Method for improving pigment gland character and gossypol character of cotton seeds and application of method

Abstract

The invention relates to a method for improving pigment gland characters and gossypol characters of cotton seeds and application of the method. Aiming at a key gene Gl2 / Gl3 for determining the development of pigment glands in cotton, a cotton seed specific promoter is used for specifically inhibiting the expression quantity of the gene or the activity of an encoded protein of the gene in the seed development process, novel cotton with seeds free of pigment glands and normal plant pigment glands can be obtained, the gossypol content of the seeds is zero, and the cotton seed has a good application prospect. The gossypol content of other tissues is normal.

Description

Technical Field

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

[0002] Cotton is not only an important natural fiber crop in the world, but also an important oil crop and high-protein food crop. China grows about 50 million mu of cotton every year, producing more than 10 million tons of seeds, containing about 2 million tons of oil and 2 million tons of protein. However, the presence of a large amount of gossypol in cotton seeds greatly limits its application as a food raw material and feed.

[0003] Currently, both upland cotton plants and cotton seeds, which are widely cultivated around the world, contain a large amount 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 cotton glands. Cottonpol 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 its safety as a food raw material, 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 mainly uses phenol-free strains as parents to introduce low-phenol traits into other excellent strains. The low-phenol varieties currently selected include Xiangmian 18, Zhongmian 22, Lumian 12, Zhejiangmian 10, Jinmian 22, Wanmian 18, Giza 77, Giza 81, etc., and their seed gossypol contents are 0.0304%, 0.0051%, 0.008%, 0.006%, 0.002%, 0.00037%, 0.023%, and 0.021%, respectively. Except for Wanmian 18, the seed gossypol content of most varieties is lower than the World Health Organization standard of ≤600mg / kg, but the gossypol content in the seeds of these low-phenol cotton varieties is higher than the first-level standard of 50mg / kg for finished cottonseed oil in the latest national standard GB / T1537-2019 issued by my country, and there are still safety risks. More importantly, since the glandless varieties do not contain cottony pol or have extremely low cottony pol 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 used seed-specific promoters to drive the dsRNA of the CAD gene, specifically silenced the seed glandularization and cottonphenol synthesis genes, and created a new cotton germplasm with normal low-phenol seeds (Sunilkumar G, Campbell L AM, 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 genes Gl2 / Gl3 of cotton glandular 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 are still present 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. Summary of the invention

[0007] The present invention provides a new method for improving the pigment gland traits and gossypol traits of cotton seeds. The method can achieve cotton seeds without pigment glands and with zero gossypol content without affecting the pigment glands and gossypol content of cotton plants. The cotton plants created by the method can normally resist pests and diseases, and the produced seeds are significantly better than "low-phenol cotton" in terms of food raw material safety because they do not contain gossypol. The new cotton has the advantages of pigment-free gland cotton and pigmented gland cotton, which does not reduce the ability of the plant to resist pests and diseases, and can completely omit the dephenolization process in the process of 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 planting.

[0008] 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 a reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

[0009] At the same time, the present invention also provides 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.

[0010] 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 only wild-type Gl2 / Gl3 proteins.

[0011] In some embodiments, the method comprises the step of introducing a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into cotton. In some embodiments, the method comprises integrating a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into the genome of cotton.

[0012] In some embodiments, compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant of the present invention has one or more amino acid substitutions, insertions and / or deletions at positions selected from the 1st-282nd and 374th-475th amino acids. In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions of the 20th-202nd or 300th-313th amino acids. In some preferred embodiments, the Gl2 / Gl3 protein mutant has one or more mutations selected from the following: the 43rd amino acid A mutates to K (A43K), the 21st-24th mutates to alanine, the 54th-59th amino acids mutate to alanine, the 43rd-52nd amino acid is deleted, the 72nd-79th amino acid is deleted, the 94th-99th amino acid is deleted, the 121st-126th amino acid is deleted, and the 300th-313th amino acid is deleted. 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 with SEQ ID NO:3-10.

[0013] 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 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.

[0014] In some embodiments, the nucleotide sequence encoding the G12 / G13 protein mutant is operably linked to a cotton seed-specific promoter.

[0015] The seed-specific promoter suitable for the method of the present invention has the following characteristics:

[0016] (1) initiating expression of the Gl2 / Gl3 protein mutant at a time no later than that of the Gl2 / Gl3 protein during seed development of wild-type cotton;

[0017] (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

[0018] (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 is substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

[0019] 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 NO: 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.

[0020] In some embodiments, the methods of the present invention include 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.

[0021] In some embodiments, the methods of the present invention include the following steps:

[0022] (1) transforming a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0023] (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.

[0024] In some embodiments, the methods of the invention result in the agenesis of pigment glands in cotton seeds, as compared to pigment glands in wild-type cotton seeds, and the development of pigment glands in cotton plants is not affected.

[0025] 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.

[0026] In some embodiments, the methods of the invention result in cotton plant tissues having gossypol levels that are no different or substantially no different than the gossypol levels of tissues in wild-type cotton plants.

[0027] 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.

[0028] In a third aspect, the present invention provides cotton cells, seeds, tissues, organs or plants, which contain 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.

[0029] 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 only wild-type Gl2 / Gl3 proteins.

[0030] 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.

[0031] In some embodiments, compared to wild-type Gl2 / Gl3, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions selected from the 1st-282nd and 374th-475th amino acids. In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at positions of the 20th-202nd or 300th-313th amino acids. In some preferred embodiments, the Gl2 / Gl3 protein mutant has one or more mutations selected from the following: the 43rd amino acid A mutates to K (A43K), the 21st-24th mutates to alanine, the 54th-59th amino acids mutate to alanine, the 43rd-52nd amino acid deletion, the 72nd-79th amino acid deletion, the 94th-99th amino acid deletion, the 121st-126th amino acid deletion, the 300th-313th amino acid deletion. 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 with SEQ ID NO: 3-10.

[0032] In some embodiments, 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 protein, the Gl2 / Gl3 protein mutant does not contain a functional transcriptional activation domain, and the Gl2 / Gl3 protein mutant comprises an amino acid sequence with 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 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 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.

[0033] 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.

[0034] The seed-specific promoter in the cotton cell, seed, tissue, organ or plant of the present invention has the following characteristics:

[0035] (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;

[0036] (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

[0037] (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 is substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

[0038] 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 NO: 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.

[0039] 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.

[0040] In some embodiments, the cotton cell, seed, tissue, organ or plant of the present invention is obtained by the following steps:

[0041] (1) transforming a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0042] (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.

[0043] 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.

[0044] In some embodiments, the seed-specific promoter in the nucleic acid construct of the present invention 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 with SEQ ID NO: 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 with SEQ ID NO: 13 or SEQ ID NO: 14.

[0045] 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.

[0046] In a fifth aspect, the present invention provides a nucleic acid construct according to the fourth aspect of the present invention, wherein the nucleic acid construct is used to inhibit the formation of pigment glands in cotton seeds.

[0047] In a sixth aspect, the present invention provides a nucleic acid construct according to the fourth aspect of the present invention, wherein the nucleic acid construct is used to reduce the content of cottonpol in cotton seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The development process of cotton pigment glands is shown. Figure 1 Figure a shows the pseudo-chronological process of glandular development in cotton immature embryos. Figure 1 Middle b shows the process of gland morphology construction of cotyledon of upland cotton immature embryo. Figure 1 Center c shows the development process of cotton immature embryo cotyledons.

[0049] Figure 2 The results of measuring the cottonpol content in cotton immature embryos at different developmental stages are shown.

