GSNOR gene and application thereof in improving cotton plants

Abstract

The invention provides a GSNOR gene and an application of the GSNOR gene in improved cotton plants. The GSNOR gene is subjected to isolation cloning and functional identification, and the expression quantity of the GSNOR gene is regulated and controlled through molecular biology and transgenic technologies, so that the regulation of plant growth and development is realized, and high-quality transgenic cotton with remarkably increased fiber quantity is obtained. The invention discloses functions and application of GSNOR protein and nitric oxide micromolecule signals, and particularly has positive effects in promoting fiber cell differentiation and improving cotton fiber density quality characters. The invention provides a new action target for variety improvement of cotton plants.

Description

Technical Field

[0001] This invention belongs to the fields of botany and molecular biology; more specifically, this invention relates to a GSNOR gene and its application in improving cotton plants. Background Technology

[0002] Cotton, a plant of the genus *Gossypium*, is an important economic crop, providing the market with a large amount of natural textile raw materials. As the world's largest producer and consumer of cotton, China faces challenges in improving the quality and increasing the yield of upland cotton, as well as optimizing the adaptability of long-staple cotton. Given the limited planting area, improving the quality of *Gossypium* plants is a pressing issue.

[0003] Cotton fibers develop from single cells in the epidermis of the cotton ovule. Their differentiation and development can be divided into four stages: fiber cell differentiation and protrusion, rapid elongation, secondary cell wall synthesis, and dehydration maturation. Fiber cell differentiation determines the final fiber density, which in turn determines the yield per unit area of ​​cotton fiber.

[0004] The length of cotton fibers depends primarily on the cotton variety, growing conditions, and processing. Longer fibers result in a softer, more comfortable fabric; for example, long-staple cotton is widely recognized as a high-end cotton fiber. The fineness of cotton fibers depends mainly on the cotton variety and growing conditions. The maturity of cotton fibers refers to the degree of cell wall thickening, i.e., the degree of fiber maturity. As maturity increases, cell walls thicken and the lumen decreases.

[0005] Therefore, increasing cotton fiber yield is an important issue facing cotton breeding. Summary of the Invention

[0006] The purpose of this invention is to provide a GSNOR gene and its application in improving cotton plants.

[0007] In a first aspect of the invention, a method for improving cotton plants is provided, comprising: downregulating GSNOR or increasing nitric oxide in cotton plants, thereby improving cotton fiber quality or yield traits.

[0008] In one or more embodiments, improving cotton fiber quality or yield includes: increasing fiber density, increasing the number of mature fibers, and increasing fiber yield.

[0009] In one or more embodiments, the GSNOR is regulated by HD1, which binds to the promoter of the GSNOR and regulates the transcription of the GSNOR.

[0010] In one or more embodiments, the GSNOR participates in regulating the synthesis of fibroblast cell walls and controls the differentiation or growth of fibroblasts.

[0011] In one or more embodiments, the GSNOR controls the differentiation and growth of fibroblasts by participating in the regulation of fibroblast cell wall synthesis; downregulating GSNOR (including knockout or knockdown) slows down fibroblast cell wall synthesis and promotes fibroblast differentiation and growth.

[0012] In one or more embodiments, increasing nitric oxide includes: reducing nitric oxide consumption or increasing the amount of nitric oxide in the body.

[0013] In one or more embodiments, a nitric oxide upregulator is applied to increase nitric oxide content; more preferably, the nitric oxide upregulator includes (but is not limited to): a nitric oxide donor (including compounds that generate nitric oxide), an agent that decomposes to produce nitric oxide, a GSNOR downregulator, or an exogenous nitric oxide synthase; preferably, the nitric oxide donor includes S-Nitrosoglutathione (GSNO).

[0014] In one or more embodiments, the GSNOR is reduced to decrease nitric oxide consumption in the plant, thereby increasing the amount of nitric oxide in the plant.

[0015] In one or more embodiments, downregulating GSNOR includes: downregulating the expression or activity of GSNOR; preferably, it includes (but is not limited to): performing gene silencing or gene knockout on the GSNOR; preferably, performing the gene knockout using gene editing methods (including gene editing based on the CRISPR system) or homologous recombination methods; more preferably, performing the gene editing using sgRNA of the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4.

[0016] In one or more embodiments, gene silencing is performed by interfering with GSNOR, including: performing gene silencing with VIGS molecules, dsRNA, antisense nucleic acids, small interfering RNA, microRNA, or constructs that can express or form said VIGS molecules, dsRNA, antisense nucleic acids, small interfering RNA, or microRNA as targets of inhibition or silencing of the GSNOR encoding gene or its transcript.

[0017] In one or more embodiments, the gossy plants include those expressing GSNOR or its homologs.

[0018] In one or more embodiments, the gossy plants include those that possess a nitric oxide metabolic system (possessing a mechanism for controlling nitric oxide synthesis).

[0019] In one or more embodiments, the *Gossypium* species includes upland cotton (*Gossypium hirsutum*).

[0020] In one or more embodiments, the GSNOR includes its homologs (protein or gene level), which are proteins or encoding genes from the same species (upland cotton) or different species (species other than upland cotton) that have sequence homology or function with SEQ ID NO:2 or the polynucleotide encoding it.

[0021] In one or more embodiments, the construct (expression vector) includes: a gossypium plant expression vector.

[0022] In one or more embodiments, the construct (expression vector) includes: a non-viral vector and a viral vector.

[0023] In one or more embodiments, the downsizing indicates a significant downsizing, such as a downsizing of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or more.

[0024] In one or more embodiments, a method for improving a cotton plant is provided, comprising preparing a transgenic plant: (1) providing a vector carrying an sgRNA with nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4; (2) providing Agrobacterium carrying the vector in step (1); (3) contacting a plant cell or tissue with the Agrobacterium in step (2) to downregulate the expression of GSNOR or disable its function; and (4) transgenic plant cells.

[0025] In another aspect of the invention, the application of GSNOR downregulators or nitric oxide upregulators (reagents that increase nitric oxide content in vivo or in vitro) is provided for improving cotton plants and enhancing cotton fiber quality or yield traits.

[0026] In one or more embodiments, the GSNOR downregulator includes (but is not limited to): a downregulator that knocks out or silences the GSNOR gene or inhibits the activity of the GSNOR protein; preferably, it includes: a reagent for knocking out the GSNOR gene based on gene editing (such as gene editing based on the CRISPR system), a reagent for knocking out the GSNOR gene based on homologous recombination, and an interfering molecule that specifically interferes with the expression of the GSNOR gene; preferably, the downregulator includes sgRNA of the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4.

[0027] In one or more embodiments, the nitric oxide upregulator includes (but is not limited to): a nitric oxide donor (including compounds that generate nitric oxide), an agent that produces nitric oxide upon decomposition, a GSNOR downregulator, or an exogenous nitric oxide synthase; preferably, the nitric oxide donor includes nitrosoglutathione.

[0028] In one or more embodiments, the interfering molecule is a VIGS molecule, dsRNA, antisense nucleic acid, small interfering RNA, or microRNA that targets the encoding gene of GSNOR or its transcript for suppression or silencing, or a construct that can express or form the VIGS molecule, dsRNA, antisense nucleic acid, small interfering RNA, or microRNA.

