain" means that the Gl2 / Gl3 protein mutant does not contain a complete transcriptional activation domain or contains a transcriptional activation domain with amino acid substitutions, as long as the transcriptional activation domain does not have or substantially does not have a transcriptional activation function. For example, for the Gl2 protein from upland cotton, its transcriptional activation domain is the amino acids 20-202 relative to SEQ ID NO: 1. Accordingly, the Gl2 protein mutant "does not contain a functional transcriptional activation domain" means that the Gl2 protein mutant has an amino acid deletion or an amino acid substitution at the amino acid positions 20-202 relative to SEQ ID NO: 1, so that the Gl2 protein mutant does not have or substantially does not have a transcriptional activation function.

[0088] The term "mutant tetramer" means that some of the Gl2 / Gl3 proteins in the tetramer are Gl2 / Gl3 protein mutants or all of the Gl2 / Gl3 proteins are Gl2 / Gl3 protein mutants. When referring to a mutant tetramer formed by a Gl2 / Gl3 protein mutant alone or with a wild-type Gl2 / Gl3 protein, the term "reduced transcriptional activation ability" means that the mutant tetramer causes the expression level of the target gene to be reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the wild-type tetramer formed by the wild-type Gl2 / Gl3 protein; and the term "abolished transcriptional activation ability" means that the mutant tetramer causes the expression level of the target gene to be lower than that of the control group or no higher than that of the control group in the absence of the wild-type tetramer by 10%, 15%, 20%, 25%, or 30%.

[0089] Method for inhibiting the formation of pigment glands in cotton seeds and method for reducing gossypol content in cotton seeds

[0090] The present invention provides a method for inhibiting the formation of pigment glands in cotton seeds, which comprises causing the cotton seeds to express a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has a reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

[0091] The present invention provides a method for reducing the gossypol content in cotton seeds, which comprises causing the cotton seeds to express a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

[0092] In some embodiments, the transcriptional activation function of the mutant tetramer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the wild-type tetramer formed by wild-type Gl2 / Gl3 protein alone.

[0093] Wild-type Gl2 / Gl3 proteins can form wild-type tetramers, which can bind to target DNA and activate the expression of downstream genes. Therefore, a nucleotide sequence containing the target DNA (e.g., the promoter of a downstream target gene of the wild-type tetramer) can be fused to a reporter gene. By detecting the expression level of the reporter gene in the presence of the wild-type tetramer or the mutant tetramer, the transcriptional activation function of the mutant tetramer can be obtained.

[0094] In the method of the present invention, the method for expressing the Gl2 / Gl3 protein mutant in the cotton seeds comprises:

[0095] 1) introducing the nucleotide sequence encoding the G12 / G13 protein mutant into cotton; or

[0096] 2) Obtaining cotton whose seeds can express the Gl2 / Gl3 protein mutant by mutagenesis.

[0097] In some embodiments, the method of the present invention comprises the step of introducing into cotton a nucleotide sequence encoding a G12 / G13 protein mutant. In some embodiments, the method of the present invention comprises integrating a nucleotide sequence encoding a G12 / G13 protein mutant into the genome of cotton.

[0098] Methods for introducing a target gene (e.g., a nucleotide sequence encoding a G12 / G13 protein mutant) into plant cells (e.g., cotton) are known in the art. For example, the target gene can be introduced into cotton by transforming a vector carrying the target gene into Agrobacterium tumefaciens, and then using Agrobacterium-mediated stem tip transformation.

[0099] The Gl2 / Gl3 protein mutants of the present invention can be obtained by performing one or more amino acid substitutions, insertions, and / or deletions on wild-type Gl2 / Gl3 proteins. The wild-type Gl2 / Gl3 proteins can be native Gl2 / Gl3 proteins derived from upland cotton, Asiatic cotton, herbaceous cotton, and / or sea island cotton. In some embodiments, the wild-type Gl2 / Gl3 proteins can be native Gl2 / Gl3 proteins derived from the following species: upland cotton (Gossypium hirsutum), Asiatic cotton (Gossypium arboretum), abnormal cotton (Gossypium anomalum), Sturtianum cotton (Gossypium sturtianum), Raymond cotton (Gossypium raimondii), Stoke cotton (Gossypium stocksii), longicalyx cotton (Gossypium longicalyx), Bick cotton (Gossypium bickii), round-leaf cotton (Gossypium rotundifolium), sea island cotton (Gossypium barbadense).

[0100] In some embodiments, the Gl2 / Gl3 protein mutants of the present invention have one or more amino acid substitutions, insertions and / or deletions at positions selected from amino acids 1-282 and 300-475 compared to the wild-type Gl2 / Gl3 protein. In some embodiments, the Gl2 / Gl3 protein mutants of the present invention have one or more amino acid substitutions, insertions and / or deletions at positions corresponding to amino acids 1-282 and / or 300-475 of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0101] In some embodiments, the G12 / G13 protein mutant has one or more amino acid substitutions, insertions, and / or deletions at amino acid positions 20 to 202. In some embodiments, the G12 / G13 protein mutant has one or more amino acid substitutions, insertions, and / or deletions at amino acid positions 20 to 282 corresponding to SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0102] In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at amino acid positions 300-313. In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at amino acid positions 300-313 corresponding to SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0103] In some preferred embodiments, the Gl2 / Gl3 protein mutant has one or more mutations selected from the following: amino acid A at position 43 is mutated to K (A43K), amino acids at positions 21-24 are mutated to alanine, amino acids at positions 54-59 are mutated to alanine, amino acids at positions 43-52 are deleted, amino acids at positions 72-79 are deleted, amino acids at positions 94-99 are deleted, amino acids at positions 121-126 are deleted, and amino acids at positions 300-313 are deleted, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0104] In some preferred embodiments, the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:3-10.

[0105] In some preferred embodiments, the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:6.

[0106] In some most preferred embodiments, the Gl2 / Gl3 protein mutant has or consists of the amino acid sequence shown in SEQ ID NO:6.

[0107] In some embodiments, compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant of the present invention is a truncated version of the N-terminus and / or C-terminus of the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant does not contain a functional transcription activation domain, and the Gl2 / Gl3 protein mutant contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence at positions 283-373 of the wild-type Gl2 / Gl3 protein. In some embodiments, the Gl2 / Gl3 protein mutant of the present invention is a truncated version of the N-terminus and / or C-terminus of SEQ ID NO:1 and / or SEQ ID NO:2, the Gl2 / Gl3 protein mutant does not contain a functional transcription activation domain, and the Gl2 / Gl3 protein mutant contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence corresponding to positions 283-373 of SEQ ID NO:1 and / or SEQ ID NO:2.

[0108] In some embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of positions 283-373 or the amino acid sequence of positions 141-475 of the wild-type Gl2 / Gl3 protein. In some embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of positions 283-373 or the amino acid sequence of positions 141-475 corresponding to SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0109] In some preferred embodiments, the Gl2 / Gl3 protein mutant comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 12. In some more preferred embodiments, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 12. In some most preferred embodiments, the Gl2 / Gl3 protein mutant has or consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0110] In some embodiments, the nucleotide sequence encoding the G12 / G13 protein mutant is operably linked to a cotton seed-specific promoter.

[0111] The seed-specific promoter suitable for the method of the present invention has the following characteristics:

[0112] (1) initiating expression of the Gl2 / Gl3 protein mutant at a time no later than that of the Gl2 / Gl3 protein during seed development in wild-type cotton;

[0113] (2) ensuring that the expression level of the Gl2 / Gl3 protein mutant is no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and

[0114] (3) The expression of the Gl2 / Gl3 protein mutant is initiated only during cotton seed development, and does not or substantially does not initiate after mature seed germination and other stages of cotton plant development.

