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. A cotton seed specific promoter is used for specifically inhibiting expression and / or activity of Gl2 / Gl3 protein 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 gossypol content of other tissues is normal.

Description

[0001] This application claims the benefit of a prior Chinese application filed on October 27, 2023, with application number 202311418083.1, and invention name “Methods and uses for improving pigment gland traits and cotton phenol traits in cotton seeds”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of plant bioengineering and specifically relates to a method and use thereof for improving cotton seed pigment gland traits and cottonpol traits by using cotton seed specific promoter. Background Art

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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, by means of RNAi, American scientists use seed-specific promoters to drive CAD gene dsRNA, specifically silence seed gland occurrence and cotton phenol synthesis genes, and create new cotton germplasm with normal seed low phenol plants (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 gene Gl2 / Gl3 of cotton gland synthesis by RNAi method, and also created similar cotton germplasm (Gao W, Zhu X, Ding L, et al. Development of the engineered "glanded plant and glandless seed" cotton. Food Chemistry: Molecular Sciences, 2022, 5: 100130.). However, the above methods can only reduce but not completely block the translation of target genes, so there are still a small amount of glands and low-content gossypol in the seeds, and other secondary metabolites in the glands still pose a great threat to the health of humans and monogastric animals.

[0007] Therefore, there is an urgent need to develop a more effective method for reducing the gossypol content in cotton seeds to achieve zero gossypol in cotton seeds while the gossypol content in other tissues of the cotton plant remains unaffected. Summary of the invention

[0008] 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 livestock, greatly improving the economic benefits of cotton planting.

[0009] Accordingly, in a first aspect, the present invention provides a method for inhibiting the formation of pigment glands in cotton seeds, the method comprising causing the cotton seeds to express an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 proteins, wherein the inhibitory molecule is expressed by a seed-specific promoter of the cotton.

[0010] 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 an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 protein, wherein the inhibitory molecule is expressed by a cotton seed-specific promoter.

[0011] Seed-specific promoters suitable for use in the method of the present invention:

[0012] (1) Initiating the expression of inhibitory molecules at a time no later than that of Gl2 / Gl3 proteins during seed development in wild-type cotton;

[0013] (2) making the expression level of the inhibitory molecule no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and

[0014] (3) The expression of inhibitory molecules is initiated only during the development of cotton seeds, and the expression of inhibitory molecules is not initiated or substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

[0015] In some embodiments, the seed-specific promoter is a promoter derived from OLE_A, OLE_D or 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: 1-3.

[0016] In some embodiments, the inhibitory molecule is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen-binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene.

[0017] In some embodiments, the inhibitory molecule is a dsRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene. In some preferred embodiments, the nucleotide sequence encoding the dsRNA 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: 28, 59 or 70.

[0018] In some embodiments, the inhibitory molecule is a microRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene. In some preferred embodiments, the nucleotide sequence encoding the microRNA 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: 44.

[0019] In some specific embodiments, the method described herein comprises integrating into the genome of the 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: 29, 45, 60, 71, 76 or 77.

[0020] In some specific embodiments, the method of the present invention comprises the following steps:

[0021] (1) transforming the nucleotide sequence containing the seed-specific promoter and encoding the inhibitory molecule into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0022] (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.

[0023] In some embodiments, the method of the present invention comprises causing cotton seeds to express two or more inhibitory molecules that inhibit the expression and / or activity of Gl2 / Gl3 proteins. In some embodiments, the two or more inhibitory molecules are expressed by the same cotton seed-specific promoter. In some embodiments, the two or more inhibitory molecules are expressed by different cotton seed-specific promoters. In some preferred embodiments, the cotton seed-specific promoters are each independently selected from promoters derived from OLE_A, OLE_D or ALB2S genes.

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

[0025] In some embodiments, the methods described herein 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 described herein result in cotton plant tissues having gossypol levels that are no different or substantially no different than the gossypol levels in tissues of 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 an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 protein, wherein the inhibitory molecule is expressed by a cotton seed-specific promoter.

[0029] In some embodiments, the seed-specific promoter:

[0030] (1) Initiating the expression of inhibitory molecules at a time no later than that of Gl2 / Gl3 proteins during seed development in wild-type cotton;

[0031] (2) making the expression level of the inhibitory molecule no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and

[0032] (3) The expression of inhibitory molecules is initiated only during the development of cotton seeds, and the expression of inhibitory molecules is not initiated or substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

[0033] In some embodiments, the seed-specific promoter is a promoter derived from OLE_A, OLE_D or 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: 1-3.

[0034] In some embodiments, the inhibitory molecule is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen-binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene.

[0035] In some embodiments, the inhibitory molecule is a dsRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene. In some preferred embodiments, the nucleotide sequence encoding the dsRNA 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: 28, 59 or 70.

[0036] In some embodiments, the inhibitory molecule is a microRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene. In some preferred embodiments, the nucleotide sequence encoding the microRNA 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: 44.

[0037] In some specific embodiments, the cotton cell, seed, tissue, organ or plant of the third aspect of the invention is 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: 29, 45, 60, 71, 76 or 77 into the genome of cotton.

[0038] In some specific embodiments, the cotton cell, seed, tissue, organ or plant of the third aspect of the present invention is obtained by the following steps:

[0039] (1) transforming the nucleotide sequence containing the seed-specific promoter and encoding the inhibitory molecule into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0040] (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.

[0041] In some embodiments, the cotton cell, seed, tissue, organ or plant of the third aspect of the present invention comprises a nucleotide sequence encoding an inhibitory molecule for the expression and / or activity of two or more Gl2 / Gl3 proteins. In some embodiments, the two or more inhibitory molecules are expressed by the same cotton seed-specific promoter. In some embodiments, the two or more inhibitory molecules are expressed by different cotton seed-specific promoters. In some preferred embodiments, the cotton seed-specific promoters are each independently selected from promoters derived from OLE_A, OLE_D or ALB2S genes.

[0042] In a fourth aspect, the present invention provides a nucleic acid construct comprising, from 5' to 3', a cotton seed-specific promoter and a nucleotide sequence encoding an inhibitory molecule that inhibits the expression and / or activity of a Gl2 / Gl3 protein. In some embodiments, the nucleic acid construct further comprises a terminator.

[0043] In some embodiments, the seed-specific promoter is a promoter derived from OLE_A, OLE_D or 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: 1-3.

[0044] In some embodiments, the inhibitory molecule is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen-binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene.

[0045] In some embodiments, the inhibitory molecule is a dsRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene. In some preferred embodiments, the nucleotide sequence encoding the dsRNA 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: 28, 59 or 70.

[0046] In some embodiments, the inhibitory molecule is a microRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene. In some preferred embodiments, the nucleotide sequence encoding the microRNA 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: 44.

[0047] In some specific 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:29, 45, 60, 71, 76 or 77.

[0048] In some embodiments, the nucleic acid construct comprises a nucleotide sequence encoding two or more inhibitory molecules that inhibit the expression and / or activity of Gl2 / Gl3 proteins. In some embodiments, the two or more inhibitory molecules are operably linked to the same cotton seed-specific promoter. In some embodiments, the two or more inhibitory molecules are operably linked to different cotton seed-specific promoters. In some preferred embodiments, the cotton seed-specific promoters are each independently selected from promoters derived from OLE_A, OLE_D or ALB2S genes.

[0049] 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.

[0050] 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

[0051] 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.

[0052] Figure 2 The results of measuring the cottonpol content in cotton immature embryos at different developmental stages are shown.

[0053] Figure 3 The gland numbers and gossypol contents of plants with different Gl2 / Gl3 genotypes are shown. Figure 3 Middle 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.

[0054] 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 measuring the cottonpol 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.

[0055] Figure 5 Shown are the expression levels of Gl2 / Gl3 genes in glandular and non-glandular tissues.

[0056] Figure 6 The expression levels of candidate promoter genes in different tissues of TM-1 are shown.

[0057] Figure 7 The gene expression levels of seed-specifically expressed genes Ghα-globulin, GhOLE1_D, GhALB2S and Gl2+Gl3 genes during seed development are shown.