[0050] Figure 3 The gland numbers and gossypol contents of plants with different Gl2 / Gl3 genotypes are shown. Figure 3Middle a shows the true leaf phenotype of plants with different Gl2 / Gl3 genotypes. Figure 3 Middle (b) shows the cottonpol content in different tissues of the Gl2 / Gl3 loss-of-function mutant (gl2gl2gl3gl3). Figure 3 Middle c shows the statistical results of the number of glands in the second true leaf of plants with different Gl2 / Gl3 genotypes after germination. Figure 3 Middle (d) shows the results of measuring the cottonpol content in the second true leaf of plants with different Gl2 / Gl3 genotypes after germination.

[0051] Figure 4 It was shown that secondary metabolites such as cottonpol were synthesized and accumulated in situ in the pigment glands. Figure 4 Middle a is a schematic diagram of non-glandular cell sampling. Figure 4 Middle b shows the results of determination of cottonypol content in immature embryos and non-glandular cells. Figure 4 Middle c shows the transcriptome expression profile of cottonpol biosynthesis pathway genes in glandular cells of TM-1 immature embryos, leaves, etc., as well as glandular mutant strains. Figure 4 D in the figure shows the GUS staining results of the glands of young embryos, leaves and roots of the transgenic lines after the promoter of the key gene CDNC in the cottonpol biosynthesis pathway was fused with the GUS gene and introduced into the plants.

[0052] Figure 5 Shown are the expression levels of Gl2 / Gl3 genes in glandular and non-glandular tissues.

[0053] Figure 6 It is shown that Gl2 / Gl3 proteins regulate the occurrence and development of glands by forming homotetramers. Figure 6 A shows a schematic diagram of the predicted structure of the Gl2 protein. Figure 6 Figure b shows the results of the Gl2 / Gl3 bimolecular fluorescence complementation experiment, proving that the Gl2 / Gl3 protein molecule can interact with itself. Figure 6 Figure c shows the results of analyzing Gl2 protein multimers using size exclusion chromatography. Figure 6 Middle (d) shows the structural model of the Gl2 protein homotetramer.

[0054] Figure 7 Demonstrated is the mining of Gl2 binding sites using ChIP-seq. Figure 7 Figure a is a Venn diagram of two ChIP-seq results for Gl2 protein, showing the overlap of the two ChIP-seq results. Figure 7 Middle b shows the distribution of Gl2 protein binding regions throughout the genome. Figure 7 Middle (c) shows the distribution of Gl2 binding regions near the gene start codon, near the stop codon and on the gene. Figure 7 Middle (d) shows the DNA motif bound by Gl2 protein predicted by MEME-ChIP. Figure 7 (e) is a Venn diagram of Gl2-bound genes and transcriptome differentially expressed gene analysis (DEG).

[0055] Figure 8 It shows that the artificial mutant protein mute9 has the ability to form tetramers and bind to DNA molecules. Figure 8 The results of the bimolecular fluorescence complementation experiment detecting the interaction between Gl2 protein and mute9 mutant protein are shown in FIG. Figure 8 Middle b shows the ability of Gl2 protein or mute9 mutant protein to bind to target DNA detected by EMSA experiment, confirming that the mutant protein can still bind to the target DNA molecule (E-box).

[0056] Fig. 9 The transcriptional activation abilities of wild-type Gl2 protein and artificial mutant proteins are shown.

[0057] Fig.10 The expression levels of candidate promoter genes in different tissues of TM-1 are shown.

[0058] Fig.11 The gene expression levels of seed-specifically expressed genes Ghα-globulin, GhOLE1_D, GhALB2S and Gl2+Gl3 genes during seed development are shown.

[0059] Fig.12 A schematic diagram of a vector containing an expression cassette of the proGhOLE1_D-mute9 mutant gene is shown.

[0060] Fig.13 The glandular phenotype and gossypol content of the novel cotton seeds are shown.

[0061] Fig.14 The plant gland phenotype of the novel cotton seeds is shown.

[0062] Fig.15 A novel cotton material fiber phenotype is shown.

[0063] Fig.16 The gossypol content of each tissue of the new cotton is shown.

[0064] Fig.17 A schematic diagram of a vector containing an expression cassette of the proGhALB2S-mute4 mutant gene is shown.

[0065] Fig.18 Shown is an amino acid sequence comparison of wild-type Gl2 / Gl3 proteins from different cotton origins. DETAILED DESCRIPTION

[0066] 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.

[0067] In order to more easily understand the present disclosure, certain terms are first defined below. The following terms and additional definitions of other terms are set forth throughout the specification.

[0068] 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.

[0069] As used herein, the terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (ie, meaning "including, but not limited to, ").

[0070] 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.

[0071] 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, as appropriate, or by variations of + / -15%, 10%, 5%, 2%. 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. When referring to measurable values ​​such as amounts or concentrations, the term "about" as used herein is meant to include variations within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0072] 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 derived nucleotide bases.

[0073] 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. The term "amino acid" as used herein refers to natural and / or non-natural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0074] When the term "encoding" is applied to a nucleotide sequence, it 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, which is said to be "encoding" RNA or "encoding" a polypeptide. The antisense strand is the complement of such nucleic acid, and the coding sequence can be deduced therefrom.

[0075] 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 described herein also includes splicing, modification, processing, etc. of mRNA molecules.

[0076] "Vector" refers to a delivery vehicle or element of genetic material. In this article, the term "vector" refers to one or a series of macromolecules comprising polynucleotides, which 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 sequences of certain proteins or polypeptides that can enhance, suppress, weaken, protect, trigger or prevent certain biological functions and physiological functions (such as the nucleotide sequences encoding the Gl2 / Gl3 protein mutants of the present invention), the coding sequences of RNAi components (for example, shRNA, siRNA, microRNA, antisense oligonucleotides), or optional markers. In the vector, the coding sequences of proteins, polypeptides, RNAi components, etc. are usually operably connected to expression control elements, such as promoters. In some embodiments, the vector can be a linear DNA molecule. In some embodiments, the vector can be a circular DNA molecule.

[0077] 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 target protein, such that transcription of the nucleic acid sequence encoding the target protein is regulated by the promoter, the promoter is said to be "operably linked" to the nucleic acid sequence encoding the target protein.

[0078] As used herein, the term "specific promoter" refers to a promoter that is capable of directing the expression of a target gene 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 its downstream gene to be specifically expressed in the seed (cottonseed) of cotton. Typically, if the mRNA level in a specific cell, tissue, or organ is at least 5 times higher than in other cells, tissues, or organs, preferably at least 10 times higher, more preferably at least 100 times higher, and most preferably at least 1000 times higher, 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.

[0079] 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 the positions in each sequence, which can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, 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. By importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / tools-bin / clustalw / ) and using ClustalW, the alignment and percent sequence identity of the nucleic acid or amino acid sequences provided herein can be determined.

[0080] 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 with a reference polypeptide (e.g., a Gl2 / Gl3 protein mutant described in the present invention); 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 with a reference polynucleotide (e.g., a polynucleotide encoding a Gl2 / Gl3 protein mutant described in the present invention).

[0081] Compared with the reference polypeptide of the present invention (such as the Gl2 / Gl3 protein mutant of the present invention), equivalents with one or more amino acid modifications are also included in the scope of the present invention, provided that the one or more amino acid modifications do not affect or substantially affect the function of the reference polypeptide. As used herein, amino acid modifications can be amino acid substitutions, amino acid deletions or amino acid insertions. Amino acid substitutions can 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 (such as charge, hydrophobicity or size). As used herein, "conservative substitutions" refer to amino acid residues that are replaced by another biologically similar residue. Examples of conservative substitutions include one hydrophobic residue such as isoleucine, valine, leucine or methionine replacing another; or one charged or polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, etc. 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.