[0115] The seed-specific promoter applicable to the present invention may be a seed-specific promoter derived from upland cotton, Asiatic cotton, herbaceous cotton and / or island cotton.

[0116] In some embodiments, the seed-specific promoter is the promoter of the OLE1_D gene, the OLE1_A gene, or the ALB2S gene. In some preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 13-15. In some more preferred embodiments, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14. In some most preferred embodiments, the seed-specific promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0117] In some embodiments, the method of the present invention comprises integrating into the genome of cotton a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 or 23. For example, a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 or 23 can be integrated into the genome of cotton by Agrobacterium-mediated shoot tip transformation.

[0118] In some embodiments, the methods of the present invention comprise the following steps:

[0119] (1) transforming a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0120] (2) contacting cotton cells, seeds, tissues, organs or plants with the Agrobacterium carrying the nucleotide sequence in step (1), thereby introducing the nucleotide sequence into the cotton cells, seeds, tissues, organs or plants.

[0121] In some embodiments, the methods of the present invention result in the abortion of pigment glands in cotton seeds compared to pigment glands in wild-type cotton seeds, and the development of pigment glands in cotton plants is not affected.

[0122] In some embodiments, the methods of the present invention reduce the gossypol content in cotton seeds by 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% compared to the gossypol content of wild-type cotton seeds, wherein the gossypol content in the cotton seeds can be determined, for example, by high performance liquid chromatography.

[0123] In some embodiments, the methods of the present invention result in gossypol levels in tissues of a cotton plant that are no different or substantially no different than the gossypol levels in tissues of a wild-type cotton plant.

[0124] The significant advantages of the method of the present invention are:

[0125] (1) It not only achieves "zero" gossypol in cotton seeds, but also removes other secondary metabolites synthesized by cotton seed glands that may affect food safety;

[0126] (2) The glandular development of tissues other than seeds can be completely maintained in the same manner as that of wild-type materials, thereby ensuring that the resulting cotton lines have no significant differences in disease and insect pest resistance compared to currently promoted cotton varieties;

[0127] (3) It does not affect the cotton fiber yield and quality of the produced cotton varieties.

[0128] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described below in conjunction with specific examples and accompanying drawings, and the advantages and features of the present invention will become clearer as the description progresses. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are carried out according to conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0129] Example

[0130] Experimental methods

[0131] (1) Shoot tip transformation method

[0132] 1. Seed germination

[0133] One day before infection, take 300 lint-free cotton seeds and place them in a sterile conical flask. Add 75% alcohol, shake for 30 seconds, and discard the waste liquid. Then add 6% H2O2 and shake on a shaker for 15 minutes. Discard the waste liquid, rinse with sterile water 4 times, and soak and culture in a dark room at 28℃ overnight for 18-24 hours.

[0134] 2. Agrobacterium Preparation

[0135] Three days before infection, use a disposable inoculating loop to dip the target glycerol bacteria and evenly spread it on YEB culture medium containing the corresponding antibiotics. Seal the edge of the culture plate with sealing film and incubate it upside down at 28℃ in a constant temperature box for 2 days to obtain activated Agrobacterium. Expand the culture of the activated Agrobacterium at 28℃ and measure the A660 value of about 0.9 using a spectrophotometer for later use.

[0136] 3. Infection and Co-cultivation

[0137] After seed culture is complete, use a scalpel to cut the seed coat. Under a dissecting microscope, peel off the cotyledons to expose the embryo apex, retaining the embryo apex and hypocotyl as the explant. Place the explant in the prepared Agrobacterium suspension, sonicate at 40 kHz for 40 seconds, and then shake at a constant speed for 50 minutes. After infection, place the explant in a co-culture medium covered with double-layer sterile filter paper and incubate in a 23°C incubator in the dark for 3-4 days.

[0138] 4. Resume Culture

[0139] After 3-4 days of co-cultivation, the explants were transferred to selective medium and placed in a culture chamber. The explants were cultured at 35°C for 3-6 days and then transferred to a culture temperature of 28°C for another 22-25 days with a photoperiod of 16 / 8 h, light / dark.

[0140] 5. Bud induction culture and resistant bud screening

[0141] After culturing on the recovery medium for 7 days, the slender roots of the cotton explants were cut off and then transferred to the bud induction medium. They were subcultured every 2 weeks for 3-4 times at 25°C and a photoperiod of 16 / 8h, light / dark, until resistant buds were obtained.

[0142] 6. Rooting culture

[0143] After resistant buds have fully expanded, transfer the explants to elongation medium and treat as for screening. Cultivate at 28°C under a 16 / 8 hour light / dark cycle. Subculture every two weeks. When resistant buds have elongated >3 cm, have >2 roots, and are >2 cm long, transplant them to a hardening room for testing and hardening.

[0144] Solution formula:

[0145] CA salt: magnesium sulfate 10 g / L, ammonium sulfate 5.36 g / L, sodium phosphite hydrate 6 g / L, calcium chloride 6 g / L, boric acid 30 mg / L, manganese sulfate 100 mg / L, zinc sulfate heptahydrate 20 mg / L, potassium iodide 7.5 mg / L, sodium molybdate dihydrate 2.5 mg / L, copper sulfate 250 μg / L, cobalt (II) chloride hexahydrate 250 μg / L, potassium nitrate 100 g / L;

[0146] Agrobacterium infection suspension: 10 ml / L CA salts, 30 g / L glucose, 4.2 g / L MES, 0.1 ml / L B5 vitamins, 1 mg / L 6-BA, 0.1 mg / L NAA, 2 ml / L 100 mM acetosyringone, pH = 5.4;

[0147] Co-culture medium: 10 ml / L CA salts, 30 g / L glucose, 4.2 g / L MES, 0.1 ml / L B5 vitamins, 1 mg / L 6-BA, 0.1 mg / L NAA, 2 ml / L 100 mM acetosyringone, 4 ml / L 50 mg / ml CYS cysteine, pH = 5.4;

[0148] Recovery medium: 4.4 g / L MS salts and B5 vitamins, 20 g / L glucose, 1.29 g / L calcium gluconate, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 4 g / L agar, pH 5.6.

[0149] Shoot induction and selection medium: 4.4 g / L mixture of MS salts and B5 vitamins, 20 g / L glucose, 1.29 g / L calcium gluconate, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L kanamycin, 4 g / L agar, pH 5.6.

[0150] Rooting medium: a mixture of MS salts and B5 vitamins at a concentration of 4.4 g / L, glucose at a concentration of 20 g / L, calcium gluconate at a concentration of 1.29 g / L, carbenicillin at a concentration of 100 mg / L, cephalosporin at a concentration of 100 mg / L, 4 g / L agar, pH = 5.6.

[0151] (2) Western Blot

[0152] 1. Tissue Protein Extraction

[0153] Take 1g of fresh embryonic tissue or embryonic tissue stored at -80°C and grind it into a powder with liquid nitrogen. Remove the PBS stored at 4°C and add 1ml of PBS to the EP tube. Shake at 4°C for 30 minutes. Centrifuge the fully lysed tissue solution at 12,000 rpm at 4°C for 10 minutes in a pre-cooled centrifuge. Collect the supernatant. Add the protein loading buffer and boil at 95°C for 10 minutes.

[0154] 2. Glue making

[0155] The present invention uses the YAZY PAGE gel rapid preparation kit. Taking a 1.0 mm gel as an example, take equal volumes of lower gel solution and lower gel buffer, 2.7 mL each, and mix them evenly.

[0156] Add 60 μL of the improved coagulant to the above mixed solution and mix well;

[0157] Pour the mixed solution into the glass plate, making sure the distance between the liquid surface and the upper edge of the short glass plate is 0.5 cm longer than the comb teeth. Add an appropriate amount of 100% ethanol to cover the lower layer of glue.