[0058] Figure 8 A schematic diagram of the expression vector containing the seed-specific promoter and the GUS gene is shown.

[0059] Fig. 9 Shown are the results of GUS staining of seed-specific promoter-GUS transgenic cotton.

[0060] Fig.10 Schematic diagrams of proGhOLE1_D::dsGhGl2-RNAi, proGhOLE1_A::dsGhGl2-RNAi and proGhALB2S::dsGhGl2-RNAi expression vectors are shown.

[0061] Fig.11The PCR identification results of proGhOLE1_D::dsGhGl2-RNAi T1 generation transgenic materials are shown.

[0062] Fig.12 The phenotypes of seeds, leaves and stem glands of proGhOLE1_D::dsGhGl2-RNAi transgenic materials are shown.

[0063] Fig.13 Schematic diagrams of proGhOLE1_A::GhGl2 / GhGl3-miRNA, proGhOLE1_D::GhGl2-miRNA and proGhALB2S::GhGl2-miRNA expression vectors are shown. DETAILED DESCRIPTION

[0064] 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.

[0065] 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.

[0066] 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.

[0067] As used herein, the terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (ie, meaning "including, but not limited to, ").

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] "Carrier" refers to a delivery vehicle or element of genetic material. In this article, the term "carrier" refers to one or a series of macromolecules comprising polynucleotides, 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, the coding sequences of RNAi components (for example, dsRNA, shRNA, siRNA, microRNA, antisense oligonucleotides), or optional markers. In the carrier, the coding sequences of proteins, polypeptides, RNAi components, etc. are usually operably connected to expression control elements, such as promoters. In some embodiments, the carrier can be a linear DNA molecule. In some embodiments, the carrier can be a circular DNA molecule.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] When referring to a specific molecule, biological material or cellular substance, the term "equivalent" or "functional variant" can be used interchangeably and refers to those with minimal homology while still maintaining 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 of 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 seed-specific promoter of the present invention).

[0079] Compared with the reference polypeptide of the present invention (such as the Gl2 / Gl3 protein 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 substitutions of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or substitutions of one charged or polar residue for another, such as substitutions of arginine for lysine, substitutions of glutamic acid for aspartic acid, substitutions of glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and the like.

[0080] 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.

[0081] 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 and Gl3 are the most important genes for controlling glandular formation, encoding Gl2 and 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.

[0082] 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 between the Gl2 and Gl3 proteins in amino acid sequence and function, the inhibitory molecules designed for the Gl2 protein or the Gl2 gene in the present invention are applicable to the Gl3 protein or the Gl3 gene. It should also be understood that the technical solutions related to the Gl2 protein or the Gl2 gene involved in the present invention are also applicable to the Gl3 protein or the Gl3 gene, and the aforementioned technical solutions can achieve similar technical effects.

[0083] The term "inhibitory molecule" refers to a molecule that can inhibit the expression or function of a biologically active substance (e.g., protein, DNA, or RNA). The inhibitory molecule can be a protein (e.g., an antibody or an antigen-binding fragment thereof), a single-stranded or double-stranded nucleotide molecule (e.g., antisense nucleic acid, siRNA, miRNA, dsRNA, or shRNA), a small molecule ligand, etc.

[0084] In this article, inhibiting the expression of Gl2 / Gl3 genes includes inhibiting the transcription process of Gl2 / Gl3 genes, inhibiting the translation process of mRNA after transcription, reducing the stability of mRNA or inducing the degradation of mRNA, etc.; inhibiting the activity of Gl2 / Gl3 proteins includes reducing the stability of proteins, inducing protein degradation, blocking the function of proteins, etc.

[0085] The term "antibody" as used herein should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0086] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., specifically bind to a G12 / G13 protein). It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.

[0087] Examples of "antigen-binding fragments" of antibodies include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region; (iv) a Fd fragment, which consists of the VH and CH1 domains; (v) a Fd' fragment, which has the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) a Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of the VH domain; (viii) a single complementarity determining region (CDR); and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by synthetic linkers using recombinant methods so that they can form a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes a "straight chain antibody" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides and the aforementioned fragments in any modified form that retain antigen-binding activity form an antigen-binding region.

[0088] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments screened for utility in the same manner as are intact antibodies.

[0089] The term "dsRNA" refers to a type of double-stranded RNA molecule that can mediate the degradation of mRNA molecules with homology thereto, thereby achieving gene silencing. dsRNA can be processed by intracellular nucleases (such as Dicer enzymes) to form siRNAs with a length of about 21 to 23 nt.

[0090] The term "siRNA" is a class of double-stranded RNA molecules comprising a sense strand and an antisense strand, which can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary or substantially complementary to the antisense strand. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA, and a sense strand complementary or substantially complementary to the antisense strand. For convenience, such mRNA is also referred to as mRNA to be silenced, and such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0091] In the context of siRNA, the term "antisense strand" refers to the strand of the siRNA that contains a region that is fully complementary or substantially complementary to the target sequence; the term "sense strand" refers to the strand of the siRNA that includes a region that is substantially complementary to a region that is defined as the antisense strand herein.

[0092] MicroRNA (miRNA for short) is a small non-coding RNA that belongs to a class of regulatory molecules found in plants and animals. In general, miRNAs control gene expression by binding to complementary sites on target messenger RNA (mRNA) transcripts. miRNAs are generated from large RNA precursors (called pri-miRNAs) and processed into pre-miRNAs of approximately 70 nucleotides in the nucleus. Pre-miRNAs fold into imperfect stem-loop structures and undergo additional processing steps in the cytoplasm, where they are excised from one side of the pre-miRNA hairpin by the RNase III enzyme Dicer to form mature miRNAs of 18-25 nucleotides in length. miRNAs that bind complementary to protein-coding mRNA sequences induce the RNA-mediated interference pathway, which causes the mRNA target to be cleaved and degraded by ribonucleases in the RISC complex. This miRNA-mediated gene silencing mechanism has been observed in plants and animals.

[0093] When referring to dsRNA, siRNA or microRNA, the term "targeting ... mRNA" means that the siRNA or the antisense strand of the siRNA generated by processing of the dsRNA or the microRNA is complementary or substantially complementary to the mRNA to be silenced (e.g., the mRNA transcribed from the Gl2 / Gl3 gene).

[0094] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours. In terms of meeting the above requirements relative to their ability to hybridize, "complementary" sequences may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0095] As used herein, the terms "complementary", "fully complementary" and "substantially complementary" may be used with respect to base pairing between the sense and antisense strands of siRNA, between the antisense strand of siRNA and the target sequence, or between microRNA and the target sequence.

[0096] Method for inhibiting the formation of pigment glands in cotton seeds and method for reducing the cottonpol content in cotton seeds

[0097] 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 an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 proteins, wherein the inhibitory molecule is expressed by a seed-specific promoter of the cotton.

[0098] 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 an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 protein, wherein the inhibitory molecule is expressed by a cotton seed-specific promoter.

[0099] Seed-specific promoters suitable for use in the method of the present invention:

[0100] (1) Initiating the expression of inhibitory molecules at a time no later than that of Gl2 / Gl3 proteins during seed development in wild-type cotton;

[0101] (2) making the expression level of the inhibitory molecule no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and

[0102] (3) The expression of inhibitory molecules is initiated only during the development of cotton seeds, and the expression of inhibitory molecules is not initiated or substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

[0103] In some embodiments, the seed-specific promoter is a promoter derived from the OLE_A, OLE_D or ALB2S gene.

[0104] The seed-specific promoter used in the method of the present invention can be derived from the following species: upland cotton (Gossypium hirsutum), Asiatic cotton (Gossypium arboretum), abnormal cotton (Gossypium anomalum), Stertian cotton (Gossypium sturtianum), Raymond's cotton (Gossypium raimondii), Stoke's cotton (Gossypium stocksii), long-calyx cotton (Gossypium longicalyx), Bick's cotton (Gossypium bickii), round-leaf cotton (Gossypium rotundifolium), sea island cotton (Gossypium barbadense).