[0082] Cotton pigment glands are a trait unique to the genus Gossypium and its closely related species, also known as cottonpol glands. The dark brown dots visible to the naked eye on the plants and seeds of cotton plants are glands. Glands are only observed in tissues such as anthers, seed coats, and fibers. Under a microscope, the glands are cavities composed of multiple cells. The central cavity is formed by the lysis of multiple cells, and the outside is composed of multiple layers of flat cells. The glands of cotyledons or leaves occur under the palisade tissue, the glands of new stems occur in the hypodermis, and the glands in the roots are distributed in the parenchyma near the cambium. The central cavity of the mature gland contains a large amount of terpene secondary metabolites such as gossypol, as well as components of the residual cell wall. The gossypol content can reach 35-50% of the total weight of the pigment gland.

[0083] Upland cotton (Gossypium hirsutum) is one of the most widely cultivated cotton species in the world, with an allotetraploid AD genome. The synthesis and accumulation of cottonphenol involves the regulation of multiple genes in cotton, among which Gl2 / Gl3 are the most important genes for controlling glandular formation, encoding Gl2 / Gl3 proteins respectively, and participating in the control of glandular formation in various tissues of cotton, while other genes can only affect the glandular formation of limited tissues, or change the number and morphology of glands. The Gl2 gene is located on the A subgenome of upland cotton, and the Gl3 gene is an allele located on the D subgenome. The Gl2 / Gl3 proteins encoded by the two genes have only two amino acid differences.

[0084] As used herein, the terms "Gl2 / Gl3" and "Gl2 and / or Gl3" can be used interchangeably to represent any one or both of the Gl2 protein and the Gl3 protein or the Gl2 gene and the Gl3 gene. It should be understood that due to the high similarity in amino acid sequence and function between the Gl2 and Gl3 proteins, the mutation mode of the protein mutant designed for the Gl2 protein in the present invention is also applicable to the Gl3 protein. It should also be understood that the technical solutions related to the Gl2 protein mutants involved in the present invention are also applicable to the Gl3 protein mutants, and those skilled in the art can understand that similar technical effects can be obtained by implementing the technical solutions described in the present invention using the Gl2 protein mutants and / or the Gl3 protein mutants.

[0085] The term "dominant negative effect" refers to the fact that certain proteins, after mutation, can inhibit or block the function or action of wild-type proteins in the same cell. Accordingly, the mutant protein obtained is a "dominant negative mutation" or a "dominant negative mutant". For example, certain proteins function in the form of polymers in cells. When a "dominant negative mutation" exists in the cell at the same time, the mutant protein can form a polymer with the wild-type protein, thereby affecting the normal function of the polymer.

[0086] When referring to the 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 20th to 202nd amino acids 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 20th to 202nd amino acid position relative to SEQ ID NO: 1, so that the Gl2 protein mutant does not have or substantially does not have a transcriptional activation function.

[0087] The term "mutant tetramer" means that part of the Gl2 / Gl3 proteins in the tetramer are Gl2 / Gl3 protein mutants or all Gl2 / Gl3 proteins are Gl2 / Gl3 protein mutants. When referring to the mutant tetramer formed by the Gl2 / Gl3 protein mutant itself or with the wild-type Gl2 / Gl3 protein, the term "reduced transcriptional activation ability" means that compared with the wild-type tetramer formed by the wild-type Gl2 / Gl3 protein, 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%; the term "eliminated transcriptional activation ability" means that compared with the control group without the wild-type tetramer, the mutant tetramer causes the expression level of the target gene to be lower than the control group or not higher than 10%, 15%, 20%, 25% or 30% of the control group.

[0088] Method for inhibiting the formation of pigment glands in cotton seeds and method for reducing the cottonpol content in cotton seeds

[0089] 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 a reduced or eliminated transcriptional activation function compared to a wild-type tetramer formed only by the wild-type Gl2 / Gl3 protein.

[0090] The present invention provides 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.

[0091] 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 only wild-type Gl2 / Gl3 proteins.

[0092] The wild-type Gl2 / Gl3 protein can form a wild-type tetramer, which can bind to the target DNA to activate the expression of downstream genes. Therefore, the nucleotide sequence containing the target DNA (e.g., the promoter of the downstream target gene of the wild-type tetramer) can be fused with the reporter gene, and the expression level of the reporter gene can be detected in the presence of the wild-type tetramer or the mutant tetramer, thereby obtaining the transcriptional activation function of the mutant tetramer.

[0093] In the method of the present invention, the method for making the cotton seeds express the Gl2 / Gl3 protein mutant comprises:

[0094] 1) introducing the nucleotide sequence encoding the Gl2 / Gl3 protein mutant into cotton; or

[0095] 2) Obtaining cotton whose seeds are capable of expressing the Gl2 / Gl3 protein mutant by a mutagenesis method.

[0096] In some embodiments, the method of the present invention comprises the step of introducing a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into cotton. In some embodiments, the method of the present invention comprises integrating a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into the genome of cotton.

[0097] Methods for introducing a target gene (e.g., a nucleotide sequence encoding a G12 / G13 protein mutant) into a plant cell (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, using the Agrobacterium-mediated stem tip transformation method.

[0098] The Gl2 / Gl3 protein mutant 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. Wild-type Gl2 / Gl3 proteins can be natural Gl2 / Gl3 proteins derived from upland cotton, Asian cotton, grass cotton and / or sea island cotton. In some embodiments, wild-type Gl2 / Gl3 proteins can be natural Gl2 / Gl3 proteins derived from the following species: upland cotton (Gossypium hirsutum), Asian cotton (Gossypium arboretum), abnormal cotton (Gossypium anomalum), Sterti cotton (Gossypium sturtianum), Raymond cotton (Gossypium raimondii), Stoke cotton (Gossypium stocksii), long calyx cotton (Gossypium longicalyx), Bick cotton (Gossypium bickii), round-leaf cotton (Gossypium rotundifolium), sea island cotton (Gossypium barbadense).

[0099] In some embodiments, compared to the wild-type Gl2 / Gl3 protein, the Gl2 / Gl3 protein mutant of the present invention has one or more amino acid substitutions, insertions and / or deletions at positions selected from amino acids 1-282 and 300-475. In some embodiments, the Gl2 / Gl3 protein mutant of the present invention has 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.

[0100] In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at the positions of amino acids 20-202. In some embodiments, the Gl2 / Gl3 protein mutant has one or more amino acid substitutions, insertions and / or deletions at the positions of amino acids 20-282 corresponding to SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0101] 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.

[0102] In some preferred embodiments, 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, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0103] 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 with SEQ ID NO: 3-10.

[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:6.

[0105] In some most preferred embodiments, the Gl2 / Gl3 protein mutant has or consists of the amino acid sequence shown in SEQ ID NO:6.

[0106] 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 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 corresponding to SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0107] 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.

[0108] 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 with 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 with SEQ ID NO: 11 or SEQ ID NO: 12. In some most preferred embodiments, the Gl2 / Gl3 protein mutant has the amino acid sequence shown in SEQ ID NO: 11 or especially consists of.

[0109] In some embodiments, the nucleotide sequence encoding the G12 / G13 protein mutant is operably linked to a cotton seed-specific promoter.

[0110] The seed-specific promoter suitable for the method of the present invention has the following characteristics:

[0111] (1) initiating expression of the Gl2 / Gl3 protein mutant at a time no later than that of the Gl2 / Gl3 protein during seed development of wild-type cotton;

[0112] (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

[0113] (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 is substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

[0114] 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 sea island cotton.

[0115] 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. 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.

[0116] In some embodiments, the methods of the present invention include 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.