[0158] After the lower layer of gel solidifies, pour off the upper layer of ethanol;

[0159] Take equal volumes of upper gel solution and colored upper gel buffer, 0.75 mL each, and mix well;

[0160] Add 15 μL of the improved coagulant to the above mixed solution and mix well;

[0161] Pour the mixed solution into a glass plate and insert the comb teeth;

[0162] After the upper layer of gel solidifies, remove the comb teeth and use it for electrophoresis.

[0163] 3. Electrophoresis: Inject the denatured protein from step 1 into the gel wells, constant voltage 90V, 90min;

[0164] 4. Transfer: Use The strips were transferred to 0.45 μm PVDF using a semi-dry transfer system at 25 V, 125 mA, for 60 min.

[0165] 5. Blocking: Place the above PVDF membrane in 5% skim milk (prepared in TBST) for 60 minutes;

[0166] 6. Primary Antibody Incubation: After blocking, wash with TBST three times for 5 minutes each time; then add 18 mL TBST, 2 mL 5% skim milk, and 1.5 μL primary antibody (full gold, HT201-01) and shake at 4°C overnight;

[0167] 7. Secondary antibody incubation: After primary antibody incubation, wash with TBST three times, 5 minutes each time; then add 18 mL TBST, 2 mL 5% skim milk, and 1.5 μL secondary antibody (full gold, HS201-01) and incubate at room temperature for 60 minutes;

[0168] 8. Chemical exposure: After secondary antibody incubation, wash three times with TBST, 5 minutes each time, and expose and observe using M5 Hiper ECL Western HRP Substrate.

[0169] (III) Chromatin immunoprecipitation sequencing (ChIP-seq)

[0170] 1. Collect fresh tissue samples in 50 mL Falcon tubes;

[0171] 2. Add 37 mL of crosslinking buffer to each sample and crosslink under vacuum at room temperature for 10 minutes;

[0172] 3. Stop the cross-linking reaction by adding 2.5 mL of 2 M glycine (final concentration 100 mM) and placing the tissue under vacuum infiltration for another 5 min at room temperature;

[0173] 4. Wash the plant tissue three times in sterile deionized water, removing as much water as possible by blotting the tissue between paper towels and snap freezing in liquid nitrogen;

[0174] 5. Take 1-2g of the cross-linked sample and grind it for about 30 minutes until it becomes a fine powder. Place the powdered sample in a 50ml enzyme-free centrifuge tube and store it in liquid nitrogen until all the sample is ground.

[0175] 6. Add 40 mL of nuclear buffer and shake at 4°C for about 30 minutes;

[0176] 7. Use a double layer of non-woven fabric to filter, mark the center with a blue pipette tip, slowly add liquid to the filter, and squeeze the filter cloth until it is clean after filtering.

[0177] 8. Centrifuge at 4°C, 1500 rpm, 15 min;

[0178] 9. Remove the supernatant and resuspend the nuclei with 5-10 mL of nuclear buffer to 40 mL.

[0179] 10. Centrifuge at 1500 rpm for 15 minutes at 4°C, discard the supernatant, and repeat 1-2 times (depending on the turbidity of the sample).

[0180] 11. Resuspend in 750 μL / times nuclear buffer to a total volume of 1-2 mL and centrifuge at 1500 rpm for 15 min at 4°C.

[0181] 12. Remove the supernatant (use a 1mL blue pipette tip covered with a white pipette tip to absorb the supernatant);

[0182] 13. Add 700 μL of nuclear lysis buffer to resuspend and dispense into glass EP tubes (no more than 300 μL per tube);

[0183] 14. Ultrasonicate at 4°C for 20 minutes, divided into two times, 10 minutes each time (maximum power, 30 seconds on, 30 seconds off);

[0184] 15. Centrifuge at 4°C, 20,000 rpm, 15 min, and remove the supernatant. If the sample is turbid, repeat the centrifugation and remove the supernatant.

[0185] 16. Take 1 / 10 of the sample as input and dilute the remaining sample 5-10 times with ChIP dilution buffer;

[0186] 17. Remove impurities, aspirate magnetic beads (5 μL per tube), place on a magnetic stand, remove storage solution, and wash once with ChIP dilution buffer;

[0187] 18. Add magnetic beads, 5 μL per tube, shake well at 4°C for about 1 hour;

[0188] 19. Place on a magnetic rack and collect the supernatant;

[0189] 20. Add 5 μL of primary antibody to each tube supernatant and shake overnight at 4°C.

[0190] 21. Take 50 μL of magnetic beads per tube, wash once with ChIP dilution buffer, and resuspend to the required volume;

[0191] 22. Add 50 μL of magnetic beads to each tube of sample and shake well at 4°C for about 1.5 hours;

[0192] 23. Centrifuge to collect the liquid on the tube wall, place it on a magnetic rack, remove the supernatant, and retain the magnetic beads;

[0193] 24. Wash with low salt solution, high salt solution, and TE in sequence, place on a magnetic stand, remove the liquid, add 250 μL elution buffer, shake at 65°C for 15 minutes, and collect the supernatant into a new centrifuge tube;

[0194] 25. Add 250 μL of elution buffer, shake at 65°C for 15 min, and combine the supernatants;

[0195] 26. Add 20 μL of 5 M NaCl and shake at 65°C for 8 hours;

[0196] 27. Add 10 μL EDTA, 20 μL Tris-HCl, 1 μL Proteinase K, and 10 μL RNase A to each tube. Incubate at 45°C, 700 rpm, and shake for 1.5 h.

[0197] 28. Add 550 μL of the 25:24:1 DNA extraction solution and shake well. Centrifuge at 20,000 rpm at 4°C for 15 min. Remove 450 μL of the supernatant and add 2.5 volumes of anhydrous ethanol, 45 μL of NaAc, and 2-4 μL of glycogen to assist in DNA precipitation. Store at -80°C overnight.

[0198] 29. Centrifuge at 4°C, wash twice with 75% alcohol, air dry, and dissolve in enzyme-free water.

[0199] Reagent formula:

[0200] Cross-linking buffer: 0.4 M sucrose, 10 mM Tris-HCl pH = 8, 1 mM PMSF, 1 mM EDTA, 1% formaldehyde;

[0201] Nuclear buffer: 0.25 M sucrose, 15 mM PIPES pH = 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, 2 mg ml –1 Pepstatin A, 2 mg ml –1 aprotinin;

[0202] Nuclear lysis buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mg ml –1 Pepstatin A, 1 mg ml – 1 aprotinin;

[0203] ChIP dilution buffer: 167 mM NaCl, 16.7 mM Tris-HCl pH = 8, 1.2 mM EDTA, 1.1% Triton X-100;

[0204] Elution buffer: 0.5% SDS, 0.1 M NaHCO3;

[0205] Low salt wash buffer: 150 mM NaCl, 20 mM Tris-HCl pH 8, 0.2% SDS, 0.5% Triton X-100, 2 mM EDTA;

[0206] High salt wash buffer: 500 mM NaCl, 20 mM Tris-HCl pH 8, 0.2% SDS, 0.5% Triton X-100, 2 mM EDTA;

[0207] LiCl wash buffer: 0.25 M LiCl, 1% sodium deoxycholate, 10 mM Tris–HCl pH 8, 1% NP-40, 1 mM EDTA;

[0208] TE buffer: 1 mM EDTA, 10 mM Tris-HCl pH=8.

[0209] The ChIP-DNA was sent to Wuhan Kangce Technology Co., Ltd. for library construction and sequencing analysis.