[0105] 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: 1-3. In some more preferred embodiments, the seed-specific promoter comprises a nucleotide sequence as shown in SEQ ID NO: 1-3. In some most preferred embodiments, the seed-specific promoter consists of a nucleotide sequence as shown in SEQ ID NO: 1-3.

[0106] It should be understood that the promoter derived from OLE_A, OLE_D or ALB2S gene provided by the present invention can specifically guide the seed-specific expression of downstream genes. Therefore, in addition to the inhibitory molecules for inhibiting the expression and / or activity of Gl2 / Gl3 proteins described in the present invention, the seed-specific promoter provided by the present invention can also be combined with inhibitory molecules for inhibiting other proteins related to gland formation or cottonphenol synthesis, thereby being used to inhibit the formation of pigment glands in cotton seeds or reduce the cottonphenol content in cotton seeds.

[0107] It should also be understood that the promoter derived from the OLE_A, OLE_D or ALB2S gene provided by the present invention can not only be used for the purposes described in the present invention, but can also be used for various application purposes (such as improving plant traits) by specifically expressing other nucleic acid molecules or proteins in cotton seeds.

[0108] In some embodiments, the inhibitory molecule of the present invention is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or its antigen-binding fragment, small molecule ligand or a combination thereof that inhibits or silences genes.

[0109] In some embodiments, the inhibitory molecule is a dsRNA that targets the mRNA transcribed from the Gl2 / Gl3 gene.

[0110] In some preferred embodiments, the nucleotide sequence encoding the dsRNA 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: 28, 59 or 70.

[0111] In some embodiments, the inhibitory molecule is siRNA that targets mRNA transcribed from the Gl2 / Gl3 gene.

[0112] In some embodiments, the siRNA comprises a sense strand and an antisense strand, and the length of the sense strand and the antisense strand are each independently 15-27 nucleotides, 18-25 nucleotides, 19-25 nucleotides, 20-25 nucleotides, 19-23 nucleotides, or 19-21 nucleotides.

[0113] In some embodiments, the sense strand and the antisense strand are the same length. In some embodiments, the sense strand and the antisense strand are different lengths.

[0114] In some preferred embodiments, the nucleotide sequence encoding the siRNA 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:28, 59 or 70.

[0115] In some embodiments, the inhibitory molecule is a microRNA that targets the mRNA transcribed from the Gl2 / Gl3 gene.

[0116] In some embodiments, the microRNA is 15-27 nucleotides, 18-25 nucleotides, 19-25 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 19-23 nucleotides, or 19-21 nucleotides in length.

[0117] In some preferred embodiments, the nucleotide sequence encoding the microRNA 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:44.

[0118] In some specific embodiments, the method described herein comprises integrating into the genome of the 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: 29, 45, 60, 71, 76 or 77.

[0119] In some embodiments, the inhibitory molecule is an anti-Gl2 / Gl3 protein antibody or an antigen-binding fragment thereof, which can specifically bind to Gl2 and Gl3 proteins and inhibit their functions (eg, transcriptional activation function).

[0120] In some embodiments, the inhibitory molecule is a small molecule ligand of the Gl2 / Gl3 protein, which can specifically bind to the Gl2 and Gl3 proteins and inhibit their functions (eg, transcriptional activation function).

[0121] In some specific embodiments, the method of the present invention comprises the following steps:

[0122] (1) transforming the nucleotide sequence containing the seed-specific promoter and encoding the inhibitory molecule into Agrobacterium to obtain Agrobacterium carrying the nucleotide sequence; and

[0123] (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.

[0124] Methods for transforming nucleotide sequences into Agrobacterium are well known to those of ordinary skill in the art, and those skilled in the art know that the nucleotide sequence carried by Agrobacterium can be introduced into cotton cells, tissues or organs by conventional methods in the art (e.g., shoot tip transformation method).

[0125] In some embodiments, the method of the present invention comprises causing cotton seeds to express two or more inhibitory molecules that inhibit the expression and / or activity of Gl2 / Gl3 proteins. In some embodiments, the two or more inhibitory molecules are expressed by the same cotton seed-specific promoter. In some embodiments, the two or more inhibitory molecules are expressed by different cotton seed-specific promoters.

[0126] In some embodiments, the two or more inhibitory molecules are selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene. In some preferred embodiments, the two or more inhibitory molecules are dsRNA and / or microRNA.

[0127] In some preferred embodiments, the cotton seed-specific promoters are each independently selected from promoters derived from OLE_A, OLE_D or ALB2S genes. 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: 1-3. In some more preferred embodiments, the seed-specific promoter comprises a nucleotide sequence as shown in SEQ ID NO: 1-3. In some most preferred embodiments, the seed-specific promoter consists of a nucleotide sequence as shown in SEQ ID NO: 1-3.

[0128] In some embodiments, the methods described herein result in the agenesis of pigment glands in cotton seeds, compared to pigment glands in wild-type cotton seeds, and the development of pigment glands in cotton plants is not affected.

[0129] In some embodiments, the methods described herein 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.

[0130] In some embodiments, the methods described herein result in cotton plant tissues having gossypol levels that are no different or substantially no different than the gossypol levels in tissues of wild-type cotton plants.

[0131] The significant advantages of the method of the present invention are:

[0132] (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;

[0133] (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;

[0134] (3) It does not affect the cotton fiber yield and quality of the produced cotton varieties.

[0135] 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.

[0136] Example

[0137] Example 1. Occurrence and development of cotton pigment glands and cottonpol accumulation

[0138] 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: 4) 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).

[0139] 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).

[0140] 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.

[0141] 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).

[0142] 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).

[0143] In summary, the Gl2 / Gl3 gene determines the development of cotton pigment glands and the accumulation of cottonpol.

[0144] Example 3. In situ synthesis and accumulation of secondary metabolites such as gossypol in pigment glands

[0145] 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).

[0146] 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).

[0147] The promoter of the key gene CDNC in the cottonphenol biosynthesis pathway (SEQ ID NO: 6) and the fusion vector of the GUS gene (SEQ ID NO: 5) 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).

[0148] In summary, the present invention confirms that secondary metabolites such as cottonpol in cotton pigment glands are synthesized and accumulated in situ in the glands.

[0149] Example 4. Gl2 / Gl3 genes are gland-specific highly expressed genes

[0150] 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.

[0151] In summary, Gl2 / Gl3 genes are gland-specific highly expressed genes.

[0152] Example 5. Screening of cotton seed development-specific promoters

[0153] 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.

[0154] 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:

[0155] 1. The candidate gene begins to express no later than Gl2 and Gl3, and continues to express until the seeds mature.

[0156] 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.

[0157] 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.

[0158] 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: 1), proGhOLE1_D (SEQ ID NO: 2), proGhALB2S (SEQ ID NO: 3) ( Figure 6 ). 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 ( Figure 7 ). Therefore, by using the above promoter to initiate the expression of inhibitory molecules of Gl2 / Gl3 genes or Gl2 / Gl3 proteins, it is possible to effectively inhibit the function of Gl2 / Gl3 proteins, thereby effectively inhibiting the occurrence of seed glands.

[0159] Example 6. proOLE1_D, proOLE1_A and proALB2S promoters direct seed tissue-specific expression of downstream genes

[0160] The proOLE1_D, proOLE1_A and proALB2S promoters from the TM-1 line of upland cotton were fused with the GUS gene to obtain the proGhOLE1_D-GUS, proGhOLE1_A-GUS and proGhALB2S-GUS expression vectors ( Figure 8 ).

[0161] The above expression vector was sent to Jiangsu Weimi Biotechnology Co., Ltd. for transformation, and the transformation background was the cotton variety "TM-1". The T0 generation transgenic cotton strain was obtained from the company, and the T1 generation was planted and harvested. The T1 positive plants were screened and the seeds were harvested, and finally the positive homozygous transgenic cotton plants containing the target gene with stable inheritance were obtained.