[0117] In some embodiments, the methods of the present invention include the following steps:

[0118] (1) transforming a nucleotide sequence encoding a Gl2 / Gl3 protein mutant into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0119] (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.

[0120] In some embodiments, the methods of the invention result in the agenesis of pigment glands in cotton seeds, as compared to pigment glands in wild-type cotton seeds, and the development of pigment glands in cotton plants is not affected.

[0121] In some embodiments, the method of the present invention reduces the cottonpol content in the cotton seeds by 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% compared to the cottonpol content in wild-type cotton seeds. The cottonpol content in the cotton seeds can be determined, for example, by high performance liquid chromatography.

[0122] In some embodiments, the methods of the invention result in cotton plant tissues having gossypol levels that are no different or substantially no different than the gossypol levels of tissues in wild-type cotton plants.

[0123] The significant advantages of the method of the present invention are:

[0124] (1) It not only achieves "zero" cottonpol in cotton seeds, but also removes other secondary metabolites synthesized by cotton seed glands that may affect food safety;

[0125] (2) The development of glands in tissues other than seeds should be completely maintained in the same manner as in wild-type materials, thereby ensuring that the cotton strains produced have no significant differences in disease and insect pest resistance from currently promoted cotton varieties;

[0126] (3) It does not affect the cotton fiber yield and quality of the produced cotton varieties.

[0127] In order to make the purpose, technical scheme and advantages of the present invention clearer, 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.

[0128] Example

[0129] Experimental methods

[0130] (I) Shoot tip transformation method

[0131] 1. Seed germination

[0132] 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.

[0133] 2. Agrobacterium Preparation

[0134] 3 days before infection, use a disposable inoculation loop to dip the target glycerol bacteria, evenly spread it on the YEB medium containing the corresponding antibiotics, seal the edge of the bacterial plate with a sealing film, and invert it in a 28℃ constant temperature box for 2 days to obtain activated Agrobacterium; expand the activated Agrobacterium at 28℃, measure A660 with a spectrophotometer to about 0.9, and set aside.

[0135] 3. Infection and Co-cultivation

[0136] After the seed culture is completed, the seed coat is cut with a scalpel, and the cotyledons are peeled off under a dissecting microscope to expose the embryo tip, and the embryo tip and hypocotyl are retained as explants. The explants are placed in the prepared Agrobacterium suspension, ultrasonicated at 40KHz for 40s, and then shaken at a constant speed for 50min. After infection, they are placed in a co-culture medium covered with double-layer sterile filter paper and co-cultured in a 23℃ constant temperature incubator in the dark for 3-4 days.

[0137] 4. Resume Culture

[0138] After 3-4 days of co-cultivation, the explants were transferred to the selection 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 (16 / 8h, light / dark).

[0139] 5. Bud induction culture and resistant bud screening

[0140] After culturing on the recovery medium for 7 days, the slender roots of the cotton explants were cut off and then transferred to the shoot induction medium and subcultured every 2 weeks at 25°C and a photoperiod of 16 / 8h, light / darkness, for a total of 3-4 times until resistant shoots were obtained.

[0141] 6. Rooting culture

[0142] After the resistant buds are fully developed, the explants are transferred to the elongation medium and treated in the same way as the screening. They are cultured at 28°C and under the conditions of a photoperiod (16 / 8h, light / dark). Subculture is performed every 2 weeks, and when the resistant buds are elongated > 3cm, the number of roots is > 2, and the root length is more than 2cm, they are transplanted to the seedling hardening room for testing and hardening.

[0143] Solution formula:

[0144] CA salt: magnesium sulfate 10g / L, ammonium sulfate 5.36g / L, sodium phosphite hydrate 6g / L, calcium chloride 6g / L, boric acid 30mg / L, manganese sulfate 100mg / L, zinc sulfate heptahydrate 20mg / L, potassium iodide 7.5mg / L, sodium molybdate dihydrate 2.5mg / L, copper sulfate 250μg / L, cobalt (II) chloride hexahydrate 250μg / L, potassium nitrate 100g / L;

[0145] 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;

[0146] 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;

[0147] Recovery 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, 4 g / L agar, pH = 5.6;

[0148] Bud induction and screening 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, 6-BA at a concentration of 1 mg / L, NAA at a concentration of 0.1 mg / L, carbenicillin at a concentration of 100 mg / L, cephalosporin at a concentration of 100 mg / L, kanamycin at a concentration of 100 mg / L, 4 g / L agar, pH = 5.6;

[0149] 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.

[0150] (2) Western Blot

[0151] 1. Tissue Protein Extraction

[0152] Take 1g of fresh embryonic tissue or embryonic tissue stored at -80℃, grind it into powder with liquid nitrogen, take out PBS stored in a 4℃ refrigerator, add 1ml PBS to the EP tube, shake at 4℃ for 30min. Precool the centrifuge, centrifuge the fully lysed tissue fluid at 4℃ and 12000rpm for 10 minutes, and take the supernatant. Add protein loading buffer and boil at 95℃ for 10min;

[0153] 2. Glue making

[0154] The present invention uses the YAZY PAGE gel rapid preparation kit, taking a 1.0 mm gel as an example, taking equal volumes of lower gel solution and lower gel buffer, 2.7 mL each, and mixing;

[0155] Add 60 μL of improved coagulant to the above mixed solution and mix well;

[0156] Pour the mixed solution into the glue-making glass plate, so that the distance between the liquid surface and the upper edge of the short glass plate is 0.5 cm longer than the comb teeth, and add an appropriate amount of 100% ethanol to cover the lower layer of glue;

[0157] After the lower layer of gel solidifies, pour off the upper layer of ethanol;

[0158] Take equal volumes of upper gel solution and colored upper gel buffer, 0.75 mL each, and mix well;

[0159] Add 15 μL of the improved coagulant to the above mixed solution and mix well;

[0160] Pour the mixed solution into a glass plate and insert the comb teeth;

[0161] After the upper layer of gel solidifies, remove the comb teeth and use it for electrophoresis.

[0162] 3. Electrophoresis: Inject the denatured protein in step 1 into the gel wells, constant voltage 90V, 90min;

[0163] 4. Transfer: Use Trans- The strips were transferred to 0.45 μm PVDF using a semi-dry transfer system, 25 V, 125 mA, 60 min;

[0164] 5. Blocking: The PVDF membrane was placed in 5% skim milk (prepared in TBST) for 60 min;

[0165] 6. Primary antibody incubation: After blocking, wash with TBST for 3 times, 5 min each time; then add 18 mL TBST, 2 mL 5% skim milk, 1.5 μL primary antibody (full gold, HT201-01), shake at 4°C overnight;

[0166] 7. Secondary antibody incubation: After the primary antibody incubation is completed, wash with TBST for 3 times, 5 minutes each time; then add 18mL TBST, 2mL 5% skim milk, 1.5μL secondary antibody (full gold, HS201-01) at room temperature for 60 minutes;

[0167] 8. Chemical exposure: After the secondary antibody incubation, wash with TBST three times, 5 minutes each time; use M5 Hiper ECLWestern HRP Substrate for exposure and observation.