[0210] (IV) Bimolecular fluorescence complementation (BIFC)

[0211] 1. Clone the CDS fragments of GhGl2 and GhGl3 genes into pxy106 and pxy104 vectors, respectively, and then transform the recombinant vectors into GV3101 Agrobacterium competent cells.

[0212] 2. Pick a single Agrobacterium clone containing the target vector and transfer it to 1 ml LB liquid medium containing spectinomycin. Culture it at 28°C and 220 rpm for 1 day.

[0213] 3. Transfer the bacterial solution to 10 ml LB medium for expansion culture and culture at 28°C and 220 rpm until the OD600 of Agrobacterium is 0.6-0.8.

[0214] 4. At room temperature, centrifuge at 8000 rpm for 5 minutes to collect the Agrobacterium cells. Resuspend the Agrobacterium cells in infection solution (containing 10 mM MgCl2, 10 mM MES, 150 μL acetosyringone, pH = 5.6) to an OD600 of 0.8. Let stand at room temperature for 2-3 hours.

[0215] 5. Combine equal volumes of two bacterial strains containing different plasmids. Use a needle to gently poke a small hole on the back of the tobacco leaf (be careful not to puncture it). Then, use a syringe with the needle removed to draw up the bacterial solution and inject it into the leaf through the wound. Use a marker to mark the water-soaked area on the tobacco leaf.

[0216] 6. After incubating the injected plants at approximately 23°C for 2 days, observe the area where the Agrobacterium was injected for fluorescence. Cut a small piece of leaf with scissors, making the surface as flat as possible, and place it backside down on a glass slide. Add a drop of water, cover with a coverslip, and gently press. Observe YFP fluorescence imaging using a laser confocal microscope.

[0217] (V) Luciferase reporter (LUC) assay

[0218] 1. Construct GhGl2-62SK, mute1-62SK, mute2-62SK, mute3-62SK, mute4-62SK, mute5-62SK, mute6-62SK, mute7-62SK, mute8-62SK, mute9-62SK, mute10-62SK, and proGhCGF1-Luc vectors, and transform the recombinant vectors into GV3101 (pSoup-P19) Agrobacterium competent cells.

[0219] 2. Prepare infection solution: Activate Agrobacterium containing the target plasmid and empty vector to a final OD600 of 0.6-0.8. Collect the cells by centrifugation, wash once with ddH2O, and resuspend in infection buffer. Mix the cultures in a 1:1 ratio according to the target and let stand in the dark for 3 hours.

[0220] Table 1. Infection solution system

[0221] 3. Injection of tobacco: Select healthy tobacco plants and inject them into the lower epidermal cells using a disposable medical syringe. Place in the dark and culture for 2 days.

[0222] 4. Fluorescence intensity and enzyme activity detection: The luciferase activity was quantitatively detected using the Dual-Luciferase Reporter Assay System (Full-Form Gold, FR201-01-V2).

[0223] (VI) Electrophoretic mobility shift assay (EMSA)

[0224] 1. Construction of prokaryotic expression vector

[0225] Gl2 and mute9 were amplified using upland cotton TM-1 cDNA as a template and ligated into pCold vector to construct a protein-inducible vector with a HIS tag. The plasmid was then transformed into the expression strain BL21.

[0226] 2. Protein Induction

[0227] Pick a monoclonal strain containing the target vector and transfer it to 50 mL of LB liquid medium containing ampicillin resistance. Culture it at 37°C for 2-3 h. Transfer 50 ml of the bacterial solution to 4 L of LB medium and culture it at 37°C with shaking at 220 rpm until OD = 0.4-0.6. Add IPTG to a final concentration of 200 μM. Induce it overnight at 16°C and 180 rpm for 18 h. Centrifuge it at 11,000 rpm for 5 min to enrich the bacteria in a 50 ml centrifuge tube. Discard the supernatant and resuspend the precipitate with 40 ml of HIS binding buffer. Use a contact sonicator to sonicate for 5 s, stop for 10 s as one cycle, and sonicate for a total of 20 cycles at a power of 200 W. Centrifuge it at 4°C and 11,000 rpm for 90 min.

[0228] Add 750 μL of Ni-NTA Agarose beads (Full Gold, DP101-02) to a column (Full Gold, affinity chromatography column, DS0130). Wash five times with HIS binding buffer. Pass the supernatant from the previous centrifugation step through a 0.45 μm filter and add it to the column. Seal the upper and lower ends of the column with sealing film and rotate overnight at 4°C to allow binding. The supernatant from the lower end of the column, the flow-through, is collected in a fresh tube and washed 10 times with HIS wash buffer, using 1 mL each time. Elute eight times with HIS elution buffer, using 500 μL each time, and collect in a 1.5 mL centrifuge tube. The supernatant, eluate, precipitate and flow-through were used for gel running to identify protein expression. 10 μL of the liquid was added with 2 μL of 6× protein loading buffer, and the precipitate was added with 20 μL of 2× protein loading buffer. The mixture was boiled at 99°C for 5 min and centrifuged at 12,000 rpm for 5 min. The supernatant was aspirated and run on the gel for identification. At the same time, 1 μg, 2 μg, 4 μg, 8 μg and 16 μg of BSA were used to roughly quantify the expression of proteins in the eluate tubes with higher concentrations. The proteins were stored in a -80°C refrigerator for later use.

[0229] 3. Annealing of Biotin-labeled Probe

[0230] Based on ChIP-seq results, a 50-bp segment of the Gl2 / Gl3 binding motif in the Gh_D11G107900 (GhMYC4) promoter region was selected. A 5'-end biotin-labeled probe (SEQ ID NO: 24), a biotin-labeled mutation probe (SEQ ID NO: 25), and a cold probe were synthesized by BGI. The probes were dissolved in ddH2O to a concentration of 100 μM. Equal volumes of the forward and reverse sequences were mixed and placed in an insulated bucket filled with boiling water and naturally cooled to anneal, thereby synthesizing double-stranded probes.

[0231] 4. Protein and probe incubation

[0232] Prepare the reaction system in a 1.5 ml centrifuge tube according to Table 3.

[0233] Table 2. Protein and probe incubation binding reaction system

[0234] Mix by pipetting, react at 25°C for 20 min, add 5 μL of 5× loading buffer to each reaction, mix by pipetting, and terminate the reaction.

[0235] 5. Non-denaturing polyacrylamide gel electrophoresis

[0236] Prepare a 5% native PAGE gel (7.3 mL of ddH2O; 1 mL of 10× TBE; 1.7 mL of 30% Acr / bis; 6 μL of TEMED) in a 50 mL tube. Add TEMED and mix immediately before placing in a gel casting mold. After gelling, place the electrophoresis tank on ice and load reactions 1-5 in sequence. Run electrophoresis at a constant voltage of 90 V, stopping when bromophenol blue reaches 3 / 4 of the gel. Run for approximately 90 minutes.

[0237] 6. Transfer

[0238] On the cathode side of the transfer mold splint, a sponge, filter paper, gel, nylon membrane (Solebo, YA1760), filter paper, and sponge were placed in sequence. The membrane and filter paper were soaked in 0.5×TBE buffer for 15 minutes and wet-transferred at 100V in an ice bath for 60 minutes.

[0239] 7. UV cross-linking

[0240] After the transfer is completed, place the nylon membrane face up on a dust-free paper and crosslink it for 15 minutes at a distance of 10-20 cm from the UV lamp on the workbench.

[0241] 8. Closed

[0242] Take a suitable container and add 15ml of blocking solution (Biyuntian, GS009-7), then place the cross-linked nylon membrane containing the sample. Slowly shake on a side-to-side shaker or horizontal shaker for 15 minutes, repeat three times. Take 7.5μl of Streptavidin-HRP Conjugate (Biyuntian, GS009-6) and add it to 15ml of blocking solution (1:2000 dilution), mix well and set aside. Remove the blocking solution used for blocking the nylon membrane and add 15ml of blocking solution containing Streptavidin-HRP Conjugate prepared in the previous step. Slowly shake on a side-to-side shaker or horizontal shaker for 15 minutes.