[0029] In one or more embodiments, the GSNOR comprises: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO:2; (b) a GSNOR derivative having the function of the polypeptide (a) formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3, or 1-2) amino acid residues of the amino acid sequence shown in (a); (c) a GSNOR derivative or active fragment having the function of the polypeptide (a) with a sequence having ≥80% homology to the amino acid sequence shown in SEQ ID NO:2 (e.g., ≥82%, ≥85%, ≥90%, ≥92%, ≥94%, ≥96%, ≥98%, or ≥99% homology); or (d) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N terminus.

[0030] In another aspect of the invention, a Gossypium cell, tissue, or organ is provided, wherein the Gossypium is a Gossypium expressing GSNOR or containing a nitric oxide metabolic system, wherein it contains an exogenous GSNOR downregulator or nitric oxide upregulator.

[0031] In one or more embodiments, the GSNOR downregulator includes (but is not limited to): a downregulator that knocks out or silences the GSNOR gene or inhibits the activity of the GSNOR protein; preferably, it includes: a reagent for knocking out the GSNOR gene based on gene editing (such as gene editing based on the CRISPR system), a reagent for knocking out the GSNOR gene based on homologous recombination, and an interfering molecule that specifically interferes with the expression of the GSNOR gene; preferably, the downregulator includes sgRNA of the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4;

[0032] In one or more embodiments, the nitric oxide upregulator includes (but is not limited to): a nitric oxide donor (such as a nitric oxide-generating compound), a GSNOR downregulator; preferably, the nitric oxide donor includes nitrosoglutathione.

[0033] In one or more embodiments, the Gossypium plant cells, tissues or organs do not directly generate living Gossypium plants, or are not used as propagation material for Gossypium plants.

[0034] In another aspect of the present invention, a method for identifying the quality or yield traits of cotton fibers in a *Gossypium* species is provided, comprising: testing the presence of GSNOR or nitric oxide in the *Gossypium* species; if the *Gossypium* species has low GSNOR expression or activity (e.g., below the average value for that species) or high nitric oxide content (e.g., above the average value for that species), then its cotton fiber quality or yield is high (including high fiber density and a large number of mature fibers); if the *Gossypium* species has high GSNOR expression or activity (e.g., above the average value for that species) or low nitric oxide content (e.g., below the average value for that species), then its cotton fiber quality or yield is low (including low fiber density and a small number of mature fibers).

[0035] In another aspect of the invention, a method is provided for screening potential substances that improve cotton fiber quality or yield traits, comprising:

[0036] (1) Treat a system of Gossypium cells or tissues (e.g., fibroblasts or fibrous tissues) with the candidate substance, the system expressing GSNOR and / or having a nitric oxide metabolic system; and

[0037] (2) Detect the expression or activity of GSNOR or the amount of nitric oxide in the system; if the candidate substance statistically reduces (e.g., by more than 5%, 10%, 15%, 20%, 30%, 50%, or 80%) the expression or activity of GSNOR and / or increases the amount of nitric oxide, it indicates that the candidate substance is a potential substance for improving the quality or yield of cotton fibers.

[0038] In one or more embodiments, the screening method further includes setting up a control group to clearly distinguish the increase or decrease in the expression or activity of GSNOR, or the increase or decrease in the amount of nitric oxide.

[0039] In one or more embodiments, the method further includes: conducting further cell experiments and / or transgenic experiments on the obtained potential substances to further identify substances from the candidate substances that are highly effective in improving cotton fiber quality or yield traits.

[0040] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as downregulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)) targeting the GSNOR protein or its encoding gene, or upstream or downstream proteins or genes thereupon, CRISPR constructs, small molecule compounds, etc.; or donors or reactants targeting nitric oxide.

[0041] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0042] Figure 1 GhGSNOR constructs carrier information.

[0043] (A) pRGEB32-GhU6.7-NPT2 vector information, the target fragment is inserted between HpaI and SbfI.

[0044] (B)35S::2301 vector information, the target fragment is inserted between XbaI and KpnI; the vector map was generated by SnapGene.

[0045] Figure 2 Analysis of GhGSNOR expression characteristics.

[0046] The phenotype of the 35S::GhGSNOR transgenic line OE13 shows that the number of fibers is reduced and the length is shortened.

[0047] The phenotype of the (B)crGhGSNOR transgenic line CR76 shows an increased number of fibers while maintaining the same length.

[0048] (C) qPCR was used to detect the expression of GhGSNOR in different transgenic lines OE13 of 35S::GhGSNOR. The expression levels of each line were higher than those of wild-type R15 cotton.

[0049] (D)qPCR detection of the expression characteristics of GhGSNOR during fiber development in wild-type R15 cotton, where -1D represents the sample 1 day before flowering, 0D represents the sample on the day of flowering, and so on.

[0050] Figure 3 Statistics on fiber quantity in GhGSNOR transgenic lines.

[0051] (A) Number of fibrous cells on the surface of ovules of different transgenic lines on the day of flowering (top) and magnified display (bottom). Among them, R15 is wild type, OE13 represents GhGSNOR overexpression line with a significantly reduced number of fibroblasts (15%), and CR76 represents GhGSNOR gene knockout line with a significantly increased number of fibroblasts (26%). Bar = 100 μm.

[0052] (B) Statistical results of surface fiber quantity in GhGSNOR transgenic lines. The error bar represents the standard deviation (STDEV); "**" indicates a student's t-test analysis with the hypothesis of equal variance between the experimental and control groups, P<0.01; "***" indicates a student's t-test analysis with the hypothesis of equal variance between the experimental and control groups, P<0.005, n≥20.

[0053] Figure 4Analysis of the positioning and functions of GhGSNOR.

[0054] (AB) Localization of GhGSNOR, A is the distribution of GFP, B is the distribution of the GhGSNOR-GFP fusion protein, Bar = 50 μm.

[0055] (C) Yeast two-hybrid experiment to verify the specific binding of GhHD1 to the promoter of GhGSNOR (the promoter was used upstream of ATG from -1 to -300 in the experiment). The left column is the control, showing that yeast can grow normally in the asexual medium. The right column shows the growth status of yeast after the addition of the self-activation inhibitor.

[0056] Figure 5 , one The effects of nitric oxide on fibroblast development.

[0057] (A) Effects of different treatments on fibroblast number in in vitro ovule culture. The first row is an electron microscope image at 50x magnification, and the second row is an electron microscope image at 200x magnification. CK is the control group, cPTIO is the nitric oxide inhibitor treatment group, and GSNO is the nitric oxide donor treatment group. Bar = 100 μm.

[0058] (B) Statistical analysis of surface fiber quantity in different treatment groups. The error bar represents the standard deviation (STDEV); "*" indicates that the experimental group and the control group were subjected to a two-sample equivariance hypothesis student's t-test analysis, P<0.05, n≥20.

[0059] Figure 6 , one Nitric oxide affects the development of fiber cell walls.

[0060] (A) Cell wall thickness under transmission electron microscopy for different treatments of nitric oxide; the cells in the second row are magnified details of the fibroblasts circled by the white curve in the first row, with the black arrow pointing to the cell wall. After inhibiting nitric oxide with cPTIO, the fibroblast cell wall became significantly thicker. After increasing the nitric oxide concentration with GSNO, the fibroblast cell wall became significantly thinner. Bar = 1 μm.