[0162] GUS staining was performed on tissues and organs (immature embryos, leaves, sepals and stems) of plants transformed with proGhOLE1_D-GUS, proGhOLE1_A-GUS and proGhALB2S-GUS expression vectors. Fig. 9As shown, the GUS gene showed a clearly observable blue color in the immature embryo of transgenic cotton, while the expression of the GUS gene was not detected in other organs. The results show that the promoter of the present invention can guide the expression of its downstream genes in the seeds of transgenic plants, and this expression has seed tissue expression specificity.

[0163] Example 7. Creating a new cotton plant using seed-specific promoters combined with dsRNA

[0164] With GhGl2 / GhGl3 as the target gene (the nucleotide sequence encoding the GhGl2 protein is shown in SEQ ID NO: 7), proGhOLE1_D::dsGhGl2-RNAi vector, proGhOLE1_A::dsGhGl2-RNAi vector and proGhALB2S::dsGhGl2-RNAi vector were constructed ( Fig.10 ).

[0165] The above vector was sent to Jiangsu Weimi Biotechnology Co., Ltd. for transformation, and the transformation background was the cotton variety "Bai Mian No. 1". The T0 generation transgenic cotton strains obtained from the company were planted and harvested for the T1 generation. For the strains transformed with the proGhOLE1_D::dsGhGl2-RNAi vector, the forward primer RNAi-JD-F (SEQ ID NO: 8): 5'-ATACATACCTACATACTTTCCATTTACTAGCA-3' and the reverse primer RNAi-JD-R (SEQ ID NO: 9): 5'-AGAAACTTTATTGCCAAATGTTTGAA-3' were used as primers to perform PCR amplification on the transgenic cotton strains, screen T1 positive plants and harvest seeds, and the PCR identification results are as follows: Fig.11 shown.

[0166] The forward primer RNAi-proGhOLE1_A-GhGl2-JD-F (SEQ ID NO: 46): 5'-CCCGACAAAAATCAACGTGTC-3', the reverse primer RNAi-proGhOLE1_A-GhGl2-JD-R (SEQ ID NO: 47): 5'-TATCAGAAGATCCCAATTCGACAA-3' and the forward primer RNAi-proGhALB2S-GhGl2-JD-F (SEQ ID NO: 48): 5'-CTACCGATCCCAGTTAAGGCAG-3', the reverse primer RNAi-proGhALB2S-GhGl2-JD-R (SEQ ID NO: 49): 5'-CTACCGATCCCAGTTAAGGCAG-3' IDNO: 49): 5'-CAAGAATCCCATCACCTACAGCG-3' The cotton lines transformed with proGhOLE1_A::dsGhGl2-RNAi vector and proGhALB2S::dsGhGl2-RNAi vector were identified, T1 positive plants were screened and seeds were harvested.

[0167] The roots, stems, leaves, petals and mature seeds of T2 transgenic lines and wild-type (non-transgenic) cotton were taken for determination of cottonphenol content. Among them, the T2 transgenic lines transformed with proGhOLE1_D::dsGhGl2-RNAi vector were named OLE1-T2-1 and OLE1-T2-3; the T2 transgenic lines transformed with proGhOLE1_A::dsGhGl2-RNAi vector were named OLE1_A-T2-2 and OLE1_A-T2-4; the T2 transgenic lines transformed with proGhALB2S::dsGhGl2-RNAi vector were named ALB2S-T2-1 and ALB2S-T2-2.

[0168] The cottonpol content was determined to be no different between the wild-type and transgenic lines in roots, stems, leaves, and petals, while the cottonpol content in mature seeds of transgenic plants was zero ( Fig.12 , Table 1-Table 3).

[0169] Table 1. Cottonpol content in various organs of RNAi interference material T2 transgenic strains (μg / mg)

[0170]

[0171] Table 2. Cottonpol content in various organs of T2 transgenic lines of RNAi interference materials (μg / mg)

[0172]

[0173] Table 3. Cottonpol content determination of each organ of T2 transgenic strains of RNAi interference materials (μg / mg)

[0174]

[0175] The effect of transgenic cotton on cotton yield was analyzed by statistically analyzing the bud and boll stage, plant height, number of fruit branches, number of bolls per plant, cotton fiber length and strength of T2 transgenic cotton and non-transgenic cotton plants. The plant height, number of fruit branches, number of bolls per plant, yield, cotton fiber length and fiber strength of transgenic cotton were similar to those of non-transgenic cotton Baimian No. 1, and there was no significant difference between the two (Tables 4-6).

[0176] Table 4. Statistics of comprehensive traits of T2 transgenic lines of RNAi interference materials

[0177]

[0178] Table 5. Statistics of comprehensive traits of T2 transgenic lines of RNAi interference materials

[0179]

[0180] Table 6. Statistics of comprehensive traits of T2 transgenic lines of RNAi interference materials

[0181]

[0182] The above results show that the method of using the cotton seed-specific promoters proOLE1_D, proOLE1_A and proALB2S combined with dsRNA of the present invention can ensure that the cottonpol content in the seeds is zero without affecting the cottonpol content in other tissues or organs of the cotton, nor affecting the comprehensive traits and final yield of the cotton.

[0183] Example 8. Creating a new cotton plant using seed-specific promoters combined with microRNA (miRNA)

[0184] Using GhGl2 / GhGl3 gene as target gene, proGhOLE1_A::GhGl2 / GhGl3-miRNA vector, proGhOLE1_D::GhGl2-miRNA vector and proGhALB2S::GhGl2-miRNA vector were constructed ( Fig.13 ).

[0185] The seed-specific miRNA vectors were introduced into the cotton variety Baimian 1 through Agrobacterium-mediated stem tip transformation to obtain transgenic strains.

[0186] The leaves, stems and mature seeds of T2 homozygous transgenic lines and wild-type (non-transgenic) cotton were taken for determination of cottonphenol content. Among them, the T2 transgenic lines transformed with proGhOLE1_A::GhGl2 / GhGl3-miRNA vector were named NSGFS-5-2 and NSGFS-5-3; the T2 transgenic lines transformed with proGhOLE1_D::GhGl2-miRNA vector were named NSGFS-6-1 and NSGFS-6-3; the T2 transgenic lines transformed with proGhALB2S::GhGl2-miRNA vector were named NSGFS-7-2 and NSGFS-7-5.

[0187] The cottonpol content was determined to be no significant difference between the wild type and transgenic lines in roots, stems, leaves and petals, while the cottonpol content in mature seeds of transgenic plants was reduced by more than 99.0% (Tables 7-9).

[0188] Table 7. Determination of gossypol content in various organs of microRNA interference material T2 generation (μg / mg)

[0189]

[0190] Table 8. Determination of gossypol content in various organs of microRNA interference material T2 generation (μg / mg)

[0191]

[0192] Table 9. Determination of gossypol content in each organ of microRNA interference material T2 generation (μg / mg)

[0193]

[0194] The effect of transgenic cotton on cotton yield was analyzed by statistically analyzing the bud and boll stage, plant height, number of fruit branches, number of bolls per plant, cotton fiber length and strength of T2 transgenic cotton and non-transgenic cotton plants. The plant height, number of fruit branches, number of bolls per plant, yield, cotton fiber length and fiber strength of transgenic cotton were similar to those of non-transgenic cotton Baimian No. 1, and there was no significant difference between the two (Tables 10-12).

[0195] Table 10. Statistics of comprehensive traits of T2 generation of microRNA interference materials

[0196]

[0197] Table 11. Statistics of comprehensive traits of T2 generation of microRNA interference materials

[0198]

[0199] Table 12. Statistics of comprehensive traits of T2 generation of microRNA interference materials

[0200]

[0201]

[0202] The above results show that the method of using the cotton seed-specific promoters proOLE1_D, proOLE1_A and proALB2S combined with microRNA of the present invention can ensure that the cottonpol content in the seeds is significantly reduced without affecting the cottonpol content in other tissues or organs of the cotton, nor affecting the comprehensive traits and final yield of the cotton. By further optimizing the microRNA sequence, it is possible to achieve the effect of zero cottonpol content in cotton seeds without affecting the cottonpol content in other tissues or organs, and without affecting the comprehensive traits and yield of cotton.