[0168] (III) Chromatin immunoprecipitation sequencing (ChIP-seq)

[0169] 1. Collect fresh tissue samples in 50 mL Falcon tubes;

[0170] 2. Add 37 mL of crosslinking buffer to each sample and crosslink under vacuum at room temperature for 10 minutes;

[0171] 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;

[0172] 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 flash freezing in liquid nitrogen;

[0173] 5. Take 1-2g of the cross-linked sample and grind it for about 30 minutes until it becomes fine powder. Place the powder sample in a 50ml enzyme-free centrifuge tube and store it in liquid nitrogen until all the samples are ground;

[0174] 6. Add 40 mL of nuclear buffer and shake at 4°C for about 30 minutes;

[0175] 7. Double-layer non-woven fabric filtration, mark the center position with a blue gun tip, slowly add filtration with a pipette, and squeeze the filter cloth until it is clean after filtration;

[0176] 8. Centrifuge at 4°C, 1500 rpm, 15 min;

[0177] 9. Remove the supernatant and resuspend the nuclei with 5-10 mL of nuclear buffer to 40 mL;

[0178] Centrifuge at 10.4°C, 1500 rpm, for 15 min, remove the supernatant, and repeat 1-2 times (depending on the turbidity of the sample);

[0179] 11. Add 750 μL / time nuclear buffer to resuspend, total volume 1-2 mL, centrifuge at 4°C, 1500 rpm, 15 min;

[0180] 12. Remove the supernatant (use a 1mL blue pipette tip covered with a white pipette tip to absorb the supernatant);

[0181] 13. Add 700 μL of cell nuclear lysis buffer to resuspend and dispense into glass EP tubes (no more than 300 μL per tube);

[0182] Ultrasonication at 14.4℃ for 20 min, divided into two times, 10 min each time (maximum power, working for 30 s, stopping for 30 s);

[0183] Centrifuge at 15.4℃, 20000rpm, 15min, take the supernatant, if it is turbid, repeat the centrifugation and take the supernatant;

[0184] 16. Take 1 / 10 of the sample as input and dilute the rest of the sample for IP treatment. Dilute 5-10 times with ChIP dilution buffer;

[0185] 17. Remove impurities, aspirate magnetic beads (5 μL per tube sample), place on a magnetic stand, remove storage solution, and wash once with ChIP dilution buffer;

[0186] 18. Add magnetic beads, 5 μL per tube, shake well at 4°C for about 1 hour;

[0187] 19. Place on a magnetic rack and collect the supernatant;

[0188] 20. Add 5 μL of primary antibody to each tube supernatant and shake overnight at 4°C;

[0189] 21. Take the magnetic beads, 50 μL per tube of sample, wash once with ChIP dilution buffer, and resuspend to the required volume;

[0190] 22. Add 50 μL magnetic beads to each tube of sample and shake at 4°C for about 1.5 hours;

[0191] 23. Centrifuge to collect the liquid on the tube wall, place it on a magnetic rack, remove the supernatant, and retain the magnetic beads;

[0192] 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 min, and collect the supernatant into a new centrifuge tube;

[0193] 25. Add 250 μL elution buffer, shake at 65°C for 15 min, and combine the supernatants;

[0194] 26. Add 20 μL of 5 M NaCl and shake at 65°C for 8 hours;

[0195] 27. Add 10 μL EDTA, 20 μL Tris-HCl, 1 μL proteinase K, and 10 μL RNase A to each tube. Shake at 45°C, 700 rpm for 1.5 h.

[0196] 28. Add 550 μL of 25:24:1 DNA extraction solution and shake well. Centrifuge at 20,000 rpm, 4°C for 15 min. Take 450 μL of the supernatant and add 2.5 times the volume of anhydrous ethanol, 45 μL of NaAC, and 2-4 μL of glycogen to assist in precipitating DNA. Store at -80°C overnight;

[0197] Centrifuge at 29.4°C, wash twice with 75% alcohol, air-dry, and dissolve in enzyme-free water.

[0198] Reagent formula:

[0199] Cross-linking buffer: 0.4 M sucrose, 10 mM Tris-HCl pH = 8, 1 mM PMSF, 1 mM EDTA, 1% formaldehyde;

[0200] 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;

[0201] 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;

[0202] ChIP dilution buffer: 167 mM NaCl, 16.7 mM Tris-HCl pH = 8, 1.2 mM EDTA, 1.1% Triton X-100;

[0203] Elution buffer: 0.5% SDS, 0.1 M NaHCO3;

[0204] Low salt wash buffer: 150 mM NaCl, 20 mM Tris-HCl pH 8, 0.2% SDS, 0.5% Triton X-100, 2 mM EDTA;

[0205] High salt wash buffer: 500 mM NaCl, 20 mM Tris-HCl pH 8, 0.2% SDS, 0.5% Triton X-100, 2 mM EDTA;

[0206] LiCl wash buffer: 0.25 M LiCl, 1% sodium deoxycholate, 10 mM Tris–HCl pH 8, 1% NP-40, 1 mM EDTA;

[0207] TE buffer: 1 mM EDTA, 10 mM Tris-HCl pH=8.

[0208] The ChIP-DNA was sent to Wuhan Kangce Technology Co., Ltd. for library construction and sequencing analysis.

[0209] (IV) Bimolecular fluorescence complementation (BIFC)

[0210] 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.

[0211] 2. Pick up a single clone of Agrobacterium containing the target vector and place it into 1 ml LB liquid medium containing spectinomycin, and culture it at 28°C and 220 rpm for 1 day.

[0212] 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.

[0213] 4. Under room temperature, centrifuge at 8000 rpm for 5 min to collect the cells, and suspend the Agrobacterium cells in infection solution (containing 10 mM MgCl2, 10 mM MES, 150 μL acetosyringone, pH=5.6) to OD600=0.8. Let stand at room temperature for 2h-3h.

[0214] 5. Combine two bacterial cells containing different plasmids in equal volumes, use a needle to gently make a small hole on the back of the tobacco leaf (be careful not to pierce it), then use a needle without the needle to absorb the bacterial liquid and inject it into the leaf from the wound. Use a marker to mark the water-stained area of ​​the tobacco leaf.

[0215] 6. After the injected plants are cultured at about 23℃ for 2 days, observe whether the area where the Agrobacterium is injected has fluorescence. Cut a small piece of leaf with scissors, make the surface as flat as possible, place the reverse side on a glass slide, drop a drop of water on it, cover it with a coverslip, press gently, and then use a laser confocal microscope to observe the fluorescence imaging of YFP.

[0216] (V) Luciferase reporter (LUC) assay

[0217] 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.

[0218] 2. Preparation of infection solution: Activate the Agrobacterium culture containing the target plasmid and the empty vector until the final OD600 is about 0.6-0.8, collect the bacteria by centrifugation, wash the bacteria once with ddH2O, and finally resuspend the strain with infection buffer. Mix the bacterial solutions in a 1:1 ratio according to the purpose and let them stand in the dark for 3 hours;

[0219] Table 1. Infection solution system

[0220]

[0221]

[0222] 3. Injection of tobacco: Select healthy tobacco plants, use a disposable medical syringe to inject the lower epidermal cells of the tobacco plants, and culture them in the dark for 2 days;

[0223] 4. Fluorescence intensity and enzyme activity detection: Use Dual-Luciferase Reporter Assay System (Full-Form Gold, FR201-01-V2) to quantitatively detect luciferase activity.

[0224] (VI) Electrophoretic mobility shift assay (EMSA)

[0225] 1. Construction of prokaryotic expression vector

[0226] Gl2 and mute9 were amplified using upland cotton TM-1 cDNA as a template and connected to the pCold vector to construct a protein inducible vector with a HIS tag. The plasmid was transformed into the expression strain BL21.

[0227] 2. Protein Induction

[0228] Pick the monoclonal strain containing the target vector to 50mL LB liquid medium containing ampicillin resistance, culture at 37℃ for 2-3h, transfer 50ml of the bacterial solution to 4L LB medium, culture at 37℃ and 220rpm with shaking until OD=0.4-0.6, add IPTG to a final concentration of 200μM, induce overnight at 16℃ and 180rpm for 18h, centrifuge at 11000rpm for 5min to enrich the bacteria in a 50ml centrifuge tube, discard the supernatant, resuspend the precipitate with 40ml HIS binding buffer, use a contact ultrasonicator to sonicate for 5s, stop for 10s as one cycle, and sonicate for a total of 20 cycles at a power of 200W, and centrifuge at 4℃ and 11000rpm for 90min.