[0061] (B) Statistical analysis of cell wall thickness under different nitric oxide treatments using transmission electron microscopy. The error bars represent the standard deviation (STDEV). “***” indicates a two-sample equivariance hypothesis Student’s T test between the experimental group and the control group, with P < 0.001. “****” indicates a two-sample equivariance hypothesis Student’s T test between the experimental group and the control group, with P < 0.0005. n = 100. Detailed Implementation

[0062] Through in-depth research, the inventors have for the first time revealed methods for improving cotton plants and enhancing cotton fiber quality or yield traits. This invention involves the isolation, cloning, and functional identification of the GSNOR gene. By regulating the expression level of the GSNOR gene through molecular biology and transgenic technology, the invention achieves regulation of plant growth and development, resulting in high-quality transgenic cotton with a significantly increased fiber quantity. This invention discloses the functions and uses of the GSNOR protein and nitric oxide small molecule signaling, particularly its positive effects on promoting fiber cell differentiation and improving cotton fiber density and quality traits. This invention provides a new target for the improvement of cotton plant varieties and has broad application value.

[0063] the term

[0064] As used herein, "plant" includes *Gossypium* species that express GSNOR or its homologs and also possess a nitric oxide metabolic system. Based on knowledge in the art, plants possessing GSNOR, nitric oxide, and the signaling pathways they participate in inherently possess the mechanisms of action claimed in this invention, and can achieve the technical effects claimed in this invention. In some preferred embodiments, the plant is an economic crop, preferably a *Gossypium* species. More specifically, upland cotton, etc.

[0065] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design and can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants can also be individuals of transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including leaves, cotton bolls, fiber cells, ovules, seeds, or parts thereof.

[0066] As used herein, “upregulation” includes: promotion, overexpression, enhancement, etc., which are statistically significant or markedly upregulated, promoted, enhanced, or increased, such as upregulation, promotion, enhancement, or increase of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or higher.

[0067] As used herein, “downregulation” includes: weakening, reducing, lowering, inhibiting; indicating significant downregulation, weakening, lowering, inhibiting, such as downregulation, weakening, lowering, inhibiting or downregulating by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or lower.

[0068] As used herein, “high expression or high activity” means that the expression or activity of a target gene / protein in a specific plant (e.g., a modified plant) is statistically significantly increased compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0069] As used herein, “low expression or low activity” means that the expression or activity of a target gene / protein in a particular plant (e.g., a modified plant) is statistically significantly reduced compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or less.

[0070] As used in this article, the terms "pathway," "signaling pathway," and "regulatory pathway" are used interchangeably.

[0071] GSNOR and its regulatory role

[0072] This invention isolates, clones, and functionally identifies the GSNOR gene. By regulating the expression level of the GSNOR gene through molecular biology and transgenic technology, the growth and development of plants are regulated, resulting in high-quality transgenic cotton with a significant increase in fiber content.

[0073] Two GSNOR homologous genes exist in allotetraploid cotton, which are key genes for scavenging nitrosylation modification. The inventors used single-cell sequencing and in situ hybridization (ISH) to verify that one copy is specifically expressed in fiber cells, and constructed GSNOR knockout and overexpression lines. In the knockout line, fiber density increased significantly, while fiber length remained relatively unchanged; however, in the overexpression line, both fiber density and length decreased significantly. This indicates that GSNOR negatively regulates fiber density, and inhibiting GSNOR function can significantly increase fiber density.

[0074] In this invention, unless otherwise specified, the GSNOR protein includes its homologs (homologous proteins). The GSNOR is a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO:2. This invention also includes sequence variants having the same function as the GSNOR protein.

[0075] The variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the GSNOR protein (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:2; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the GSNOR protein is also included in this invention.

[0076] In this invention, the term "GSNOR protein" also includes its homologs. It should be understood that while this invention preferably studies GSNOR proteins derived from specific species, other polypeptides or genes derived from the same or other species, particularly Malvaceae plants, that are highly homologous to the said GSNOR protein (e.g., having more than 70%, more particularly 80%, 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of this invention.

[0077] This invention also relates to a polynucleotide sequence encoding the GSNOR protein of this invention or a sequence variant thereof. The polynucleotide may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO:2 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:2, but differing from the gene sequence shown in SEQ ID NO:1. This invention also relates to variants (variants) of the aforementioned polynucleotide that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention.

[0078] Based on the inventor's new discovery, a use is provided for GSNOR protein or its regulatory molecule (upregulator) for: improving cotton plants, enhancing cotton fiber quality or yield traits; preparing preparations to enhance cotton fiber quality or yield traits; or, as a molecular marker for identifying cotton fiber quality or yield traits in cotton plants.

[0079] Once the function of the GSNOR is known, various methods well known to those skilled in the art can be used to implement regulation. The downregulators of the GSNOR or its encoding gene include inhibitors, antagonists, blockers, and blocking agents, and these terms are used interchangeably.

[0080] The downregulator of GSNOR or its encoding gene refers to any substance that can reduce the activity of GSNOR, reduce the stability of GSNOR or its encoding gene, downregulate GSNOR expression, reduce the effective duration of GSNOR, or inhibit the transcription and translation of the GSNOR gene. These substances can all be used in this invention as substances useful for downregulating GSNOR, thereby improving cotton fiber quality or yield traits. For example, the downregulator is: interfering RNA molecules or antisense nucleotides that specifically interfere with GSNOR gene expression; antibodies or ligands that specifically bind to the protein encoded by the GSNOR gene; etc.

[0081] As an optional embodiment of the present invention, the downregulator can be a GSNOR-specific interfering RNA molecule. Those skilled in the art will understand that such interfering RNA molecules can be prepared using the GSNOR sequence information provided in this invention. There are no particular limitations on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis, in vitro transcription, etc. The interfering RNA can be delivered into cells using appropriate transfection reagents, or it can be delivered into cells using various techniques known in the art.

[0082] As an alternative approach to this invention, RNAi is used to suppress GSNOR. RNAi is an evolutionarily conserved cellular defense mechanism used to control the expression of exogenous genes in most eukaryotes, including humans. RNAi is typically triggered by double-stranded RNA (dsRNA) and induces sequence-specific mRNA degradation of the single-stranded target RNA. The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), typically produced by the enzymatic cleavage of long dsRNA within the cell. siRNAs are typically about 21 nucleotides long (e.g., 21-23 nucleotides). After the small RNA or RNAi is introduced into the cell, the sequence is believed to be delivered to an enzyme complex called the RISC (RNA-induced silencing complex). The RISC recognizes the target and cleaves it with a nuclease. Notably, if a larger RNA sequence is delivered to the cell, the RNase III enzyme (Dicer) converts the longer dsRNA into a 21-23 nt ds-siRNA fragment.

[0083] As an optional embodiment of the present invention, the downregulator can be the VIGS (virus-induced gene silencing) method, which inhibits GSNOR and thus exerts a regulatory effect. The VIGS method is a genetic technique that uses a recombinant virus containing a target gene fragment to suppress the expression of endogenous genes in plants.

[0084] As an alternative approach to this invention, shRNA technology is used for interference. shRNA is an RNA sequence that can rotate a tight hairpin, which can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into the cell and utilizes a promoter (such as U6) to ensure that shRNA is always expressed. This vector is typically delivered to daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure is cleaved into siRNA by cellular mechanisms and then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the bound siRNA. shRNA is transcribed by RNA polymerase III.