[0243] 9. Washing the membrane

[0244] Take 25 ml of washing solution (5X) (Biyuntian, GS009-8), add 100 ml of redistilled water or Milli-Q grade pure water, mix well to make 125 ml of washing solution. Transfer the nylon membrane to another container containing 15-20 ml of washing solution and rinse for 1 minute. Remove the washing solution, add 15-20 ml of washing solution, and wash slowly on a side-swing shaker or horizontal shaker for 5 minutes. Repeat three times, with each wash time being about 5 minutes. Transfer the nylon membrane to another container containing 20-25 ml of detection balance solution and shake slowly on a side-swing shaker or horizontal shaker for 5 minutes.

[0245] 10. Color rendering

[0246] BeyoECL Moon A solution (Biyuntian, P0018FM) and BeyoECL Moon B solution (Biyuntian, P0018FM) were mixed in equal volumes to prepare 1 mL of color development solution. After removing the membrane, 1 mL of color development solution was evenly added dropwise. The reaction was carried out in the dark for 5 minutes, and imaging was performed using a BIO-RAD multifunctional chemiluminescence imager.

[0247] Example 1. Occurrence and development of cotton pigment glands and gossypol accumulation

[0248] The development of pigment glands proceeds through four stages: glandular cell initiation, glandular development, secondary metabolite synthesis, and internal cell lysis. The present invention utilized the cotton Gl2 gene promoter (SEQ ID NO: 16) to construct a proGhGl2::GUS cotton transgenic line. GUS staining of cotton true leaves and immature embryos revealed that glandular cells arise from pseudo-meristems expressing the Gl2 / Gl3 genes. After their fate is determined, these glandular cells undergo multiple divisions to form visible glands (as shown in Figure 1, a).

[0249] By observing the embryos at different developmental stages, it was found that glandular progenitors began to appear around 14 days after the embryos developed. At this time, the glands appeared white under an optical microscope, but no gossypol accumulated in the embryos at this time; gossypol began to accumulate in the embryos around 17-18 days after the embryos developed, and the glands appeared light yellow under an optical microscope (as shown in Figure 1c and Figure 2); as the glands developed, the cells in the center of the glands began to lyse, and eventually a mature pigment gland could be seen with an internal cavity filled with secondary metabolites such as gossypol wrapped by tightly arranged sheath cells on the outside (as shown in Figure 1b).

[0250] In summary, gossypol only begins to appear when the gland develops to a certain stage, and there is no gossypol accumulation in the immature embryo in the early stage of gland development.

[0251] Example 2. Alleles Gl2 and Gl3 are key genes regulating glandular development and gossypol synthesis

[0252] Gl2 and its alleles (including the Gl3 allele on the D subgenome) are necessary and sufficient for gland development. Plants with a simultaneous loss-of-function Gl2 / Gl3 mutant (gl2gl2gl3gl3) lack any pigmented glands (as shown in Figure 3a), and gossypol is undetectable in any of the mutant's tissues and organs (as shown in Figure 3b). Furthermore, as the wild-type Gl2 and Gl3 copy numbers decrease, the number of true leaf glands and gossypol content decrease significantly (as shown in Figure 3c and d).

[0253] The Gl2 and Gl3 proteins differ only in two amino acids (Gl3 has N207H and S281G substitutions compared to Gl2). Genetic analysis indicates that the Gl2 and Gl3 genes function identically in glandular development. The difference between the two is that Gl2 expression is significantly higher than Gl3, with Gl2 expression approximately double that of Gl3. Compared to the wild type (Gl2Gl2Gl3Gl3), the gl2 mutant (gl2gl2Gl3Gl3) exhibited significantly lower glandular number and gossypol content than the gl3 mutant (Gl2Gl2gl3gl3) (as shown in Figure 3, c and d).

[0254] In summary, the Gl2 / Gl3 genes determine the development of cotton pigment glands and the accumulation of gossypol.

[0255] Example 3. In situ synthesis and accumulation of gossypol and other secondary metabolites in pigment glands

[0256] Non-glandular cells of immature embryos (30 days, 40 days, and 50 days) of the upland cotton genetic standard line TM-1 were obtained, and their gossypol content was determined by high performance liquid chromatography (HPLC). The results showed that no gossypol was detected in the non-glandular cells of the immature embryos (as shown in Figure 4a and b).

[0257] In addition, glandular cells from TM-1 immature embryos, leaves, and other parts were obtained for transcriptome sequencing. Combined with the transcriptome expression profile of immature embryos of the Gl2 / Gl3 mutant strain, it was found that genes in the gossypol biosynthesis pathway were gland-specifically expressed genes (as shown in Figure 4c).

[0258] A vector encoding the CDNC promoter (SEQ ID NO: 17), a key gene in the gossypol biosynthesis pathway, and the GUS gene (SEQ ID NO: 18) was constructed and introduced into cotton. GUS staining of T1-positive plants revealed that glands in embryos, leaves, and roots of the transgenic lines exhibited distinct blue color (as shown in Figure 4(d)).

[0259] In summary, the present invention confirms that secondary metabolites such as gossypol in cotton pigment glands are synthesized and accumulated in situ in the glands.

[0260] Example 4. Gl2 / Gl3 genes are gland-specific highly expressed genes

[0261] Glandular cell-specific transcriptome analysis revealed that the expression levels of the Gl2 / Gl3 genes in glandular cells of cotton embryos, leaves, and stems were much higher than in non-glandular cells (Figure 5). ProGhGl2::GUS transgenic cotton lines were constructed, and GUS staining revealed that glands in embryos, leaves, and other parts of the transgenic lines exhibited a distinct blue color, while non-glandular tissues did not.

[0262] In summary, Gl2 / Gl3 genes are gland-specific highly expressed genes.

[0263] Example 5. Gl2 / Gl3 regulates glandular development and growth by forming homotetramers

[0264] Bioinformatics analysis of the Gl2 protein (SEQ ID NO: 1) from upland cotton revealed that it belongs to the bHLH transcription factor family and contains a conserved basic helix loop helix (bHLH) domain and a relatively conserved N-terminal region (as shown in Figure 6a). The bHLH domain of the Gl2 protein (amino acids 300-373 of SEQ ID NO: 1) is composed of more than 70 amino acids and is divided into an HLH region and an alkaline region. The alkaline region (amino acids 300-313 of SEQ ID NO: 1) is the region that binds to DNA; the HLH region is primarily composed of hydrophobic amino acids and is the region that forms homologous or heterologous multimers. The N-terminal region of Gl2 contains the bHLH_MYC_N domain, which is responsible for forming a complex with other transcription-related proteins that mediates transcription initiation, thereby regulating the initiation of its downstream genes.

[0265] Bimolecular fluorescence complementation experiments confirmed that the Gl2 / Gl3 protein molecule can interact with itself (as shown in Figure 6b), and further analysis by size exclusion chromatography determined that the Gl2 / Gl3 protein molecule exists in the form of a homotetramer (as shown in Figure 6c and d).