[0203] Experimental methods

[0204] (I) Shoot tip transformation method

[0205] 1. Seed germination

[0206] 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.

[0207] 2. Agrobacterium Preparation

[0208] 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.

[0209] 3. Infection and Co-cultivation

[0210] 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.

[0211] 4. Resume Culture

[0212] 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).

[0213] 5. Bud induction culture and resistant bud screening

[0214] 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.

[0215] 6. Rooting culture

[0216] 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.

[0217] Solution formula:

[0218] 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.

[0219] 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;

[0220] 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;

[0221] 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;

[0222] 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;

[0223] 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.

[0224] (ii) GUS staining

[0225] The material was quickly cut with a sharp blade and placed in pre-cooled 90% acetone for fixation for 20 minutes; the acetone was discarded and 1 mL of GUS washing solution was added to wash away the residual acetone, and washed twice; 1 mL of GUS staining solution (Solebo GUS staining kit - ready-to-use, item number: G3061) was added, and the sample was vacuumed on ice in the dark for 15-20 minutes; staining was carried out at 37°C in the dark for several hours or overnight; the staining solution was aspirated and 1 mL of 70% ethanol was added to stop the staining reaction and decolorization; 75% ethanol, 90% ethanol, and 100% ethanol were replaced until decolorization was complete; photographed and observed under a dissecting microscope and microscope.

[0226] (III) Construction of proGhOLE1_D-GUS, proGhOLE1_A-GUS and proGhALB2S-GUS expression vectors

[0227] 1) The VM062 plant expression vector was double-digested with restriction endonucleases HindⅢ and BamHI to linearize the circular vector.

[0228] 2) Synthesis of GUS gene: The GUS gene sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and the GUS gene sequence was amplified using primers with homology arms. The amplification primers of the GUS gene are as follows:

[0229] GUS-F forward primer (SEQ ID NO: 10):

[0230] cagtgccaagctaagcttATGTTACGTCCTGTAGAAACCCCA

[0231] GUS-R reverse primer (SEQ ID NO: 11):

[0232] ctaggctacgtaggatccTCATTGTTTGCCTCCCTGCT

[0233] Amplification system: KOD One TM PCR Master Mix (Toyobo, KMM-101) 20 μL; upstream primer (10 μM) 1 μL; downstream primer (10 μM) 1 μL; template 1.5 μL; sterile water 16.5 μL; total volume 40 μL.

[0234] The PCR reaction program was 98°C for 5 min; 98°C for 30 s; 55°C for 30 s; 68°C for 1 min, 35 cycles; then 68°C for 10 min and stored at 4°C.

[0235] 3) Detection and recovery of vector enzyme digestion products and PCR products: Recover PCR products and enzyme digestion products, and then use The target fragment was purified and recovered using Quick Gel Extraction Kit (Quanshi Gold, EG101-01).

[0236] 4) Recombination of the recovered products: Seamless cloning and Assembly Kit (Quanshijin, CU201-02) was used for homologous recombination. The reaction system was: 2×Basic Assembly Mix 5μL; enzyme-cut vector XμL; PCR product YμL; sterile water to 10μL; gently mix and react at 50℃ for 15 minutes.

[0237] 5) Transform E. coli DH5α and use KOD One TM PCR Master Mix (Toyobo, KMM-101) was used for PCR detection, and positive clones were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the intermediate vector VM062-GUS (SEQ ID NO: 12) was successfully obtained, and the plasmid was extracted and stored for later use.

[0238] 6) The VM062-GUS vector from the previous step was digested with restriction endonuclease HindⅢ.

[0239] 7) Using genomic DNA from leaves of upland cotton TM-1 as a template, primers with homology arms were used to amplify the proGhOLE1_A, proGhOLE1_D and proGhALB2S promoter sequences.

[0240] Amplification primers for proGhOLE1_A:

[0241] GUS-proGhOLE1_A-F forward primer (SEQ ID NO: 13):

[0242] 5'-cagtgccaagctaagcttTGAGTTTGGGAAATTAGGCATCC-3';

[0243] GUS-proGhOLE1_A-R reverse primer (SEQ ID NO: 14):

[0244] 5'-aggacgtaacataagcttTGATTAAAAAAACAAAGAAAAGAACTTG-3';

[0245] Amplification primers for proGhOLE1_D:

[0246] GUS-proGhOLE1_D-F forward primer (SEQ ID NO: 15):

[0247] 5'-cagtgccaagctaagcttCTGATCAGTCTGCTGTCAAATCG-3';

[0248] GUS-proGhOLE1_D-R reverse primer (SEQ ID NO: 16):

[0249] 5'-aggacgtaacataagcttTGCCTAAAAAAACAAAGAAAAGAACT-3'

[0250] Amplification primers for proGhALB2S:

[0251] GUS-proGhALB2S-F forward primer (SEQ ID NO: 17):

[0252] 5'-cagtgccaagctaagcttGCGACTCGACGATTGTTCCT-3'

[0253] GUS-proGhALB2S-R reverse primer (SEQ ID NO: 18):

[0254] 5'-aggacgtaacataagcttTGTTGATTTATGCTGGTATGGAGAA-3'

[0255] The amplification system and PCR reaction procedure were the same as above.

[0256] 8) Detection and recovery of vector enzyme digestion products and PCR products: PCR products and enzyme digestion products were detected by agarose gel electrophoresis, and then The target fragment was purified and recovered using Quick Gel Extraction Kit.

[0257] 9) Recombination of the recovered products: Homologous recombination was performed using the Seamless cloning and Assembly Kit.

[0258] 10) Transform E. coli DH5α and use KOD One TM PCR Master Mix was used for PCR detection, and positive clones were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0259] (IV) Construction of proGhOLE1_D::dsGhGl2-RNAi vector

[0260] 1) Linearization of WMV076 (plant RNAi vector provided by Jiangsu Weimi Biotechnology Co., Ltd.): Double-digest the WMV076 vector with HindⅢ and XbaⅠ restriction endonucleases to linearize the circular vector.

[0261] 2) Construction of proGhOLE1_D-RNAi intermediate vector: Using upland cotton DNA as a template, primers with linkers were designed using one-step cloning technology to amplify the promoter proGhOLE1_D.

[0262] Forward primer sequence RNAi-proGhOLE1_D-F (SEQ ID NO: 19):

[0263] 5'-gatcccccgaattaAAGCTTCTGATCAGTCTGCTGTCAAATCGGTG-3';

[0264] Reverse primer sequence RNAi-proGhOLE1_D-R (SEQ ID NO: 20):

[0265] 5'-TCCCCGGTCTAGATGCCTAAAAAAACAAAGAAAAGAACTTGAAG-3'.

[0266] The amplification system and PCR reaction procedure were the same as above.

[0267] Detection and recovery of vector enzyme digestion products and PCR products: PCR products and enzyme digestion products were detected by agarose gel electrophoresis, and then The target fragment was purified and recovered using Quick Gel Extraction Kit.

[0268] Recombination of the recovered products: Homologous recombination was performed using the Seamless cloning and Assembly Kit.

[0269] The recombinant product was transformed into Escherichia coli DH5α and KOD One TM PCR Master Mix was used for PCR detection, and positive clones were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After sequence comparison, plasmids were extracted from the clones with correct sequencing using a plasmid extraction kit to obtain the proGhOLE1_D-RNAi intermediate vector.

[0270] 3) Construction of proGhOLE1_D::dsGhGl2-F-RNAi intermediate vector: The proGhOLE1_D-RNAi vector was digested with BamHI restriction endonuclease to linearize the circular vector.