[0229] Add 750μL beads (Ni-NTA Agarose, Full Gold, DP101-02) to the column (Full Gold, affinity chromatography column, DS0130), wash 5 times with HIS binding buffer, filter the supernatant from the previous step through a 0.45μm filter membrane, add it to the column, seal the upper and lower parts of the column with sealing film, and rotate at 4℃ overnight for binding. The supernatant in the column flows out from the bottom, which is the flow-through liquid. Collect it in a new tube and wash it 10 times with HIS washing buffer, using 1mL each time. Elute 8 times with HIS elution buffer, 500μL each time, and collect it in a 1.5mL 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 12000 rpm for 5 min. The supernatant was aspirated and gel run was performed 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 higher concentrations in the eluate. The protein was stored in a -80°C refrigerator for later use.

[0230] 3. Annealing of biotin-labeled probe

[0231] According to the ChIP-seq results, a binding motif segment of about 50 bp of Gl2 / Gl3 in the promoter region of Gh_D11G107900 (GhMYC4) 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 for natural cooling and annealing to synthesize double-stranded probes.

[0232] 4. Protein and probe incubation

[0233] Prepare the reaction system in a 1.5 ml centrifuge tube according to Table 3.

[0234] Table 2. Protein and probe incubation binding reaction system

[0235]

[0236] Mix by pipetting, react at 25°C for 20 min, add 5 μL 5× loading buffer to each reaction, mix by pipetting, and terminate the reaction.

[0237] 5. Non-denaturing polyacrylamide gel electrophoresis

[0238] Prepare 5% non-denaturing PAGE gel (ddH2O 7.3mL; 10×TBE 1mL; 30% Acr / bis1.7mL; TEMED 6μL) in a 50mL tube, mix well immediately after adding TEMED, and add to the gel-making mold. After gelation, place the electrophoresis tank on ice, load reactions 1-5 in sequence, and perform electrophoresis at 90V constant voltage. Stop electrophoresis when bromophenol blue reaches 3 / 4 of the gel, about 90 minutes.

[0239] 6. Transfer

[0240] 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 min, and the membrane was transferred by wet transfer at 100V in an ice bath for 60 min.

[0241] 7. UV cross-linking

[0242] After the transfer is completed, place the nylon membrane with the front side facing up on a dust-free paper and cross-link it for 15 minutes at a distance of 10-20 cm from the UV lamp on the workbench.

[0243] 8. Closure

[0244] Take a suitable container and add 15 ml of blocking solution (Biyuntian, GS009-7), and then put in the cross-linked nylon membrane containing the sample. Slowly shake on a side-swing shaker or a horizontal shaker for 15 minutes, and repeat three times. Take 7.5 μl of Streptavidin-HRPConjugate (Biyuntian, GS009-6) and add it to 15 ml of blocking solution (1:2000 dilution), mix well and set aside. Remove the blocking solution used for nylon membrane blocking, and add 15 ml of blocking solution containing Streptavidin-HRP Conjugate prepared in the previous step. Slowly shake on a side-swing shaker or a horizontal shaker for 15 minutes.

[0245] 9. Wash the membrane

[0246] 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 with 15-20 ml of washing solution and rinse for 1 minute. Remove the washing solution, add 15-20 ml of washing solution, wash slowly on a side-swing shaker or horizontal shaker for 5 minutes, repeat three times, and each washing time is about 5 minutes. Transfer the nylon membrane to another container with 20-25 ml of detection balance solution, and shake slowly on a side-swing shaker or horizontal shaker for 5 minutes.

[0247] 10. Color rendering

[0248] BeyoECL Moon A solution (Biyuntian, P0018FM) and BeyoECL Moon B solution (Biyuntian, P0018FM) were mixed in equal volumes, and 1 mL of color developing solution was prepared. After taking out the membrane, 1 mL of color developing solution was evenly added. The reaction was carried out in the dark for 5 minutes, and imaging was performed using a BIO-RAD multifunctional chemiluminescence imager.

[0249] Example 1. Occurrence and development of cotton pigment glands and cottonpol accumulation

[0250] The development of pigment glands goes through four stages: glandular cell initiation, glandular development, secondary metabolite synthesis, and internal cell lysis. The present invention uses the cotton Gl2 gene promoter (SEQ ID NO: 16) to construct a proGhGl2::GUS cotton transgenic line. Through GUS staining of cotton true leaves and immature embryos, it is found that glandular cells occur in pseudo-meristems expressing Gl2 / Gl3 genes. After the fate is determined, the glandular cells undergo multiple divisions to form glands visible to the naked eye (such as Figure 1 (shown in a).

[0251] 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 there was no cottonpol accumulation in the embryos. Cottonpol began to accumulate in the embryos around 17-18 days after the embryos developed. At this time, the glands appeared light yellow under an optical microscope (such as Figure 1 Middle c, Figure 2 As the gland develops, the cells in the center of the gland begin to lyse, and eventually a mature pigment gland (such as Figure 1 (as shown in b).

[0252] In summary, cottonypol begins to appear when the gland develops to a certain stage, and there is no cottonypol accumulation in the immature embryo in the early stage of gland development.

[0253] Example 2. Alleles Gl2 and Gl3 are key genes for regulating the development of glands and cottonpol synthesis. Gl2 and its alleles (including the allele Gl3 on the D subgenome) are necessary and sufficient for the development of glands. The Gl2 / Gl3 loss-of-function mutant (gl2gl2gl3gl3) has no pigment glands in the whole plant (e.g. Figure 3 ), and no cottonpol was detected in the tissues and organs of the mutant plants (as shown in a Figure 3 As the wild-type Gl2 and Gl3 copy numbers decreased, the number of true leaf glands and the cottonpol content decreased significantly (as shown in Figure 3 (as shown in c and d).

[0254] There are only two amino acid differences between Gl2 and Gl3 proteins (compared to Gl2, Gl3 has N207H and S281G substitutions). Genetic analysis shows that the functions of Gl2 and Gl3 genes in gland development are consistent. The difference between the two is that the expression level of Gl2 is significantly higher than that of Gl3, and the expression level of Gl2 is about twice that of Gl3. Compared with the wild type (Gl2Gl2Gl3Gl3), the number of glands and the content of cottonpol in the gl2 mutant (gl2gl2Gl3Gl3) are significantly lower than those in the gl3 mutant (Gl2Gl2gl3gl3) (e.g. Figure 3 (as shown in c and d).

[0255] In summary, the Gl2 / Gl3 gene determines the development of cotton pigment glands and the accumulation of cottonpol.

[0256] Example 3. In situ synthesis and accumulation of secondary metabolites such as gossypol in pigment glands

[0257] Non-glandular cells of immature embryos (30 days, 40 days, and 50 days) of the genetic standard line TM-1 of upland cotton were obtained, and the cottonpol content was determined by high performance liquid chromatography (HPLC). The results showed that no cottonpol was detected in the non-glandular cells of the immature embryos (e.g. Figure 4 (as shown in a and b).

[0258] In addition, the glandular cells of TM-1 immature embryos and leaves were obtained for transcriptome sequencing. Combined with the transcriptome expression profile of immature embryos of the Gl2 / Gl3 mutant strain, it was found that the genes of the cottonpol synthesis pathway are gland-specifically expressed genes (such as Figure 4 (as shown in c).