[0266] Example 6. Gl2 / Gl3 activates target gene expression by binding to target sequence

[0267] In this study, the proGhGl2:GhGl2-3*FLAG vector was constructed using the proGhGl2 promoter and the gene encoding the Gl2 protein (SEQ ID NO:19). Transgenic lines were obtained by shoot apex transformation, and positive plants were confirmed by PCR and Western blot analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed using early developmental embryos (13-21 dpa, with anthesis designated 0 dpa). Analysis of the two ChIP-seq results revealed 5627 Gl2 protein-bound regions throughout the genome, with 991 regions overlapping between the two experiments (shown in Figure 7a). Binding peaks located upstream of the transcription start site (TSS), i.e., within the core promoter region (3000 bp upstream of the TSS), accounted for 52.89% (shown in Figure 7b and c). The MEME-ChIP program was used to search for the DNA motifs bound by Gl2, and it was found that the E-box motif (NACGTG) was significantly enriched (as shown in Figure 7d), further confirming that Gl2 transactivated the expression of target genes by binding to the E-box motif.

[0268] Transcriptome analysis was used to further analyze the co-expression regulatory relationship between Gl2 / Gl3 and its downstream target genes, and ultimately 612 high-confidence potential target genes of Gl2 were identified (as shown in Figure 7e), including a gene CGF1 that regulates gland development, and two genes 2-ODD-1 and CYP736A12 in the gossypol biosynthesis pathway (Table 3).

[0269] Table 3. Downstream target genes regulated by Gl2 protein

[0270] Example 7. Design and functional verification of Gl2 / Gl3 protein mutants

[0271] Based on the above experimental results, Gl2 is a key gene for regulating the development of glands. The present invention is based on the mechanism of action of a dominant negative effect. That is, by artificially introducing or designing mutation sites, a wild-type Gl2 protein mutant is obtained while retaining the ability to form a tetramer and bind to the promoter of its downstream target gene. The mutant protein can form a tetramer without transcriptional function with the wild-type Gl2 / Gl3 protein or the artificial mutant protein itself, and competitively bind to the downstream target gene promoter with the wild-type Gl2 / Gl3 tetramer, thereby interfering with the function of the wild-type Gl2 / Gl3. At the same time, by fusing the Gl2 / Gl3 mutant protein gene with a cotton seed-specific promoter, a new cotton material with normal plant growth and zero cottonpol in cotton seeds is created. Because the mutant protein gene is only expressed in cotton seeds, it only interferes with the development of cotton seed glands without affecting the development of glands in other tissues.

[0272] Combined with Gl2 protein structural analysis, it was found that mutations in the Gl2 N-terminal activation domain (amino acids 20-202 of the Gl2 protein) or the C-terminal DNA binding domain (amino acids 300-313 of the Gl2 protein) can cause the Gl2 protein to lose its ability to activate downstream target genes. Therefore, the present invention mainly targets these regions for mutation and protein truncation.

[0273] The Gl2 protein mutants designed and constructed by the present invention are shown in Table 4.

[0274] Table 4. Gl2 protein mutants

[0275] Bimolecular fluorescence complementation (BIFC) assay (as shown in FIG8 a) confirmed that the mute9 (SEQ ID NO: 11) mutant protein could interact with the wild-type Gl2 protein and itself, and EMSA experiments confirmed that the protein could still bind to the DNA molecule (E-box) (as shown in FIG8 b).

[0276] Luciferase assays were used to investigate the differences in the transcriptional activation abilities of the mutant proteins mute1 to mute10. At 25°C, 62sk-Gl2, 62sk-mute1, 62sk-mute2, 62sk-mute3, 62sk-mute4, 62sk-mute5, 62sk-mute6, 62sk-mute7, 62sk-mute8, 62sk-mute9, 62sk-mute10, and the 62sk empty vector (control) were co-injected with pCGF1-LUC into tobacco leaves. Enzyme activity assays revealed that, compared to wild-type Gl2, the artificial mutant proteins almost completely lost their transcriptional activation abilities (Figure 9).

[0277] It can be seen from this that the mutant of the present invention loses or almost loses the ability to activate transcription while retaining the ability to bind to the target DNA. The mutant tetramer formed by the mutant of the present invention can be used to compete with the wild-type tetramer formed by the wild-type Gl2 / Gl3 protein for binding to the target DNA, thereby inhibiting the expression of downstream genes related to cotton gland development and gossypol synthesis.

[0278] Example 8. Screening of cotton seed development-specific promoters

[0279] Gl2 / Gl3 gene expression begins after cotton flowering, from the 12th day of embryonic development until seed maturity. Given the Gl2 / Gl3 gene's function in regulating glandularization, only by completely inhibiting the Gl2 / Gl3 protein function during cotton seed development can cotton seeds be completely gland-free and have zero gossypol. However, Ghα-globulin, a cotton seed-specific gene commonly used in the development of low-phenol cotton, only reaches a significantly higher expression level than the combined expression of the Gl2 and Gl3 genes after 16 days of embryonic development. Therefore, the introduction of a Gl2 / Gl3 gene silencing expression cassette driven by the Ghα-globulin promoter into plants cannot completely eliminate seed glands, thus failing to achieve "zero gossypol" in seeds.

[0280] The present invention uses high-resolution spatiotemporal transcriptome technology to screen genes specifically expressed during cotton seed development, and the screening is performed according to the following principles:

[0281] 1. The candidate gene begins to express no later than the expression time of Gl2 and Gl3, and continues to express until the seeds mature.

[0282] 2. The expression level of the candidate gene in cotton seed glands is not lower than the sum of the expression levels of Gl2 and Gl3.

[0283] 3. The candidate gene is expressed only during cotton seed development, with very low or no expression at other stages of cotton plant development, and very low or no expression after mature seeds germinate. Seed development here refers to the process from fertilized egg to seed maturity, which for cotton is from flowering to boll opening.

[0284] The present invention screened for multiple cotton seed-specifically expressed genes and obtained the corresponding gene promoters, proGhOLE1_A (SEQ ID NO: 15), proGhOLE1_D (SEQ ID NO: 13), and proGhALB2S (SEQ ID NO: 14) ( FIG10 ). The expression levels of the cotton seed-specific genes GhOLE1_A, GhOLE1_D, and GhALB2S, screened according to the aforementioned principles, were significantly higher throughout seed development than the combined expression levels of the Gl2 and Gl3 genes ( FIG11 ). Therefore, introducing an expression cassette fused to the Gl2 / Gl3 mutant promoter into cotton completely inhibited the transcriptional activation function of the wild-type Gl2 / Gl3 protein, thereby completely suppressing the development of seed glands.

[0285] Example 9. Creation of a novel cotton plant with zero gossypol in seeds and unchanged gossypol content in plant glands using the seed-specific promoter proGhOLE1_D and the mutant protein mute9

[0286] A plant expression cassette (Figure 12) was constructed using the seed-specific expression gene GhOLE1_D promoter (proGhOLE1_D) (SEQ ID NO:13) fused to the coding sequence of the Gl2 / Gl3 mutant mute9 (SEQ ID NO:22). The nucleotide sequence of proGhOLE1_D-mute9 is shown in SEQ ID NO:23. The expression cassette was introduced into cotton plants using Agrobacterium transformation using cotton stem tips as explants. Positive homozygous plants harboring the expression cassette (designated NSGFC-1-1, NSGFC-1-3, and NSGFC-1-7) were screened and identified. A cotton recipient material (TM-1) harboring an empty vector was used as a control. The positive homozygous plants were tested for traits such as gossypol content, fiber yield and quality, boll number, flowering time, insect resistance, and Verticillium wilt resistance in their seeds and various plant organs.

[0287] The results showed that no glands were observed in the mature seeds of the transgenic materials introduced with the mutant protein expression cassette, and the gossypol content was zero (Figures 13 and 16), while the comprehensive traits such as the gossypol content in other organs of the plants and the length of cotton fibers were not significantly different from those of the control materials (Figures 14-16, Table 5).