[0271] GhGl2 / GhGl3 gene positive dsRNA chain amplification:

[0272] Using forward primer RNAi-GhGl2-F1 (SEQ ID NO: 21): 5'-CATCTAGACCGGGGATCCCAGTGTGAGCGTGTTAGAGAAGCT-3'; reverse primer RNAi-GhGl2-R1 (SEQ ID NO: 22): 5'-gtatatacgtacGGATCCTGCTTCAACGTGGTTTATAGGGG-3' as a primer pair, and upland cotton DNA as a template, PCR amplification was performed to obtain the GhGl2 / GhGl3 gene positive interference fragment 1 (SEQ ID NO: 23); the GhGl2 / GhGl3 gene positive interference fragment 1 was purified and recovered, and the linearized proGhOLE1_D-RNAi vector was ligated and transformed using a one-step cloning enzyme to finally obtain the correctly sequenced positive clone proGhOLE1_D::dsGhGl2-F-RNAi, and the plasmid was extracted and stored for later use.

[0273] 4) Construction of proGhOLE1_D::dsGhGl2-RNAi vector: proGhOLE1_D::dsGhGl2-F-RNAi was double-digested with AscⅠ and SpeⅠ restriction endonucleases to linearize the circular vector.

[0274] GhGl2 / GhGl3 gene antisense dsRNA strand amplification:

[0275] Using forward primer RNAi-GhGl2-F2 (SEQ ID NO: 24): 5'-acgtgtgcagGGCGCGCCTGCTTCAACGTGGTTTATAGGGG-3'; reverse primer RNAi-GhGl2-R2 (SEQ ID NO: 25): 5'-aaattcgagctcactagtCAGTGTGAGCGTGTTAGAGAAGCT-3' as a primer pair, upland cotton DNA was used as a template for PCR amplification to obtain the GhGl2 / GhGl3 gene antisense interference fragment 1, the sequence of which is shown in SEQ ID NO: 26; the GhGl2 / GhGl3 gene antisense interference fragment 1 was purified and recovered, and the linearized proGhOLE1_D::dsGhGl2-F-RNAi vector was used for enzyme ligation and transformation to finally obtain the positive clone proGhOLE1_D::dsGhGl2-RNAi (SEQ ID NO: 27) with correct sequencing. Among them, the nucleotide sequence encoding the dsRNA targeting the GhGl2 / GhGl3 gene is shown in SEQ ID NO:28, and the nucleotide sequence of the seed-specific promoter proGhOLE1_D fused with the coding sequence of the dsRNA targeting the GhGl2 / GhGl3 gene is shown in SEQ ID NO:29.

[0276] (V) Construction of proGhOLE1_A::dsGhGl2-RNAi vector

[0277] 1) Linearization of WMV076 (plant RNAi vector provided by Jiangsu Weimi Biotechnology Co., Ltd.): Double-digest the WMV076 vector with HindⅢ and XbaⅠ restriction endonucleases to linearize the circular vector.

[0278] 2) Construction of proGhOLE1_A-RNAi intermediate vector: Using upland cotton DNA as a template, primers with linkers were designed using one-step cloning technology to amplify the promoter proGhOLE1_A.

[0279] Forward primer sequence RNAi-proGhOLE1_A-F (SEQ ID NO: 50):

[0280] 5'-gatcccccgaattaAAGCTTtgagttgggaaattaggcatcc-3';

[0281] Reverse primer sequence RNAi-proGhOLE1_A-R (SEQ ID NO: 51):

[0282] 5'-gtacggatccccggtctagaTGATTAAAAAAACAAAGAAAAGAACTTG-3'.

[0283] The amplification system and PCR reaction procedure were the same as above.

[0284] Detection and recovery of vector enzyme digestion products and PCR products: PCR products and enzyme digestion products were detected by agarose gel electrophoresis, and then The target fragment was purified and recovered using Quick Gel Extraction Kit.

[0285] Recombination of the recovered products: Homologous recombination was performed using the Seamless cloning and Assembly Kit.

[0286] The recombinant product was transformed into Escherichia coli DH5α, and PCR detection was performed using KOD OneTM PCR Master Mix. Positive clones were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After sequence alignment, the plasmids of the clones with correct sequencing were extracted using a plasmid extraction kit to obtain the proGhOLE1_A-RNAi intermediate vector.

[0287] 3) Construction of proGhOLE1_A::dsGhGl2-F-RNAi intermediate vector: The proGhOLE1_A-RNAi vector was cut with BamHI restriction endonuclease to linearize the circular vector.

[0288] GhGl2 / GhGl3 gene positive dsRNA chain amplification:

[0289] Using forward primer RNAi-proGhOLE1_A-GhGl2-F1 (SEQ ID NO: 52): 5'-tctagaccggggatccGGGGCGATGGCTATTTTCG-3'; reverse primer RNAi-proGhOLE1_A-GhGl2-R1 (SEQ ID NO: 53): 5'-cggtatatacgtacggatccTATCAGAAGATCCCAATTCGACAA-3' as a primer pair, and upland cotton DNA as a template, PCR amplification was performed to obtain the GhGl2 / GhGl3 gene positive interference fragment 2 (SEQ ID NO: 54); the GhGl2 / GhGl3 gene positive interference fragment 2 was purified and recovered, and the linearized proGhOLE1_A-RNAi vector was used for enzyme ligation and transformation, and finally the positive clone proGhOLE1_A::dsGhGl2-F-RNAi with correct sequencing was obtained, and the plasmid was extracted and stored for later use.

[0290] 4) Construction of proGhOLE1_A::dsGhGl2-RNAi vector: proGhOLE1_A::dsGhGl2-F-RNAi was double-digested with AscⅠ and SpeⅠ restriction endonucleases to linearize the circular vector.

[0291] GhGl2 / GhGl3 gene antisense dsRNA strand amplification:

[0292] Using forward primer RNAi-proGhOLE1_A-GhGl2-F2 (SEQ ID NO: 55): 5'-gtacgtgtgcagggcgcgccTATCAGAAGATCCCAATTCGACAA-3'; reverse primer RNAi-proGhOLE1_A-GhGl2-R2 (SEQ ID NO: 56): 5'-ggaaattcgagctcactagtGGGGCGATGGCTATTTTCG-3' as a primer pair, and upland cotton DNA as a template, PCR amplification was performed to obtain the GhGl2 / GhGl3 gene antisense interference fragment 2, the sequence of which is shown in SEQ ID NO:57; GhGl2 / GhGl3 gene antisense interference fragment 2 was purified and recovered, and the linearized proGhOLE1_A::dsGhGl2-F-RNAi vector was subjected to enzyme ligation and transformation using a one-step cloning enzyme, and finally the positive clone proGhOLE1_A::dsGhGl2-RNAi (SEQ ID NO:58) with correct sequencing was obtained. Among them, the nucleotide sequence encoding the dsRNA targeting the GhGl2 / GhGl3 gene is shown in SEQ ID NO:59, and the nucleotide sequence of the seed-specific promoter proGhOLE1_A fused with the coding sequence of the dsRNA targeting the GhGl2 / GhGl3 gene is shown in SEQ ID NO:60.

[0293] (VI) Construction of proGhALB2S::dsGhGl2-RNAi vector

[0294] 1) Linearization of WMV076 (plant RNAi vector provided by Jiangsu Weimi Biotechnology Co., Ltd.): Double-digest the WMV076 vector with HindⅢ and XbaⅠ restriction endonucleases to linearize the circular vector.

[0295] 2) Construction of proGhALB2S-RNAi intermediate vector: Using upland cotton DNA as a template, primers with linkers were designed using one-step cloning technology to amplify the promoter proGhALB2S.

[0296] Forward primer sequence RNAi-proGhALB2S-F (SEQ ID NO: 61):

[0297] 5'-gatcccccgaattaAAGCTTgcgactcgacgattgttccttg-3';

[0298] Reverse primer sequence RNAi-proGhALB2S-R (SEQ ID NO: 62):

[0299] 5'-gtacGGATCCCCGGTCTAGAtgttgatttatgctggtatggagaaaataatcagatgag-3'.

[0300] The amplification system and PCR reaction procedure were the same as above.

[0301] Detection and recovery of vector enzyme digestion products and PCR products: PCR products and enzyme digestion products were detected by agarose gel electrophoresis, and then The target fragment was purified and recovered using Quick Gel Extraction Kit.