[0259] The promoter of the key gene CDNC in the cottonphenol biosynthesis pathway (SEQ ID NO: 17) and the fusion vector of the GUS gene (SEQ ID NO: 18) were constructed and introduced into cotton. The positive plants of the T1 generation were stained with GUS and it was found that the glands in the young embryos, leaves and roots of the transgenic lines showed obvious blue (such as Figure 4 (shown in d).

[0260] 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.

[0261] Example 4. Gl2 / Gl3 genes are gland-specific highly expressed genes

[0262] Through the glandular cell-specific transcriptome, it was found that the expression level of Gl2 / Gl3 genes in cotton embryos, leaves, and stem glandular cells was much higher than that in non-glandular cells ( Figure 5). The proGhGl2::GUS cotton transgenic lines were constructed, and GUS staining revealed that the glands in the young embryos and leaves of the transgenic lines showed obvious blue, while the non-glandular tissues did not show obvious blue.

[0263] In summary, Gl2 / Gl3 genes are gland-specific highly expressed genes.

[0264] Example 5. Gl2 / Gl3 regulates the occurrence and development of glands by forming homotetramers

[0265] 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 (e.g. Figure 6 As shown in a). The bHLH domain of Gl2 protein (amino acids 300-373 of SEQ ID NO: 1) is composed of more than 70 amino acids and is divided into the HLH region and the alkaline region, wherein the alkaline region (amino acids 300-313 of SEQ ID NO: 1) is the region that binds to DNA; the HLH region is mainly composed of hydrophobic amino acids and is the region that forms homologous or heterologous multimers. There is a bHLH_MYC_N domain in the N-terminal region of Gl2, which is responsible for forming a complex that mediates transcription initiation with other transcription-related proteins, thereby regulating the initiation of its downstream genes.

[0266] Bimolecular fluorescence complementation experiments confirmed that Gl2 / Gl3 protein molecules can interact with themselves (e.g. Figure 6 b), and further analysis by size exclusion chromatography confirmed that the Gl2 / Gl3 protein molecule exists in the form of a homotetramer (as shown in Figure 6 (as shown in c and d).

[0267] Example 6. Gl2 / Gl3 activates target gene expression by binding to target sequence

[0268] In this study, the proGhGl2 promoter and the gene encoding the Gl2 protein (SEQ ID NO: 19) were used to construct the proGhGl2: GhGl2-3*FLAG vector, and the transgenic lines were obtained by shoot tip transformation. The positive plants were confirmed by PCR and Western Blot detection. Chromatin immunoprecipitation sequencing analysis (ChIP-seq) was performed using young embryos in the early developmental stage (13dpa~21dpa, with the day of flowering as 0dpa) as the material. By analyzing the results of two ChIP-seq, a total of 5627 Gl2 protein-bound regions were identified in the whole genome, and 991 regions overlapped in the two experiments (such as Figure 7Among them, the binding peak located upstream of the transcription start site (TSS), namely the core promoter region (3000 bp upstream of TSS), accounted for 52.89% (as shown in Figure 1). Figure 7 Using the MEME-ChIP program to search for Gl2-binding DNA motifs, it was found that the E-box motif (NACGTG) was significantly enriched (as shown in Figure 2A). Figure 7 This further confirmed that Gl2 transactivated the expression of target genes by binding to the E-box motif.

[0269] Transcriptome analysis was used to further analyze the co-expression regulatory relationship between Gl2 / Gl3 and its downstream target genes, and finally 612 high-confidence potential target genes of Gl2 were identified (such as Figure 7 The genes of the cotton phenol synthesis pathway were 2-ODD-1 and CYP736A12 (Table 3).

[0270] Table 3. Downstream target genes regulated by Gl2 protein

[0271]

[0272] Example 7. Design and functional verification of Gl2 / Gl3 protein mutants

[0273] 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 dominant negative effect, that is, by artificially introducing or designing mutation sites, under the premise of retaining the tetramer formation and the binding ability of the downstream target gene promoter, a wild-type Gl2 protein mutant is obtained. The mutant protein can form a tetramer without transcription function with the wild-type Gl2 / Gl3 protein or the artificial mutant protein itself, and competitively binds to the downstream target gene promoter with the wild-type Gl2 / Gl3 tetramer, thereby achieving the function of interfering with the wild-type Gl2 / Gl3. At the same time, the Gl2 / Gl3 mutant protein gene is fused with the cotton seed specific promoter, and introduced into cotton to create a new material with normal plants and zero cottonpol in cottonpol seeds. Since the mutant protein gene is only expressed in cotton seeds, it only interferes with the development of cotton seed glands, and does not affect the development of glands in other tissues.

[0274] Combined with the Gl2 protein structure 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) would cause the Gl2 protein to lose its ability to activate downstream target genes. Therefore, the present invention mainly mutates and truncates the above regions.

[0275] The Gl2 protein mutants designed and constructed by the present invention are shown in Table 4.

[0276] Table 4. Gl2 protein mutants

[0277]

[0278]

[0279] By bimolecular fluorescence complementation (BIFC) assay (e.g. Figure 8 (a) confirmed that the mutant protein mute9 (SEQ ID NO: 11) can interact with the wild-type Gl2 protein and itself, and EMSA experiments confirmed that the protein can still bind to the DNA molecule (E-box) (as shown in Figure 8 (as shown in b).

[0280] Through luciferase system analysis experiments, the differences in the transcriptional activation ability of mutant proteins mute1 to mute10 were explored. Under normal temperature conditions of 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 62sk empty vector (control) were co-injected with pCGF1-LUC into tobacco leaves. Enzyme activity assays showed that compared with the wild-type protein Gl2, the artificial mutant proteins almost completely lost their transcriptional activation ability ( Fig. 9 ).

[0281] It can be seen that the mutant of the present invention has lost or almost lost 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 to bind to the target DNA, thereby inhibiting the expression of downstream genes related to cotton gland development and cottonpol synthesis.

[0282] Example 8. Screening of cotton seed development-specific promoters

[0283] The expression of Gl2 / Gl3 genes begins from the 12th day after the cotton flowering when the embryo develops until the seeds mature. According to the function of Gl2 / Gl3 genes in regulating the occurrence of glands, only by completely inhibiting the function of Gl2 / Gl3 proteins in the development of cotton seeds can the cotton seeds be completely gland-free and the cottonphenol content be zero. However, the expression level of Ghα-globulin, a cotton seed-specific expression gene commonly used in the creation of low-phenol cotton, is significantly higher than the sum of the expression levels of Gl2 and Gl3 genes only after the embryo develops to the 16th day. Therefore, after the silencing expression box of the Gl2 / Gl3 gene driven by the Ghα-globulin promoter is introduced into the plant, the glands in the seeds cannot be completely eliminated, so "zero" cottonphenol in the seeds cannot be achieved.

[0284] The present invention uses high-resolution spatiotemporal transcriptome technology to screen genes specifically expressed during cotton seed development, and the screening is carried out according to the following principles:

[0285] 1. The candidate gene begins to express no later than Gl2 and Gl3, and continues to express until the seeds mature.

[0286] 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.

[0287] 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. The seed development process here refers to the development from fertilized eggs to seed maturity, which for cotton is from flowering to boll opening.

[0288] The present invention screened a plurality of cotton seed-specific expression genes and obtained the promoters of the corresponding genes, proGhOLE1_A (SEQ ID NO: 15), proGhOLE1_D (SEQ ID NO: 13), proGhALB2S (SEQ ID NO: 14) ( Fig.10 ). The expression levels of cotton seed-specific genes GhOLE1_A, GhOLE1_D, and GhALB2S screened according to the above principles were significantly higher than the sum of the expression levels of Gl2 and Gl3 genes during the entire seed development stage ( Fig.11 ). Therefore, by introducing the above promoter fused with the expression cassette of the Gl2 / Gl3 mutant into cotton, the transcriptional activation function of the wild-type Gl2 / Gl3 protein can be completely inhibited, thereby completely inhibiting the occurrence of seed glands.