[0085] As an alternative aspect of this invention, GSNOR expression is modulated using antisense compounds that specifically hybridize with one or more nucleic acids encoding GSNOR. The specific hybridization of oligomers with their target nucleic acids interferes with the normal function of the nucleic acids. This modulation of target nucleic acid function by compounds that specifically hybridize with the target nucleic acid is commonly referred to as "antisense."

[0086] As a particularly preferred embodiment of the present invention, a CRISPR / Cas (e.g., Cas9) system can be used for targeted gene editing to knock out the GSNOR gene in the target disease region. Common methods for knocking out the GSNOR gene include co-transferring sgRNA or a nucleic acid capable of forming said sgRNA, Cas9 mRNA or a nucleic acid capable of forming said Cas9 mRNA, to the target region or target cells. After identifying the target site, known methods can be used to introduce sgRNA and Cas9 into the cells. The nucleic acid capable of forming said sgRNA is a nucleic acid construct or expression vector, or the nucleic acid capable of forming said Cas9 mRNA is a nucleic acid construct or expression vector. These expression vectors are introduced into the cells, thereby forming active sgRNA and Cas9 mRNA within the cells.

[0087] As an optional approach of this invention, homologous recombination can be used to specifically target GSNOR, causing expression defects or deletions. Alternatively, Cre and loxp methods can be applied to selectively knock out, reduce, or inactivate related genes in the genome of animals or cells.

[0088] In one embodiment of the present invention, the downregulator is a small molecule compound targeting GSNOR. Those skilled in the art can employ methods suitable for screening small molecule compounds to perform such screening. The screening can rely on various existing or future compound libraries in the art, or new compound libraries can be established independently.

[0089] The above are some representative methods for downregulating GSNOR. It should be understood that the present invention provides a novel target. After those skilled in the art understand the overall scheme of the present invention, other methods known in the art or methods under development can also be used to regulate GSNOR, and these methods are also included in the present invention.

[0090] The present invention also includes plants obtained using any of the foregoing methods, said plants comprising: transgenic plants into which the coding nucleic acid of the polypeptide has been transferred.

[0091] Nitric oxide and its regulatory role

[0092] Given the correlation between GSNOR and nitrosylation modification, this invention also discloses the function and uses of nitric oxide small molecule signaling, especially its positive role in promoting fibroblast differentiation and improving cotton fiber density and quality traits, and has broad application value.

[0093] Nitric oxide (NO) is a gaseous signaling molecule that plays a vital role in the growth and development of both plants and animals. It is particularly important in the medical field, especially in its significant contributions to cardiovascular disease treatment. In plants, NO plays a role in protecting against adverse environmental conditions such as drought and cold. Besides signaling, NO affects plant development primarily through nitrosation, controlling protein function and thus regulating plant development. However, research on the effects of NO on cotton fiber development in *Gossypium* species remains lacking.

[0094] The inventors used DAF-FM DA to trace nitric oxide. In the early stages of fiber development, nitric oxide signals were specifically distributed in fibroblasts. After exogenous application of nitric oxide, the density of fibroblasts increased significantly, while the addition of nitric oxide inhibitors significantly reduced the density of fibroblasts. This further demonstrates that nitric oxide can promote fibroblast differentiation and increase fibroblast density.

[0095] The inventors' research results have pioneeringly demonstrated that nitric oxide and its regulatory gene GSNOR play an important role in regulating fiber cell density and have great application value in improving fiber yield and quality.

[0096] Any substance that can increase the supply of nitric oxide or increase the amount of nitric oxide in the body of gossy plants can be used in this invention.

[0097] As a preferred approach, a nitric oxide upregulator is administered to increase the supply of nitric oxide; more preferably, the nitric oxide upregulator includes (but is not limited to): a nitric oxide donor (including compounds that generate nitric oxide), an agent that decomposes to produce nitric oxide, a GSNOR downregulator, or an exogenous nitric oxide synthase. As a preferred example, the nitric oxide donor includes S-Nitrosoglutathione (GSNO).

[0098] As another preferred approach, the GSNOR is lowered to reduce the consumption of nitric oxide in the body, thereby increasing the amount of nitric oxide in the body.

[0099] Plant breeding screening or targeted screening of regulatory molecules

[0100] Based on the inventors' new discoveries, this invention provides molecular markers suitable for analyzing cotton fiber quality or yield traits in Gossypium species, including the GSNOR gene. This invention also relates to specific detection molecules designed for said GSNOR or nitric oxide, and identification strategies. This enables early determination of cotton fiber quality or yield traits, and identification of plants with superior traits.

[0101] Therefore, this invention provides a method for specifically identifying the cotton fiber quality or yield traits of *Gossypium* plants, comprising: testing the presence of GSNOR or nitric oxide in the *Gossypium* plant; if the *Gossypium* plant has low GSNOR expression or activity, or high nitric oxide content, its cotton fiber quality or yield is high; if the *Gossypium* plant has high GSNOR expression or activity, or low nitric oxide content, its cotton fiber quality or yield is low. According to this method, early seed selection can be carried out to achieve efficient and high-quality production.

[0102] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0103] The identification method of this invention only requires PCR reaction and / or agarose gel electrophoresis, and by judging the length of the corresponding PCR product, the phenotype of the sample can be accurately and quickly determined. It is low-cost, suitable for large-scale identification, and requires very little sample. If needed, those skilled in the art can design primers for identifying the molecular markers.

[0104] Methods for obtaining DNA from the sample to be tested are well-known to those skilled in the art, such as the traditional phenol / chloroform / isoamyl alcohol method, or commercially available DNA extraction kits. Polymerase chain reaction (PCR) is also well-known to those skilled in the art; its basic principle is the in vitro enzymatic synthesis of specific DNA fragments. The method of this invention can be performed using conventional PCR techniques.

[0105] This invention has promising applications in molecular design breeding and crop variety improvement using genetic engineering technology.

[0106] After learning about the functions of GSNOR or nitric oxide, it can be used as a molecular marker for targeted screening of plants. This new discovery can also be used to screen for substances or potential substances that can target and regulate cotton fiber quality or yield traits by modulating this mechanism.

[0107] The present invention provides a method for screening potential substances to improve cotton fiber quality or yield traits, comprising: (1) treating a cotton plant cell or tissue (such as fiber cells or fibrous tissue) system with a candidate substance, the system expressing GSNOR and / or having a nitric oxide metabolism system; and (2) detecting the expression or activity of GSNOR or the amount of nitric oxide in the system; if the candidate substance statistically reduces the expression or activity of GSNOR and / or increases the amount of nitric oxide, it indicates that the candidate substance is a potential substance to improve cotton fiber quality or yield traits.

[0108] Furthermore, proteins or genes that interact with GSNOR as discovered in this invention, including HD1, can also be used in the screening process. The GSNOR is regulated by HD1, which binds to the GSNOR promoter and regulates GSNOR transcription.

[0109] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0110] The detection of protein-protein interactions and their strength can be achieved using a variety of techniques well-known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques.

[0111] Through large-scale screening, a class of substances that specifically act on GSNOR proteins or their encoding genes, or regulate nitric oxide levels in plants, can be obtained, thus having a regulatory effect on cotton fiber quality or yield.