[0302] Recombination of the recovered products: Homologous recombination was performed using the Seamless cloning and Assembly Kit.

[0303] The recombinant product was transformed into Escherichia coli DH5α, and PCR detection was performed using KOD OneTM PCR Master Mix. Positive clones were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After sequence alignment, the plasmids of the clones with correct sequencing were extracted using a plasmid extraction kit to obtain the proGhALB2S-RNAi intermediate vector.

[0304] 3) Construction of proGhALB2S::dsGhGl2-F-RNAi intermediate vector: The proGhALB2S-RNAi vector was cut with BamHI restriction endonuclease to linearize the circular vector.

[0305] GhGl2 / GhGl3 gene positive dsRNA chain amplification:

[0306] Using forward primer RNAi-proGhALB2S-GhGl2-F1 (SEQ ID NO: 63): 5'-aacatctagaccggggatccATGTCTTCCTCTTCTTCGTCTTCTCT-3'; reverse primer RNAi-proGhALB2S-GhGl2-R1 (SEQ ID NO: 64): 5'-cggtatatacgtacggatccCAAGAATCCCATCACCTACAGCG-3' as a primer pair, and upland cotton DNA as a template, PCR amplification was performed to obtain the GhGl2 / GhGl3 gene positive interference fragment 3 (SEQ ID NO: 65); the GhGl2 / GhGl3 gene positive interference fragment 3 was purified and recovered, and the linearized proGhALB2S-RNAi vector was used for enzyme ligation and transformation, and finally the positive clone proGhALB2S::dsGhGl2-F-RNAi with correct sequencing was obtained, and the plasmid was extracted and stored for later use.

[0307] 4) Construction of proGhALB2S::dsGhGl2-RNAi vector: proGhALB2S::dsGhGl2-F-RNAi was double-digested with AscⅠ and SpeⅠ restriction endonucleases to linearize the circular vector.

[0308] GhGl2 / GhGl3 gene antisense dsRNA strand amplification:

[0309] Using forward primer RNAi-proGhALB2S-GhGl2-F2 (SEQ ID NO: 66): 5'-gtacgtgtgcagGGCGCGCCCAAGAATCCCATCACCTACAGCG-3'; reverse primer RNAi-proGhALB2S-GhGl2-R2 (SEQ ID NO: 67): 5'-GGAAATTCGAGCTCACTAGTATGTCTTCCTCTTCTTCGTCTTCTCT-3' as a primer pair, and upland cotton DNA as a template, PCR amplification was performed to obtain the GhGl2 / GhGl3 gene antisense interference fragment 3, the sequence of which is shown in SEQ ID NO:68; GhGl2 / GhGl3 gene antisense interference fragment 3 was purified and recovered, and the linearized proGhALB2S::dsGhGl2-F-RNAi vector was subjected to enzyme ligation and transformation using a one-step cloning enzyme, and finally the positive clone proGhALB2S::dsGhGl2-RNAi (SEQ ID NO:69) with correct sequencing was obtained. Among them, the nucleotide sequence encoding the dsRNA targeting the GhGl2 / GhGl3 gene is shown in SEQ ID NO:70, and the nucleotide sequence of the seed-specific promoter proGhALB2S fused with the coding sequence of the dsRNA targeting the GhGl2 / GhGl3 gene is shown in SEQ ID NO:71.

[0310] (VII) Construction of proGhOLE1_A::GhGl2 / GhGl3-miRNA, proGhOLE1_D::GhGl2-miRNA and proGhALB2S::GhGl2-miRNA vectors

[0311] The miRNA silencing vector is modified based on the Arabidopsis miR319a gene silencing sequence. The Arabidopsis miR319a gene silencing sequence is divided into the first segment, the tail segment and the core silencing segment.

[0312] Arabidopsis miR319a gene silencing sequence (SEQ ID NO: 30):

[0313]

[0314] First fragment (SEQ ID NO:31):

[0315]

[0316] Tail fragment (SEQ ID NO: 32):

[0317]

[0318]

[0319] Core Silencing Fragment (SEQ ID NO: 33):

[0320]

[0321] The specific construction method of proGhOLE1_A::GhGl2 / GhGl3-miRNA vector is as follows:

[0322] 1) First, the Arabidopsis miR319a first fragment and tail fragment fusion fragment Median Vector (SEQ ID NO: 34) was synthesized by Sangon Biotechnology Co., Ltd., and StuI and SnaBI restriction sites were added between the first fragment and the tail fragment for the subsequent addition of the core silencing fragment. Primers with adapters were designed using one-step cloning technology to amplify the sequence Median Vector.

[0323] Forward primer sequence Median Vector-F: 5'-CAAACACACGCTCGGACGC-3' (SEQ ID NO: 35);

[0324] Reverse primer sequence Median Vector-R: 5′-tcctaggctacgtaggatccCATGGCGATGCCTTAAATAAAGA-3′ (SEQ ID NO: 36).

[0325] 2) Linearization of VM062 (plant overexpression vector provided by Jiangsu Weimi Biotechnology Co., Ltd.): VM062 vector was double-digested with HindⅢ and BamHI restriction enzymes to linearize the circular vector.

[0326] 3) Amplification of seed-specific promoters proGhOLE1_A, proGhOLE1_D and proGhALB2S: PCR amplification was performed using upland cotton DNA as a template to obtain seed-specific promoters proGhOLE1_A, proGhOLE1_D and proGhALB2S. The amplification primers are as follows:

[0327] Seed-specific promoter proGhOLE1_A amplification primers:

[0328] Forward primer miRNA-proGhOLE1_A-F:

[0329]

[0330] Reverse primer miRNA-proGhOLE1_A-R:

[0331]

[0332] Seed-specific promoter proGhOLE1_D amplification primers:

[0333] Forward primer miRNA-proGhOLE1_D-F:

[0334]

[0335] Reverse primer miRNA-proGhOLE1_D-R:

[0336]

[0337] Seed-specific promoter proGhALB2S amplification primers:

[0338] Forward primer miRNA-proGhALB2S_A-F:

[0339]

[0340] Reverse primer miRNA-proGhALB2S_A-R:

[0341]

[0342] The Median Vector and proGhOLE1_A, proGhOLE1_D, and proGhALB2S fragments were obtained by PCR amplification. After purification and recovery, they were ligated with the linearized VM062 vector using a one-step cloning enzyme and transformed to obtain the correctly sequenced positive clones proGhOLE1_A-Median Vector, proGhOLE1_D-Median Vector, and proGhALB2S-Median Vector. The plasmids were extracted and stored for later use.

[0343] 4) Construction of proGhOLE1_A::GhGl2 / GhGl3-miRNA, proGhOLE1_D::GhGl2-miRNA and proGhALB2S::GhGl2-miRNA vectors: First, the GhGl2 / GhGl3 artificial miRNA sequence was designed at http: / / wmd3.weigelworld.org / (Warthman et al., 2008). The designed artificial miRNA fragment was reverse complemented, and positions 12 and 16 were transposed to GA, CA, TA or AT to obtain the forward sequence of GhGl2 / GhGl3 artificial miRNA (SEQ ID NO: 39, TGATACGGTAT T GTA A CGCTC) and reverse sequence (SEQ ID NO:40, GAGCG T TAC A ATACCGTATCA), where the underline indicates the replaced position. The forward and reverse sequences of the artificial GhGl2 / GhGl3 miRNA were fused with the core silencing fragment of Arabidopsis miR319a by gene synthesis by Sangon Biotech (Shanghai) Co., Ltd. and named Stem-loop: TGATACGGTAT T GTA A CGCTCcacaggtcgtgatatgattcaattagcttccgactcattcatccaaataccgagtcgccaaaattcaaactagactcgttaaatgaatgaatgatgcggtagacaaattggatcattgattctcttt gaGAGCG T TAC A ATACCGTATCA (SEQ ID NO: 41). Primers with adapters were designed using one-step cloning technology to amplify the Stem-loop containing the core silencing fragment of the GhGl2 / GhGl3 gene.