[0289] Example 9. Creation of a new type of cotton with “zero” cottonpol in seeds and unchanged cottonpol content in plant glands using seed-specific promoter proGhOLE1_D and mutant protein mute9

[0290] The plant expression cassette was constructed by using the seed-specific expression gene GhOLE1_D promoter proGhOLE1_D (SEQ ID NO: 13) fused with the Gl2 / Gl3 mutant mute9 coding sequence (SEQ ID NO: 22). Fig.12 ), the nucleotide sequence of proGhOLE1_D-mute9 is shown in SEQ ID NO:23. The cotton stem tip was used as an explant, and the expression cassette was introduced into cotton by Agrobacterium transformation. Positive pure plants with the expression cassette were screened and identified (named NSGFC-1-1, NSGFC-1-3, and NSGFC-1-7, respectively). The cotton recipient material introduced with an empty vector was used as a control (TM-1), and the seeds and various organs of the positive pure plants were tested for cotton phenol content, fiber yield and quality, boll number, flowering period, insect resistance, Verticillium wilt resistance and other traits.

[0291] The results showed that no glands were observed in mature seeds of transgenic materials into which the mutant protein expression cassette was introduced, and the cottonpol content was zero ( Fig.13 , Fig.16 ), while the comprehensive traits such as cotton phenol content in other organs of the plant and cotton fiber length had no significant difference with the control material ( Figure 14-16 , Table 5).

[0292] Table 5. Statistics of comprehensive traits of new cotton NSGFC-1

[0293]

[0294] 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, there is no significant difference in the comprehensive traits of the new type of cotton compared with the control material.

[0295] Example 10. Creation of a new cotton with “zero” cottonpol in seeds and unchanged cottonpol content in plant glands using seed-specific promoter proGhALB2S and mutant protein mute4

[0296] The seed-specific expression gene ALB2S promoter proGhALB2S (SEQ ID NO: 14) was fused with the Gl2 / Gl3 mutant mute4 coding sequence (SEQ ID NO: 20) to construct a plant expression cassette ( Fig.17), the nucleotide sequence of proGhALB2S-mute4 is shown in SEQ ID NO:21. The cotton stem tip was used as an explant, and the expression cassette was introduced into cotton by Agrobacterium transformation. Positive pure plants with the expression cassette were screened and identified (named NSGFC-2-4 and NSGFC-2-5, respectively). The cotton recipient material introduced with an empty vector was used as a control (TM-1), and the seeds and various organs of the positive pure plants were tested for cotton phenol content, fiber yield and quality, boll number, flowering period, insect resistance, Verticillium wilt resistance and other traits.

[0297] The results showed that the cottonpol content in mature seeds of transgenic materials introduced with mutant protein expression cassette was zero (Table 6), while the cottonpol content in other organs of the plant, cotton fiber length and other comprehensive traits had no significant difference from those of the control materials (Table 7).

[0298] Table 6. Cottonpol content in various organs of new cotton NSGFC-2 (μg / mg)

[0299]

[0300] Table 7. Statistics of comprehensive traits of new cotton NSGFC-2

[0301]

[0302] From the above results, it can be seen that the new cotton seeds created by the method of the present invention do not contain gossypol and other secondary metabolites, which greatly improves the safety of the seeds as food raw materials and feed applications; at the same time, the gossypol content of the new cotton plants is not affected, and they can resist pests and diseases, and the comprehensive traits such as plant yield and cotton fiber length are not affected, so the production of raw cotton is not affected. In summary, the new cotton created by the method of the present invention greatly improves the comprehensive utilization value of cotton.

[0303] Example 11. Application of the method for creating new cotton using seed-specific promoter and Gl2 / Gl3 mutant protein in various cotton lines

[0304] The plant expression cassette used in Example 9 was introduced into Lumianyan 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 selected from the F2 generation for field trials.

[0305] Field trial design: randomized block arrangement, 3 replications, 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.

[0306] Field management: Follow local conventional cultivation and management measures, and the field management is consistent. The density per mu is 2100-3500 plants, and the sowing period is concentrated in mid-to-late April. The sowing method is direct seeding. Control cutworms, cotton aphids, cotton bollworms, whiteflies, etc. 4-16 times, water 0-1 times, drain 1-4 times, chemically regulate 2-6 times, and cultivate and weed 1-11 times. The base fertilizer is mainly compound fertilizer, and some pilot projects apply organic fertilizer. Topdressing is mainly urea and compound fertilizer.

[0307] The experimental results show that in the main cotton-growing areas of my country, the seeds of the new cotton created by Lumianyan No. 37 and Xinluzhong 67 have no glands and zero cottonphenol content (Table 8, Table 9), the glands and cottonphenol content of other organs of the plant are stable, and other comprehensive traits have no significant differences from the control materials (Table 10, Table 11). It can be seen that the method of creating a new cotton with "zero" cottonphenol in seeds, unchanged glands and cottonphenol content in plants of the present invention is universal in a variety of cotton strains.

[0308] Table 8. Cottonpol content in various organs of the new transgenic material Lumianyan 37 (μg / mg)

[0309]

[0310] Table 9. Cottonpol content in various organs of Xinluzhong 67 transgenic new material (μg / mg)

[0311]

[0312] Table 10. Statistics of traits of new transgenic materials of Lumianyan 37

[0313]

[0314] Table 11. Statistics of traits of new transgenic material Xinluzhong 67

[0315]

[0316] Example 12. Comparison of amino acid sequences of Gl2 / Gl3 proteins from different cotton species

[0317] The amino acid sequences of wild-type Gl2 / Gl3 proteins from different cotton species are highly conserved. By comparing the amino acid sequences of wild-type Gl2 and Gl3 proteins from Gossypium arboretum (A2 genome), Gossypium anomalum (B1 genome), Gossypium sturtianum (C1 genome), Gossypium raimondii (D5 genome), Gossypium stocksii (E1 genome), Gossypium longicalyx (F1 genome), Gossypium bickii (G1 genome), Gossypium rotundifolium (K2 genome), Gossypium hirsutum (AD1 genome), and Gossypium barbadense (AD2 genome), we found that there was little difference between the amino acid sequences of Gl2 and Gl3 proteins from different cotton species ( Fig.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 are used in the method of the present invention for creating a new type of cotton with "zero" cottonpol in seeds and unchanged plant glands and cottonpol content.

[0318] Equivalent solutions

[0319] 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 functions and / or obtaining 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 appreciate that all parameters, dimensions, materials, etc. described herein are merely exemplary, and 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 experiments. 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.

[0320] It should be understood that according to the disclosure of the claims and the specification of the present invention, Gl2 and / or Gl3 proteins from different cotton sources can be used to construct mutant proteins, thereby creating new cotton with zero cottonpol content in seeds and normal cottonpol content in plants. In addition, the mutation types provided by the present invention are only exemplary, and other mutation types that can achieve the purpose of the present invention are also included in the scope of the present invention.

[0321] 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. Equivalent solutions having the same effect as the Gl2 / Gl3 mutant protein of the present invention or the combination of the Gl2 / Gl3 mutant protein of the present invention and a seed-specific promoter can be obtained by various mutagenesis methods known in the art (e.g., physical mutagenesis, chemical mutagenesis). Those of ordinary skill in the art will appreciate that these equivalent solutions are also encompassed within the scope of the present invention.

[0322] Sequence Listing

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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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