[0288] Table 5. Statistics of comprehensive traits of new cotton NSGFC-1

[0289] The experimental results show that the use of seed-specific promoter proGhOLE1_D fused with Gl2 / Gl3 mutant protein mute9 can successfully create a new type of cotton with "zero" gossypol in seeds and unchanged gland and gossypol content in other organs of the plant. In addition, the comprehensive traits of the new type of cotton have no significant differences compared with the control material.

[0290] Example 10. Creation of a novel cotton plant with zero gossypol in seeds and unchanged gossypol content in plant glands using the seed-specific promoter proGhALB2S and the mutant protein mute4

[0291] A plant expression cassette (Figure 17) was constructed by fusion of the seed-specific ALB2S promoter, proGhALB2S (SEQ ID NO:14), with the coding sequence of the Gl2 / Gl3 mutant mute4 (SEQ ID NO:20). The nucleotide sequence of proGhALB2S-mute4 is shown in SEQ ID NO:21. Cotton stem tips were used as explants and the expression cassette was introduced into cotton plants using Agrobacterium tumefaciens transformation. Positive homozygous plants harboring the expression cassette (designated NSGFC-2-4 and NSGFC-2-5, respectively) were screened and identified. A cotton recipient material (TM-1) harboring an empty vector was used as a control. The positive homozygous plants were tested for traits such as gossypol content, fiber yield and quality, boll number, flowering time, insect resistance, and Verticillium wilt resistance in their seeds and various plant organs.

[0292] The results showed that the gossypol content in mature seeds of the transgenic material with the mutant protein expression cassette was zero (Table 6), while the gossypol content in other organs of the plant, cotton fiber length and other comprehensive traits had no significant differences from those of the control material (Table 7).

[0293] Table 6. Gossypol content in various organs of new cotton NSGFC-2 (μg / mg)

[0294] Table 7. Statistics of comprehensive traits of new cotton NSGFC-2

[0295] The above results demonstrate that the novel cotton seeds produced using the method of the present invention are free of gossypol and other secondary metabolites, significantly improving the safety of the seeds for use as food and feed. Furthermore, the gossypol content of the novel cotton plants remains unaffected, enabling them to resist pests and diseases. Comprehensive traits such as plant yield and cotton fiber length remain unaffected, thus maintaining the production of raw cotton. In summary, the novel cotton produced using the method of the present invention significantly enhances the comprehensive utilization value of cotton.

[0296] Example 11. Application of the method for creating new cotton using seed-specific promoters and Gl2 / Gl3 mutant proteins in various cotton lines

[0297] The plant expression cassette used in Example 9 was introduced into Lumianyan No. 37 and Xinluzhong 67 to create new cotton. The cotton recipient material introduced with an empty vector was used as a control. Positive pure plants were screened from the F2 generation for field trials.

[0298] Field experiment design: randomized block arrangement, 3 replicates, plot area 20m 2 There are about 3-6 rows in each plot, and no less than 4 protective rows planted around the experimental area.

[0299] Field Management: Follow local conventional cultivation and management practices, with consistent field management. Plant density is 2,100-3,500 plants per mu, with sowing concentrated in mid-to-late April, using direct seeding. Control cutworms, aphids, bollworms, and whiteflies 4-16 times. Water 0-1 times, drain 1-4 times, perform chemical conditioning 2-6 times, and cultivate and weed 1-11 times. Base fertilizer is primarily compound fertilizer, with some pilot projects applying organic fertilizer. Topdressing primarily consists of urea and compound fertilizer.

[0300] The experimental results showed that in my country's main cotton-growing areas, the seeds of the new cotton varieties Lumianyan 37 and Xinluzhong 67 were gland-free and had zero gossypol content (Tables 8 and 9). The characteristics of other plant organs, such as glandular content and gossypol content, were stable, and other comprehensive traits showed no significant differences from the control materials (Tables 10 and 11). This shows that the method of the present invention for creating new cotton with "zero" gossypol in seeds and unchanged glandular content and gossypol content in plants is universally applicable to a variety of cotton strains.

[0301] Table 8. Determination of gossypol content in various organs of the new transgenic material Lumianyan 37 (μg / mg)

[0302] Table 9. Determination of gossypol content in various organs of the new transgenic material Xinluzhong 67 (μg / mg)

[0303] Table 10. Statistics of traits of new transgenic material of Lumianyan 37

[0304] Table 11. Statistics of traits of the new transgenic material Xinluzhong 67

[0305] Example 12. Comparison of amino acid sequences of Gl2 / Gl3 proteins from different cotton species

[0306] The amino acid sequences of wild-type Gl2 / Gl3 proteins from different cotton species are highly conserved. By performing a full-length alignment of the amino acid sequences of wild-type Gl2 and Gl3 proteins from Asian cotton (Gossypium arboretum, A2 genome), abnormal cotton (Gossypium anomalum, B1 genome), Sturtianum cotton (Gossypium sturtianum, C1 genome), Raymond cotton (Gossypium raimondii, D5 genome), Stocks cotton (Gossypium stocksii, E1 genome), longicalyx cotton (Gossypium longicalyx, F1 genome), Bick cotton (Gossypium bickii, G1 genome), round-leaf cotton (Gossypium rotundifolium, K2 genome), upland cotton (Gossypium hirsutum, AD1 genome), and sea island cotton (Gossypium barbadense, AD2 genome), it was found that there were very few differences between the amino acid sequences of Gl2 and Gl3 proteins from different cotton sources (Figure 18). Therefore, the mutation method of the present invention can be used to transform Gl2 / Gl3 proteins from various cotton sources to obtain corresponding Gl2 / Gl3 protein mutants, which can be used in the method of the present invention to create a new type of cotton with "zero" gossypol in seeds and unchanged plant glands and gossypol content.

[0307] Equivalent solutions

[0308] Although several embodiments have been described and shown herein, a person of ordinary skill in the art will readily imagine a variety of other alternatives for performing the functions and / or obtaining the results and / or obtaining one or more of the advantages described herein, and each of such variations and / or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, a person of ordinary skill in the art will readily understand that all parameters, dimensions, materials, etc. described herein are merely exemplary, and that the actual parameters, dimensions, materials will depend on one or more specific applications for which the inventive teachings are used. A person of ordinary skill in the art will recognize or be able to confirm many equivalents of the specific inventive embodiments described herein using only routine experimentation. Therefore, the foregoing embodiments are presented by way of example only, and within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced in a manner different from that specifically described and required.

[0309] It should be understood that, according to the claims and disclosure of the present invention, G12 and / or G13 proteins from various cotton sources can be used to construct mutant proteins, thereby creating new cotton plants with zero gossypol content in seeds and normal gossypol content in plants. Furthermore, the mutant types provided herein are merely exemplary, and other mutant types that can achieve the objectives of the present invention are also within the scope of the present invention.

[0310] It should also be understood that the method for creating the novel cotton of the present invention is not limited to the artificial construction method of the present invention. Various mutagenesis methods known in the art (e.g., physical mutagenesis, chemical mutagenesis) can be used to produce equivalent solutions having the same effect as the G12 / G13 mutant protein of the present invention or the combination of the G12 / G13 mutant protein of the present invention and a seed-specific promoter. Those skilled in the art will understand that these equivalent solutions are also encompassed within the scope of the present invention.

[0311] Sequence Listing

Claims

1. A method for inhibiting the formation of pigment glands in cotton seeds, the method comprising causing the cotton seeds to express a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has a reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

2. A method for reducing the cottonpol content in cotton seeds, the method comprising causing the cotton seeds to express a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has a reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

3. The method of claim 1 or 2, wherein the transcriptional activation function of the mutant tetramer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared to the wild-type tetramer.

4. The method according to any one of claims 1 to 3, comprising the step of introducing into said cotton a nucleotide sequence encoding said Gl2 / Gl3 protein mutant.