[0112] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0113] Sequence Description

[0114] GhGSNOR gene nucleotide sequence (SEQ ID NO:1)

[0115] ATGGCGACTCAAGGGCAAGTCATAACTTGCAAAGGTATTTCTCTTTATTTATTTATTGATGTG

[0116] TTTGCCAGTTGTTTTAAAACCTTATTAATTCTAATGTGCTTGTGATTGTGAAGCGGCGGTGGC

[0117] TTATGAACCGAACAAGCCACTGGTCATTGAGGATGTCCAGGTGGCTCCGCCACAAGCCGGT

[0118] GAGGTTCGGATCAAGATTCTCTTCACTGCTCTCTGCCACACTGATGCCTACACTTGGAGCGG

[0119] AAAGGTTTTTCCGTTTTCTTTTCATTTCCATTTTCTATATCTACATGTTTCCTTTTTTGGTTTGC

[0120] AAAATATATCAGTGCCAATTGCTTATCTTGAACAAGGAGCTGTTTAAGATGAATAAAAAAA

[0121] AAGGGATTATTTTAGCGTTATTTGCTTTTCCATCCTAACGAACACTTCTTTTTCTGTAGGATA

[0122] ATGATTTTTTTTTTTGGTTCAAAAAATGCCATGTAAACTTGATTGCTAGGAACGAATAAAAA

[0123] GTGGAAATTCCATGCTAAATGGGAGTATTTTGTTGATTTTGTATTATTGATTTAAAATAGGA

[0124] AAGATATGTGTATAATGTTTCAAATTGGAGTGAGATGCTATTGATAAGAATATAATTTGATG

[0125] TTCTTTGTGTATCCTCTTGCAGTGACAGTGCTAACACCTAGTTTGATCTGGTTCTAGTTTTTCT

[0126] TTTCTTCTTTGATATTTTTCTAATCGGCTGTTTAATTATGATGTCATGCTTGATTTACCTTTTT

[0127] AAGGATCCTGAGGGTCTATTCCCGTGCATTCTTGGACATGAAGCTGCTGGGTATGTTTTTTCC

[0128] TTTTAGTTCTTACAGTTTATGAGCCTCTTGGTTGGTAAAGAGTGAGAAATGTTTGAATTTTTT

[0129] AGTGCAGAATTGTAGAAAGTGTTGGTGAAGGTGTAACTGAAGTTCAACCAGGTGACCATGT

[0130] CATCCCTTGTTACCAGGCAGAGTGCAGAGAATGCAAGTTCTGCAAATCAGGTAAGACTAAC

[0131] CTATGCGGCAAAGTTAGGACAGCCACTGGAGCTGGAGTCATGATGAATGATCGCAAGAGCC

[0132] GCTTCTCAATTAATGGGAAACCTATCTACCACTTCATGGGCACCTCAACATTTAGCCAGTAC

[0133] ACGGTTGTGCATGATGTTAGTGTTGCAAAGATTGATCCACAGGCTCCTTTGGACAAAGTATG

[0134] CCTTCTTGGTTGTGGTGTTCCAACTGGTAAGTGCTTGTTTAGCTTTCTCTTCATTTTATGAATC

[0135] TGTAATTGCATATACAATCATATAGCAATTGGATTTTTTTTTATTTGACTGTATATATATTAT

[0136] TTATTTTGTGATGCCTTTGTACACAACTTTCATTTCATACGTACTTTGGTGTTGGATAGGACT

[0137] TGGAGCAGTTTGGAACACAGCCAAAGTTGAACCAGGGGCCATTGTTGCTATTTTTGGCCTTG

[0138] GGACTGTTGGTCTTGCTGTAAGTGTCAAATATGCATCTTGCGTAGAAATTTGTTGATATTGTT

[0139] TATTATGAAGTCCTATGCACATTTTGAGCTGCCTTTTAAAAATATTTGTTATTCTCATCGTTA

[0140] TTCTCTTTGTGGTGCTTGTTTGTTTGATATTAAATTGTTTTTGTGATCCTGGAATGCAATGTTG

[0141] CAGGTCGCAGAGGGTGCAAAGTCAGCTGGTGCTTCAAGAATAATTGGCGTTGATATTGATA

[0142] GCAAAAAGTTTGATGTAGGCAAGTAATTTACAAATGATAATGCTTACAAAGAAGAATTTGG

[0143] TAAATATTAATATGAAATATTCAATAAACAATGTGTAATGTGAATAATGAAATAAATTGTAA

[0144] GAAAATGGGTGGATATTGTTTTTCTTTTCTTATTTTTTGTAATAAATCTAAACCATTCATTATT

[0145] TCTATTTCTATATTCTGATTTCTTTCTTTCTTTCTTTTTGGTGAAATGCAACTTGCAGCAAAGA

[0146] ATTTTGGAGTTACAGAGTTTGTGAATCCAAAGGACTATGATAAACCAATTCAGCAGGTCCTT

[0147] GTTGATCTCACAGATGGTGGTGTTGATTACAGTTTCGAGTGTACGGAAATGTCTCTGTCAT

[0148] GAGGGCTGCTTTGGAATGCTGTCACAAGGTATGTGGGTGCCTGAATTTGATGAGCACTAATGA

[0149] CTGCATACCGGAAGACCACTGCTAAAGTGCCCGAGAATTTTCAGGGCTGGGGAACATCA

[0150] GTTATCATAGGTGTTGCCGCATCAGGTCAGGAGATATCTACACGACCTTTCCAATTGGTGAC

[0151] TGGCCGTGTTTGGAAAGGGACAGCTTTTGGGTGGTTTCAAGAGTCGTTCACAAGTGCCTTGGC

[0152] TTGTGGAAAAATACATGAAGAAGGTAAACGTAGTTTGTAGGCGAATCTATATTTAAGCCTGT

[0153] AGAATGATCAGGATATTATTATACTCTCCTTATCATTGCAGGAATCAAAATAGATGAGTAC

[0154] ATCACCCACAATTTGACCCTTGGAGATATTAACAAGGCATTTGATCTGATGCATGAAGGGGG

[0155] TTGCCTCCGATGCGTGCTTAAGATGAATGAATGA

[0156] The amino acid sequence of the protein encoded by GhGSNOR (SEQ ID NO:2)

[0157] MATGVTCKAAVAYNKVDVVAAGVRKTACHTDAYTWSGKDGCGHAAGVSVGGVTVGDHVCY

[0158] ACRCKCKSGKTNCGKVRTATGAGVMMNDRKSRSNGKYHMGTSTSYTVVHDVSVAKDADKVC

[0159] GCGVTGGAVWNTAKVGAVAGGTVGAVAGAKSAGASRGVDDSKKDVAKNGVTVNKDYDKVV

[0160] DTDGGVDYSCGNVSVMRAACCHKGWGTSVGVAASGSTRVTGRVWKGTAGGKSRSVWVKYM

[0161] KKKDYTHNTGDNKADMHGGCRCVKMN

[0162] CRISPR knockout target for GhGSNOR:

[0163] Sg1: GCAATGTTGCAGGTCGCAGA (SEQ ID NO: 3);

[0164] Sg2: GCTTCATGTCCAAGAATGCACGG (SEQ ID NO: 4).

[0165] Example 1: Cloning of the full-length GhGSNOR gene

[0166] 1. RNA extraction from upland cotton

[0167] Ovules of upland cotton R15 (upland cotton variety "Jinmian R15", purchased from the Cotton Research Institute of Shanxi Academy of Agricultural Sciences) were collected on the day of flowering and ground into powder in liquid nitrogen.

[0168] RNA extraction was performed using a polysaccharide and polyphenol total RNA extraction kit (purchased from Tiangen Biotech Co., Ltd.).