[0344] Forward Primer Stem-loop-F:

[0345]

[0346] Reverse primer Stem-loop-R:

[0347]

[0348] The proGhOLE1_A-Median Vector, proGhOLE1_D-Median Vector and proGhALB2S-Median Vector were double-digested with StuⅠ and SnaBI restriction endonucleases to linearize the circular vectors. The Stem-loop core silencing fragment with the adapter was purified and recovered, and then ligated and transformed with the linearized vectors using a one-step cloning enzyme, and finally the positive clones proGhOLE1_A::GhGl2 / GhGl3-miRNA, proGhOLE1_D::GhGl2-miRNA and proGhALB2S::GhGl2-miRNA with correct sequencing were obtained. Among them, the nucleotide sequence encoding the microRNA targeting the GhGl2 / GhGl3 gene (first fragment-GhGl2 / GhGl3 core silencing fragment-tail fragment) is shown as SEQ ID NO:44, and the nucleotide sequences of proOLE1_A-first fragment-GhGl2 / GhGl3 core silencing fragment-tail fragment, proOLE1_D-first fragment-GhGl2 / GhGl3 core silencing fragment-tail fragment and proALB2S-first fragment-GhGl2 / GhGl3 core silencing fragment-tail fragment are shown as SEQ ID NOs:45, 76 and 77, respectively.

[0349] Sequence Listing

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

Claims

1. A method for inhibiting the formation of pigment glands in cotton seeds, the method comprising causing the cotton seeds to express an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 proteins, wherein the inhibitory molecule is expressed by a seed-specific promoter of the cotton.

2. A method for reducing the cottonpol content in cotton seeds, the method comprising causing the cotton seeds to express an inhibitory molecule that inhibits the expression and / or activity of Gl2 / Gl3 proteins, wherein the inhibitory molecule is expressed by a cotton seed-specific promoter.

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

4. The method according to any one of claims 1 to 3, wherein the seed-specific promoter is a promoter derived from the OLE_A, OLE_D or 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: 1-3.

5. The method of any one of claims 1 to 4, wherein the inhibitory molecule is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen-binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene.

6. The method of claim 5, wherein the inhibitory molecule is dsRNA, which targets mRNA transcribed from the Gl2 / Gl3 gene.

7. The method of claim 6, wherein the nucleotide sequence encoding the dsRNA 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: 28, 59 or 70.

8. The method of claim 5, wherein the inhibitory molecule is a microRNA, and the microRNA targets the mRNA generated by transcription of the Gl2 / Gl3 gene; Preferably, the nucleotide sequence encoding the microRNA 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:

44.

9. The method of any one of claims 6-8, comprising integrating into the genome of the 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: 29, 45, 60, 71, 76 or 77.

10. The method according to any one of claims 1 to 9, comprising the steps of: (1) transforming the nucleotide sequence containing the seed-specific promoter and encoding the inhibitory molecule 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.

11. The method according to any one of claims 1 to 10, comprising causing the cotton seeds to express two or more inhibitory molecules that inhibit the expression and / or activity of Gl2 / Gl3 proteins, wherein the two or more inhibitory molecules are expressed by the same or different cotton seed-specific promoters, preferably the cotton seed-specific promoters are each independently selected from promoters derived from OLE_A, OLE_D or ALB2S genes.

12. The method of any one of claims 1-11, wherein the method results in the absence of pigment glands in the cotton seeds compared to pigment glands in wild-type cotton seeds, and the occurrence and development of pigment glands in the cotton plants is not affected.

13. The method of any one of claims 1-12, 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 seeds.

14. The method of any one of claims 1 to 13, 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.

15. Cotton cell, seed, tissue, organ or plant obtained by the method of any one of claims 1 to 14.

16. A cotton cell, seed, tissue, organ or plant comprising a nucleotide sequence encoding an inhibitory molecule that inhibits the expression and / or activity of a G12 / G13 protein, wherein the inhibitory molecule is expressed from a cotton seed-specific promoter.

17. The cotton cell, seed, tissue, organ or plant of claim 16, wherein the seed-specific promoter: (1) initiating the expression of the inhibitory molecule no later than the expression time of Gl2 / Gl3 protein during seed development of wild-type cotton; (2) making the expression level of the inhibitory molecule no less than the sum of the expression levels of the wild-type Gl2 and Gl3 proteins; and (3) The expression of the inhibitory molecule is initiated only during the development of cotton seeds, and the expression of the inhibitory molecule is not initiated or substantially not initiated after the germination of mature seeds and at other stages of cotton plant development.

18. The cotton cell, seed, tissue, organ or plant of claim 16 or 17, wherein the seed-specific promoter is a promoter derived from the OLE_A, OLE_D or 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: 1-3.

19. The cotton cell, seed, tissue, organ or plant of any one of claims 16-18, wherein the inhibitory molecule is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen-binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene.

20. The cotton cell, seed, tissue, organ or plant of claim 19, wherein the inhibitory molecule is dsRNA, which targets mRNA transcribed from the Gl2 / Gl3 gene.

21. The cotton cell, seed, tissue, organ or plant of claim 20, wherein the nucleotide sequence encoding the dsRNA 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:28, 59 or 70.

22. The cotton cell, seed, tissue, organ or plant of claim 19, wherein the inhibitory molecule is a microRNA, and the microRNA targets the mRNA generated by transcription of the Gl2 / Gl3 gene; Preferably, the nucleotide sequence encoding the microRNA 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:

44.

23. The cotton cell, seed, tissue, organ or plant of any one of claims 20-22, 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: 29, 45, 60, 71, 76 or 77 into the genome of cotton.

24. The cotton cell, seed, tissue, organ or plant of any one of claims 16 to 23, obtained by the following steps: (1) transforming the nucleotide sequence containing the seed-specific promoter and encoding the inhibitory molecule 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.

25. The cotton cell, seed, tissue, organ or plant of any one of claims 16 to 24, comprising a nucleotide sequence encoding an inhibitory molecule for the expression and / or activity of two or more Gl2 / Gl3 proteins, wherein the two or more inhibitory molecules are expressed by the same or different cotton seed-specific promoters, preferably the cotton seed-specific promoters are each independently selected from a promoter derived from the OLE_A, OLE_D or ALB2S gene.

26. A nucleic acid construct comprising, from 5' to 3', a cotton seed-specific promoter and a nucleotide sequence encoding an inhibitory molecule that inhibits the expression and / or activity of a G12 / G13 protein.

27. The nucleic acid construct of claim 26, wherein the seed-specific promoter is a promoter derived from the OLE_A, OLE_D or 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: 1-3.

28. The nucleic acid construct of claim 26 or 27, wherein the inhibitory molecule is selected from: dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or antigen-binding fragment thereof, small molecule ligand or a combination thereof that inhibits or silences a gene.

29. The nucleic acid construct of claim 28, wherein the inhibitory molecule is a dsRNA that targets the mRNA generated by transcription of the Gl2 / Gl3 gene.

30. The nucleic acid construct of claim 29, wherein the nucleotide sequence encoding the dsRNA 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: 28, 59 or 70.

31. The nucleic acid construct of claim 28, wherein the inhibitory molecule is a microRNA that targets mRNA generated by transcription of the Gl2 / Gl3 gene; Preferably, the nucleotide sequence encoding the microRNA 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:

44.

32. The nucleic acid construct of any one of claims 29-31, 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:29, 45, 60, 71, 76 or 77.

33. The nucleic acid construct of any one of claims 26 to 32, comprising a nucleotide sequence encoding two or more inhibitory molecules that inhibit the expression and / or activity of Gl2 / Gl3 proteins, wherein the two or more inhibitory molecules are operably linked to the same or different cotton seed-specific promoters, preferably the cotton seed-specific promoters are each independently selected from promoters derived from OLE_A, OLE_D or ALB2S genes.

34. The nucleic acid construct of any one of claims 26 to 33 for use in inhibiting pigment gland formation in cotton seeds.

35. The nucleic acid construct of any one of claims 26 to 33, for use in reducing the content of gossypol in cotton seeds.

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