5. The method of claim 4, wherein the method comprises integrating the nucleotide sequence encoding the Gl2 / Gl3 protein mutant into the genome of the cotton.

6. The method of any one of claims 1 to 5, wherein compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions selected from amino acids 1-282 and 300-475; preferably, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions 20-202 or 300-313; More preferably, the Gl2 / Gl3 protein mutant has one or more mutations selected from the following: amino acid A at position 43 mutates to K (A43K), amino acids at positions 21-24 mutate to alanine, amino acids at positions 54-59 mutate to alanine, amino acids at positions 43-52 are deleted, amino acids at positions 72-79 are deleted, amino acids at positions 94-99 are deleted, amino acids at positions 121-126 are deleted, and amino acids at positions 300-313 are deleted.

7. The method of claim 6, wherein the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:3-10.

8. The method of any one of claims 1 to 5, wherein compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant is a truncated version of the N-terminus and / or C-terminus of the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant does not comprise a functional transcriptional activation domain, and the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence at positions 283-373 of the wild-type Gl2 / Gl3 protein; Preferably, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of positions 283-373 or the amino acid sequence of positions 141-475 of the wild-type Gl2 / Gl3 protein; More preferably, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:11 or SEQ ID NO:

12.

9. The method of any one of claims 1 to 8, wherein the nucleotide sequence encoding the Gl2 / Gl3 protein mutant is operably linked to a seed-specific promoter of cotton.

10. The method of claim 9, wherein the seed-specific promoter: (1) initiating expression of the Gl2 / Gl3 protein mutant at a time no later than the expression time of the Gl2 / Gl3 protein during seed development of wild-type cotton; (2) making the expression level of the Gl2 / Gl3 protein mutant no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and (3) The expression of the Gl2 / Gl3 protein mutant is initiated only during the development of cotton seeds, and the expression of the Gl2 / Gl3 protein mutant is not initiated or substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

11. The method of claim 9 or 10, wherein the seed-specific promoter is a promoter of the OLE1_D gene, the OLE1_A gene or the ALB2S gene; Preferably, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:13-15; more preferably, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:13 or SEQ ID NO:

14.

12. The method of any one of claims 1-11, wherein the method comprises integrating into the genome of cotton a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 21 or 23.

13. The method of any one of claims 1 to 12, comprising the steps of: (1) transforming the nucleotide sequence encoding the Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and (2) contacting cotton cells, seeds, tissues, organs or plants with the Agrobacterium carrying the nucleotide sequence in step (1), thereby introducing the nucleotide sequence into the cotton cells, seeds, tissues, organs or plants.

14. The method of any one of claims 1-13, wherein the method results in the absence of pigment gland development in the cotton seed, compared to pigment glands in wild-type cotton seeds, and the occurrence and development of pigment glands in the cotton plant is not affected.

15. The method of any one of claims 1-14, wherein the method reduces the gossypol content in the cotton seed by 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% compared to the gossypol content of wild-type cotton seed.

16. The method of any one of claims 1 to 15, wherein the method results in a cotton plant having a gossypol content in each tissue that is no different or substantially no different than the gossypol content in each tissue of a wild-type cotton plant.

17. Cotton cell, seed, tissue, organ or plant obtained by the method of any one of claims 1 to 16.

18. Cotton cells, seeds, tissues, organs or plants, comprising a nucleotide sequence encoding a Gl2 / Gl3 protein mutant, wherein the Gl2 / Gl3 protein mutant forms a mutant tetramer by itself or with a wild-type Gl2 / Gl3 protein, and the mutant tetramer has reduced or eliminated transcriptional activation function compared to the wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

19. The cotton cell, seed, tissue, organ or plant of claim 18, wherein the transcriptional activation function of the mutant tetramer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared to the wild-type tetramer.

20. The cotton cell, seed, tissue, organ or plant of claim 18 or 19, wherein the nucleotide sequence encoding the Gl2 / Gl3 protein mutant is integrated into the genome of the cotton cell, seed, tissue, organ or plant.

21. The cotton cell, seed, tissue, organ or plant of any one of claims 18 to 20, wherein compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions selected from amino acids 1-282 and 300-475; preferably, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions 20-202 or 300-313; More preferably, the Gl2 / Gl3 protein mutant has one or more mutations selected from the following: amino acid A at position 43 mutates to K (A43K), amino acids at positions 21-24 mutate to alanine, amino acids at positions 54-59 mutate to alanine, amino acids at positions 43-52 are deleted, amino acids at positions 72-79 are deleted, amino acids at positions 94-99 are deleted, amino acids at positions 121-126 are deleted, and amino acids at positions 300-313 are deleted.

22. The cotton cell, seed, tissue, organ or plant of claim 21, wherein the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NOs: 3-10.

23. The cotton cell, seed, tissue, organ or plant of any one of claims 18-20, wherein the Gl2 / Gl3 protein mutant is a truncated version of the N-terminus and / or C-terminus of the wild-type Gl2 / Gl3 protein compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant does not comprise a functional transcriptional activation domain, and the Gl2 / Gl3 protein mutant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of positions 283-373 of the wild-type Gl2 / Gl3 protein; Preferably, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of positions 283-373 or the amino acid sequence of positions 141-475 of the wild-type Gl2 / Gl3 protein; More preferably, the amino acid sequence of the Gl2 / Gl3 protein mutant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:11 or SEQ ID NO:

12.

24. The cotton cell, seed, tissue, organ or plant of any one of claims 18-23, wherein the nucleotide sequence encoding the Gl2 / Gl3 protein mutant is operably linked to a cotton seed-specific promoter.

25. The cotton cell, seed, tissue, organ or plant of claim 24, wherein the seed-specific promoter: (1) initiating expression of the Gl2 / Gl3 protein mutant at a time no later than the expression time of the Gl2 / Gl3 protein during seed development of the wild type; (2) making the expression level of the Gl2 / Gl3 protein mutant no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and (3) The expression of the Gl2 / Gl3 protein mutant is initiated only during the development of cotton seeds, and the expression of the Gl2 / Gl3 protein mutant is not initiated or substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

26. The cotton cell, seed, tissue, organ or plant of claim 24 or 25, wherein the seed-specific promoter is a promoter of the OLE1_D gene, the OLE1_A gene or the ALB2S gene; Preferably, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:13-15; more preferably, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:13 or SEQ ID NO:

14.

27. The cotton cell, seed, tissue, organ or plant of any one of claims 18-26, obtained by integrating a nucleotide sequence having at least 60%, at least 70%, 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 21 or 23 into the genome of cotton.

28. The cotton cell, seed, tissue, organ or plant of any one of claims 18 to 27, obtained by the following steps: (1) transforming the nucleotide sequence encoding the Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and (2) contacting cotton cells, seeds, tissues, organs or plants with the Agrobacterium carrying the nucleotide sequence in step (1), thereby obtaining the cotton cells, seeds, tissues, organs or plants.

29. A nucleic acid construct comprising, from 5' to 3', a cotton seed-specific promoter and a gene encoding a Gl2 / Gl3 protein mutant.

30. The nucleic acid construct of claim 29, wherein the seed-specific promoter is a promoter of the OLE1_D gene, the OLE1_A gene or the ALB2S gene; Preferably, the seed-specific promoter comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NOs: 13-15; More preferably, the seed-specific promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO:

14.

31. The nucleic acid construct of claim 29 or 30, comprising a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:21 or 23.

32. The nucleic acid construct of any one of claims 29 to 31 for use in inhibiting pigment gland formation in cotton seeds.

33. The nucleic acid construct of any one of claims 29 to 31 for use in reducing the content of gossypol in cotton seeds.

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