[0169] The process is briefly described as follows:

[0170] (1) Take an appropriate amount of sample and add 700 μL of lysis buffer SL (add 1580 μL of β-mercaptoethanol before use);

[0171] (2) Centrifuge at 10000×g for 2 minutes at low temperature;

[0172] (3) Carefully aspirate the supernatant into the CS filter column and centrifuge at 10000×g for 1 minute at low temperature;

[0173] (4) Transfer the filtered solution to a clean RNAse-free centrifuge tube, add 0.4 times the volume of anhydrous ethanol, mix quickly and gently, and transfer to the adsorption column CR3.

[0174] (5) Centrifuge at 10000×g at low temperature for 30s, and discard the waste liquid;

[0175] (6) Add 350 μL of protease removal solution RW1 to the adsorption column CR3;

[0176] (7) Centrifuge at 10000×g at low temperature for 30s, and discard the waste liquid;

[0177] (8) Add at least 80 μL of DNAes I to the adsorption column CR3 to remove residual DNA and make the RNA purer. Let stand at room temperature for 15 minutes.

[0178] (9) Add 350 μL of protease removal solution RW1 to the adsorption column CR3;

[0179] (10) Centrifuge at 10000×g at low temperature for 30s, and discard the waste liquid;

[0180] (11) Add 500 μL of rinsing solution RW to the adsorption column CR3;

[0181] (12) Centrifuge at 10000×g at low temperature for 30s, and discard the waste liquid;

[0182] (13) Repeat steps (12) and (13);

[0183] (14) Centrifuge at 10000×g for 2 minutes at low temperature;

[0184] (15) Add an appropriate amount of RNAse-free H2O, centrifuge at 10000×g for 1 minute at low temperature;

[0185] (16) The concentration and status of RNA extraction were detected using instruments such as NanoDrop.

[0186] 2. Preparation, acquisition, and detection of GhGSNOR templates

[0187] The reverse transcription kit (purchased from Aikerui Biotechnology) was used, and the procedure is briefly described below:

[0188] Take 1 mg of the above RNA, add 2 μL of reverse transcription MIX, and make up the volume to 10 μL with dd H2O. After gently mixing, transfer the solution to a PCR instrument and use the following reaction procedure: react at 37℃ for 15 minutes, heat to 85℃ for 5 seconds to end the reaction, and cool to 4℃ to store the reverse transcription product.

[0189] Based on the sequence of GhGSNOR cDNA, synthesize specific primers:

[0190] GhGSNOR-F: 5'-ATGGCGACTCAAGGGCAAG-3' (SEQ ID NO: 5);

[0191] GhGSNOR-R: 5'-TCATTCATTCATCTTAAGCACGCATCG-3' (SEQ ID NO: 6).

[0192] Using the above reverse transcription product as a template, 25 μL of high-fidelity enzyme MIX (purchased from Yisheng Biotechnology) and upstream and downstream primers were added, and the system was brought up to 50 μL with dd H2O. PCR reaction was performed under the following conditions: 94℃ pre-denaturation for 5 min; then 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 38 cycles; and finally 72℃ extension for 10 min.

[0193] After electrophoretic purification and recovery of the PCR product, it was subcloned into the T vector pEASY-blunt Zero (purchased from Beijing TransGen Biotech). After sequencing to confirm the sequence was correct, the full-length transcriptome sequence of GhGSNOR was obtained.

[0194] Example 2: Construction of crGhGSNOR and 35S::GhGSNOR expression vectors and Agrobacterium transformation

[0195] Construction of a.crGhGSNOR plant expression vector

[0196] Analyzing the GhGSNOR sequence, a specific knockout site was identified. The target site was synthesized and recombined into the pRGEB32-GhU6.7-NPT2 vector between the restriction enzyme sites HpaI and SbfI, resulting in the crGhGSNOR vector for GhGSNOR knockout. Figure 1 A).

[0197] Construction of b.35S::GhGSNOR expression vector

[0198] Using the correctly sequenced T vector as a template, 25 μL of high-fidelity enzyme MIX (purchased from Yisheng Biotechnology) was added. A homologous arm of 35S::2301 was introduced at the 5' end of the primers. After adding forward and reverse primers, the volume was brought up to 50 μL with dd H2O. PCR was then performed under the following conditions: 94℃ pre-denaturation for 5 min; followed by 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 1.5 min, for a total of 38 cycles; and a final extension at 72℃ for 10 min. The PCR product was purified by electrophoresis and recovered to obtain the cloned fragment.

[0199] The 35S::2301 vector was double-digested with restriction endonucleases XbaI and KpnI, purified by electrophoresis, and recovered to obtain the vector fragment. The cloned fragment and the vector fragment were recombined with homologous recombinase, and the result was confirmed by sequencing to obtain the 35S::GhGSNOR vector for overexpressing GhGSNOR. Figure 1 B).

[0200] Primer sequences:

[0201] GhGSNOR-2301-F(SEQ ID NO:7):

[0202] 5'-GAGAGAACAGTCGACTCTAGAATGGCGACTCAAGGGCAAG-3';

[0203] GhGSNOR-2301-R(SEQ ID NO:8):

[0204] 5'-GAACGATCGAGCTCGGTACCTCATTCATTCATCTTAAGCACGCATCG-3'.

[0205] c. Agrobacterium tumefaciens transformation

[0206] Transformation of Agrobacterium tumefaciens was performed using the freeze-thaw method. A single colony of LBA4404 or GV3101 (Invitrogen) was incubated overnight at 28°C and 220 rpm in 3 ml LB medium (25 μg / ml rifamycin Rif and 50 μg / ml kanamycin Kan or gentamicin Gen). Alternatively, 2 ml of the bacterial culture was incubated in 50 ml LB medium (25 μg / ml Rif and 50 μg / ml Gen) at 28°C and 220 rpm until OD500 was reached. 600=0.5 (approximately 6 hours). Place on ice for 30 minutes, centrifuge at 5000g for 5 minutes at 4°C. Resuspend in 10 ml of 0.15M NaCl. Centrifuge at 5000g for 5 minutes at 4°C. Resuspend in 1 ml of 20mM CaCl2, aliquot 50 μl / tube, flash freeze in liquid nitrogen, and store competent cells at -70°C. Mix the binary vector containing the target gene with 50 μl / tube of competent cells, place on ice for 30 minutes, and flash freeze in liquid nitrogen for 1 minute. Thaw the bacterial culture in a 37°C water bath for 5 minutes, add 1 ml of LB medium, and incubate at 28°C and 220 rpm for 2–4 hours. Spread 50–100 μl onto LB agar plates (25 μg / ml Rif, 50 μg / ml Gen, and 50 μg / ml Kanamycin or Hygromycin Hyg), and pick single colonies for PCR identification after 2 days. Obtain the corresponding Agrobacterium clones for both vectors.

[0207] Example 3: Plant transformation and screening of transgenic progeny

[0208] a. Genetically modified cotton

[0209] Agrobacterium containing the vector plasmids crGhGSNOR and 35S::GhGSNOR was cultured on YEB bacterial medium supplemented with kanamycin 50 mg / L, rifampin 100 mg / L, and streptomycin 300 mg / L for 2–3 days. Single colonies were then inoculated into YEB liquid medium containing the same antibiotics and cultured overnight on a shaker at 28°C and 200 rpm / min. The bacterial suspension was centrifuged at 4000 rpm / min for 10 min, and the precipitate was resuspended in 1 / 2 MS liquid medium containing glucose 30 g / L and acetylsyleugenol 100 μmol / L. OD was adjusted. 600 The value is around 0.4 to 0.6, and it is used as a backup for infection.

[0210] Cotton R15 seeds (Jinmian R15 variety) were disinfected and placed in 1 / 2MS0 (1 / 2MS salt + 5g / L glucose + 7g / L agar powder, pH 6.0) medium for germination culture in the dark. After 5-7 days, the hypocotyls of the sterile seedlings were cut into segments of about 1.0cm as explants for transformation.

[0211] Explants were soaked in Agrobacterium tumefaciens solution for 15–20 min to infect them, then transferred to co-culture medium MSB1 (MS salt + B5 organic + 30 g / L glucose + 0.1 mg / L KT + 0.1 mg / L 2,4-D + 2.2 g / L Gelrite, pH 6.0) and cultured in the dark at 22°C for 2 days. After that, the explants were transferred to medium MSB2 (MSB1 + 500 mg / L cephalosporin + 80 mg / L kanamycin) to induce callus formation. Explants underwent induction of resistant callus, callus proliferation, and embryogenic callus induction (medium MSB3: MS salt + B5 organic + 30 g / L glucose + 2.5 g / L gelrite, pH 6.0), followed by somatic embryogenesis (medium MSB4: MS salt + B5 organic + 30 g / L glucose + 1.0 g / L asparagine + 2.0 g / L glutamine + 3.0 g / L gelrite, pH 6.0; KNO3 in MS salt was doubled, and NH4NO3 was removed), resulting in the regeneration of resistant in vitro plantlets. When the plantlets reached 3-4 true leaves, they were transplanted into flowerpots and placed in an artificial climate chamber for growth. At least five transgenic lines were obtained, including the GhGSNOR overexpression line OE13 and the GhGSNOR knockout line CR76.

[0212] Example 4: Molecular biological identification of transgenic plants

[0213] a.PCR

[0214] DNA extraction was performed using the cold phenol method. 2g of material was ground into powder in liquid nitrogen and transferred to a 50ml centrifuge tube. 8ml of extraction buffer (1M Tris-HCl, 50mM EDTA, 1% SDS, pH 9.0) and an equal volume of water-saturated phenol:chloroform:isoamyl alcohol (25:24:1) were added. The mixture was vortexed and incubated on ice for 1 hour, mixing every 10 minutes. The tube was then centrifuged at 13000g for 20 minutes at 4°C. The phenol:chloroform:isoamyl alcohol extraction was repeated 2–4 times, with a final extraction using chloroform:isoamyl alcohol (24:1). The supernatant was collected, and half a volume of high-salt solution (0.8M sodium citrate, 1.2M NaCl) and half a volume of isopropanol were added. The mixture was incubated at -70°C for 1 hour. Centrifuge at 13000g for 20 min at 4℃, discard the supernatant, wash the precipitate twice with 1 ml of 70% ethanol, blow under the air at room temperature for 20 min, and dissolve the precipitate in 1 ml of sterile water. Centrifuge at 13000g for 10 min at 4℃. Take the supernatant, add 5-10 μl of RNase (10 mg / ml), and digest at 37℃ for 30 min.

[0215] PCR identification uses NPT II specific primers:

[0216] CRGhGSNOR-1F (SEQ ID NO:9):

[0217] 5'-TAGGAAAGATATGTGTATAATGTTTCAAATTGGAGTGAG-3';

[0218] CRGhGSNOR-1R (SEQ ID NO:10):

[0219] 5'-AAGCGGCTCTTGCGATCA-3';

[0220] CRGhGSNOR-2F (SEQ ID NO:11):

[0221] 5'-TGGAACACAGCCAAAGTTGAACC-3';

[0222] CRGhGSNOR-2R (SEQ ID NO:12):

[0223] 5'-TCCGGTTATGCAGTCATTAGTGC-3';

[0224] The reaction conditions were as follows: pre-denaturation at 94℃ for 5 min; then pre-denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, extension at 72℃ for 1 min, for a total of 35 cycles; and finally extension at 72℃ for 10 min. The amplified fragment size was 510 bp.

[0225] After sequencing, the CR76 line with successfully knocked-out genes was detected and obtained, and then subcultured.

[0226] b. Quantitative Real-Time RT-PCR Analysis

[0227] (i) RT-PCR analysis

[0228] 1 μg of total RNA was reverse transcribed using Oligo(dT) primers, and 20 μl of reaction solution was added. The mixture was incubated at 42°C for 30 minutes to reverse transcribe first-strand cDNA, and then incubated overnight. 0.5 μl of the reverse transcription product was added to a 25 μl PCR reaction system to detect the expression of the target gene. The PCR primers were GhTCP4-specific primers.

[0229] GhGSNOR-qRT-F(SEQ ID NO:13):

[0230] 5'-TGTTACCAGGCAGAGTGCAG-3';

[0231] GhGSNOR-qRT-R(SEQ ID NO:14):

[0232] 5'-ACACCACAACCAAGAAGGCA-3'.

[0233] The cotton gene Histone3 (AF024716) was used as an internal standard to correct the template amount for the RT-PCR reaction. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 minutes; then 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 30 cycles; and finally 72℃ extension for 10 minutes.

[0234] (ii) Quantitative RT-PCR analysis

[0235] Quantitative RT-PCR detection was performed using the chimeric fluorescence method ( Premix Ex Taq TM II (Perfect RealTime), TaKaRa, DRR041A). The reaction system is as follows:

[0236]

[0237] The cotton gene Histone3 (AF024716) was used as an internal standard. Data analysis was performed using Realplex v2.0 (purchased from Eppendorf, Hamburg, NSW, Germany). The experiment was repeated three times, and the mean and variance of each group were calculated to create graphs.

[0238] Transgenic line OE13, whose expression level was at least 10 times higher than that of wild-type R15, was selected and subcultured.

[0239] Example 5: Analysis of fiber length and gene expression in transgenic cotton

[0240] 1. Trait analysis of genetically modified cotton

[0241] transgenic cotton varieties crGhGSNOR and 35S::GhGSNOR, along with wild-type R15, were planted on farms in Shanghai and Hainan under conventional field management. During the cotton's maturity and boll-opening stage, mature bolls were harvested individually, ensuring uniformity in harvesting location. A random number of seeds were then combed to smooth their fibers, and their length was measured for statistical analysis.

[0242] The results showed that the phenotype of crGhGSNOR transgenic cotton (cr76) with reduced GhGSNOR expression was stably inherited, with a significant increase in the number of mature fibers (26%) compared to the wild type, while the fiber length remained unchanged. Figure 2 A, Figure 3 ).

[0243] The results also showed that the phenotype of the 35S::GhGSNOR transgenic cotton (OE13) was stably inherited, with a significant reduction in the number of mature fibers compared to the wild type, a reduction of 15%, and shorter fiber length. Figure 2 B, Figure 3 ).

[0244] The above results indicate that decreased GhGSNOR expression can significantly increase fiber density, thereby significantly increasing cotton yield.

[0245] 2. Gene expression analysis of GhGSNOR

[0246] Quantitative analysis showed that GhGSNOR expression was low in the early stages of fiber development, increased rapidly 3 days after flowering, and then decreased slowly. Figure 2 D). This shows that nitric oxide signaling is very important in the early stages of fiber development, and that reducing GhGSNOR expression to decrease nitric oxide consumption or increasing nitric oxide content can significantly increase fiber density and improve cotton yield.

[0247] After overexpression of GhGSNOR ( Figure 2 C) Fiber length and density were significantly reduced. This suggests that nitric oxide plays a crucial role in fiber development, especially in the early stages, maintaining fiber length while increasing fiber cell density, thereby improving cotton yield.

[0248] Using fluorescent protein tracing technology, GhGSNOR was located near the cell wall. Figure 4 AB), GhGSNOR controls fibroblast differentiation and growth by participating in the regulation of fibroblast cell wall synthesis.

[0249] Through in-depth analysis, the inventors discovered that the expression of the GhGSNOR gene is regulated by the key transcription factor GhHD1 (Gh_A06G1710) in the early development of cotton fibers. Yeast two-hybrid experiments confirmed that GhHD1 specifically binds to the promoter of GhGSNOR (1 to 300 bp upstream of the ATG). Figure 4 C), which further illustrates that GhGSNOR plays an important role in regulating fiber development.

[0250] Example 6: Molecular mechanisms by which nitric oxide and GhGSNOR regulate cotton fiber development

[0251] In vitro culture samples treated with the nitric oxide inhibitor cPTIO and the nitric oxide donor S-Nitrosoglutathione (GSNO) showed, according to cryo-transmission field emission electron microscopy, a significant decrease in the number of fibroblasts was observed (53%) in cPTIO-treated samples, while a significant increase (62%) was observed in GSNO-treated samples. Figure 5This indicates that an increase or decrease in nitric oxide content can indeed regulate fiber cell density. Within a certain range, nitric oxide concentration is directly proportional to fiber cell density, and it also confirms that GhGSNOR is inversely proportional to fiber cell density. Increased nitric oxide content leads to increased cotton fiber density and improved cotton yield.

[0252] Ultrathin transmission electron microscopy results showed that cPTIO treatment significantly thickened the fibroblast cell walls, while GHSNOR treatment significantly thinned the fibroblast cell walls. Figure 6 This indicates that nitric oxide can affect cell wall synthesis and promote fibroblast development. This is consistent with the localization signal of GhGSNOR mentioned above, suggesting that GhGSNOR affects fibroblast differentiation by promoting cell wall synthesis, inhibiting fibroblast processes, and thus affecting fibroblast differentiation.

[0253] In summary, as an example, this invention constructed two transgenic vectors, 35S::GhGSNOR and crGhGSNOR, and corresponding Agrobacterium strains. Overexpression and knockout lines of GhGSNOR were established, and it was verified that this gene and its signal increase fibroblast density by controlling fibroblast cell wall synthesis. Therefore, this invention discloses for the first time a method to increase fibroblast density: by inhibiting GhGSNOR expression or applying nitric oxide.

[0254] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for improving Gossypium plants, comprising: In cotton plants, downregulating GSNOR or increasing nitric oxide can improve cotton fiber quality or yield traits.

2. The method as described in claim 1, characterized in that, The improvement of cotton fiber quality or yield includes: increasing fiber density, increasing the number of mature fibers, and increasing fiber yield; Preferably, the GSNOR is regulated by HD1, which binds to the promoter of the GSNOR and regulates the transcription of the GSNOR; Preferably, the GSNOR participates in regulating the synthesis of fibroblast cell walls and controls the differentiation or growth of fibroblasts.

3. The method as described in claim 1, characterized in that, The increase in nitric oxide includes: reducing nitric oxide consumption or increasing the amount of nitric oxide in the body; Preferably, a nitric oxide upregulator is applied to increase nitric oxide content; more preferably, the nitric oxide upregulator includes: a nitric oxide donor, an agent that produces nitric oxide after decomposition, a GSNOR downregulator, or an exogenous nitric oxide synthase; preferably, the nitric oxide donor includes nitrosoglutathione. Preferably, the GSNOR is reduced to decrease the consumption of nitric oxide in the plant, thereby increasing the amount of nitric oxide in the plant.

4. The method as described in claim 1, characterized in that, The downregulation of GSNOR includes: downregulating the expression or activity of GSNOR; preferably, it includes: performing gene silencing or gene knockout on the GSNOR; preferably, the gene knockout is performed by gene editing or homologous recombination; more preferably, the gene editing is performed by sgRNA containing the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:

4.

5. The application of GSNOR downregulators or nitric oxide upregulators is used to improve cotton plants and enhance cotton fiber quality or yield traits.

6. The application as described in claim 5, characterized in that, The GSNOR downregulator comprises: a downregulator that knocks out or silences the GSNOR gene or inhibits the activity of the GSNOR protein; preferably, it comprises: a reagent for knocking out the GSNOR gene based on gene editing, a reagent for knocking out the GSNOR gene based on homologous recombination, or an interfering molecule that specifically interferes with the expression of the GSNOR gene; preferably, the downregulator comprises sgRNA with the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4; or The nitric oxide upregulator includes: a nitric oxide donor, an agent that produces nitric oxide after decomposition, a GSNOR downregulator, or an exogenous nitric oxide synthase; preferably, the nitric oxide donor includes nitrosoglutathione.

7. The method as described in any one of claims 1-4 or the application as described in any one of claims 5-6, characterized in that, The GSNOR comprises: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO:2; (b) a GSNOR derivative having the function of the polypeptide (a) formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence shown in (a); (c) a GSNOR derivative or active fragment having the function of the polypeptide (a) with a sequence having ≥80% homology to the amino acid sequence shown in SEQ ID NO:2; or (d) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N terminus.

8. A Gossypium cell, tissue or organ, wherein the Gossypium is a Gossypium expressing GSNOR or containing a nitric oxide metabolism system, wherein it contains an exogenous GSNOR downregulator or nitric oxide upregulator. Preferably, the GSNOR downregulator comprises: a downregulator that knocks out or silences the GSNOR gene or inhibits the activity of the GSNOR protein; preferably, it comprises: a reagent for knocking out the GSNOR gene based on gene editing, a reagent for knocking out the GSNOR gene based on homologous recombination, and an interfering molecule that specifically interferes with the expression of the GSNOR gene; preferably, the downregulator comprises sgRNA with the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4; Preferably, the nitric oxide upregulator includes: a nitric oxide donor and a GSNOR downregulator; preferably, the nitric oxide donor includes nitrosoglutathione.

9. A method for identifying cotton fiber quality or yield traits in plants of the genus *Gossypium*, the method comprising: The test measures the presence of GSNOR or nitric oxide in cotton plants. If the tested cotton plant has low GSNOR expression or activity or high nitric oxide content, its cotton fiber quality or yield is high. If the tested cotton plant has high GSNOR expression or activity or low nitric oxide content, its cotton fiber quality or yield is low.

10. A method for screening potential substances that improve cotton fiber quality or yield traits, comprising: (1) Treat a Gossypium cell or tissue system with candidate substances, the system expressing GSNOR and / or having a nitric oxide metabolism system; and (2) Detect the expression or activity of GSNOR or the amount of nitric oxide in the system; if the candidate substance statistically reduces the expression or activity of GSNOR and / or increases the amount of nitric oxide, it indicates that the candidate substance is a potential substance for improving the quality or yield of cotton fibers.

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