CoNOS gene and genetic engineering method thereof for improving cotton fiber traits

Abstract

The invention provides a coNOS gene and a gene engineering method thereof for improving cotton fiber traits. The invention discloses a new target, namely coNOS, which is a nitric oxide (NO) synthase, which can be used for improving fiber traits and / or yield traits of gossypium plants. Functional identification is carried out on the coNOS gene, and cotton plants with significantly increased fiber density, significantly increased fiber quantity and significantly increased yield are obtained through a transgenic technology. The invention also discloses the coNOS for increasing the content of nitric oxide in the fiber and controlling the synthesis of the cell wall of the fiber cell.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of botany and molecular biology; more particularly, the present application relates to a coNOS gene and a genetic engineering method for improving cotton fiber traits. BACKGROUND

[0002] At present, the quality of Gossypium hirsutum still needs to be improved, the yield is insufficient, long-staple cotton has poor adaptability, and the planting area is limited.

[0003] The level of cotton fiber quality is mainly determined by three aspects: first, the genetic quality of cotton, which is affected by the cotton genetic genes or the variety itself; second, the production quality of cotton, which is affected by cultivation management measures and climate environment; and third, the processing quality of cotton, which is affected by the cotton harvesting and processing process.

[0004] The genetic characteristics of the variety greatly determine the cotton fiber quality. In the existing cotton production, there are still problems such as poor fiber strength and fineness, which cannot meet the requirements of high-quality cotton, and the adaptability and stability of the variety are poor, which cannot meet the demand of production and market for high-quality cotton. Therefore, improving the yield of cotton fiber is an important issue faced by cotton breeding.

[0005] Cotton fiber is developed from the single cell of the epidermal layer of cotton ovule, and its differentiation and development process can be divided into four periods: fiber cell differentiation and protrusion, rapid elongation, secondary wall synthesis and dehydration maturation. Fiber cell differentiation determines the final density of fiber, and the fiber density determines the unit yield of cotton fiber.

[0006] Nitric oxide (NO) is a gaseous signal molecule, which plays a very important role in the growth and development of animals and plants. Due to its important application in the medical field, especially its outstanding contribution in cardiovascular diseases, its discovery won the Nobel Prize in Physiology or Medicine in 1998. NO also plays a very important role in plant development, especially in resisting adverse environments. SUMMARY

[0007] The purpose of the present application is to provide a coNOS gene and its application in improving Gossypium plants.

[0008] In the first aspect of the present application, a method for improving Gossypium plants is provided, comprising: up-regulating coNOS in Gossypium plants, thereby improving the fiber quality or yield traits of cotton.

[0009] In one or more embodiments, the improvement of the fiber quality or yield of cotton includes: improving fiber cells (including increasing the content of nitric oxide (NO) in fiber cells), increasing fiber density, increasing the number of mature fibers, and improving fiber yield.

[0010] In one or more embodiments, the coNOS controls fiber cell cell wall synthesis, reduces fiber cell cell wall thickness, modulates fiber cell differentiation or growth.

[0011] In one or more embodiments, the reducing fiber cell cell wall thickness is reducing fiber cell initial stage cell wall thickness.

[0012] In one or more embodiments, the upregulating coNOS expression or activity comprises:

[0013] introducing (exogenous introduction) a coding sequence of coNOS into a plant; preferably, introducing the coding sequence of coNOS into a plant with a construct (e.g., an expression vector) containing the coding sequence of coNOS;

[0014] engineering an endogenous coNOS gene in a plant, thereby upregulating coNOS expression or activity; preferably, engineering is performed by a method of gene editing;

[0015] enhancing coNOS gene promoter driving ability;

[0016] modulating with an upregulating molecule that interacts with coNOS, thereby increasing coNOS expression or activity; or

[0017] introducing (exogenous introduction) a nitric oxide synthesis pathway that increases nitric oxide content into a plant.

[0018] In one or more embodiments, the construct (expression vector) comprises: a Gossypium plant expression vector.

[0019] In one or more embodiments, the construct (expression vector) comprises: a non-viral vector, a viral vector.

[0020] In one or more embodiments, the upregulating means significant upregulation, such as upregulation of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or more.

[0021] In one or more embodiments, a method for improving a Gossypium plant is provided, comprising making a transgenic plant: (1) providing a vector carrying a NOS nucleotide sequence; (2) providing Agrobacterium carrying the vector in step (1); (3) contacting plant cells or tissues with the Agrobacterium in step (2), thereby introducing the coNOS into the plant cells or tissues.

[0022] In another aspect of the present application, a use of coNOS or an upregulator thereof is provided, for: improving a Gossypium plant, increasing cotton fiber quality or yield traits; making a preparation for increasing cotton fiber quality or yield traits; or, as a molecular marker for identifying cotton fiber quality or yield traits of a Gossypium plant.

[0023] In one or more embodiments, the improving cotton fiber quality or yield comprises: improving fiber cell (including increasing NO content in fiber cell), increasing fiber density, increasing number of mature fiber, increasing fiber yield; more preferably, the coNOS controls fiber cell cell wall synthesis, decreases fiber cell cell wall thickness, regulates fiber cell differentiation or growth.

[0024] In one or more embodiments, the decreasing fiber cell cell wall thickness is decreasing fiber cell initial stage cell wall thickness.

[0025] In one or more embodiments, the up-regulator comprises (but not limited to): polynucleotide or expression construct encoding coNOS, gene editing reagent promoting coNOS gene expression (such as endogenous gene expression), up-regulator promoting coNOS gene promoter driving ability, up-regulator interacting with coNOS protein, thereby increasing its expression or activity, down-regulator of coNOS gene specific microRNA, chemical up-regulator of coNOS, or combination thereof.

[0026] In one or more embodiments, the coNOS comprises: (a) polypeptide with amino acid sequence as shown in SEQ ID NO: 2; (b) coNOS derivative with function of (a) polypeptide formed by substitution, deletion or addition of one or more (such as 1-20, 1-10, 1-5, 1-3 or 1-2) amino acid residues to the amino acid sequence shown in (a), or active fragment thereof; (c) coNOS derivative or active fragment with function of (a) polypeptide, with homology of ≥80% (such as homology of ≥82%, ≥85%, ≥90%, ≥92%, ≥94%, ≥96%, ≥98% or ≥99%) compared with the amino acid sequence shown in (b); (d) polypeptide formed by adding tag sequence or enzyme cutting site sequence to N or C terminal of polypeptide with amino acid sequence shown in (b), or adding signal peptide sequence to N terminal of polypeptide.

[0027] In one or more embodiments, the Gossypium plant comprises Gossypium plant with manipulated increased NO synthesis.

[0028] In one or more embodiments, the Gossypium plant comprises Gossypium hirsutum.

[0029] In one or more embodiments, the Gossypium plant before modification is Gossypium plant with insufficient expression or activity of coNOS.

[0030] In another aspect of the present application, a Gossypium plant cell, tissue or organ is provided, wherein the Gossypium plant is a Gossypium plant expressing exogenous coNOS.

[0031] In one or more embodiments, the Gossypium plant comprises a nitric oxide synthesis pathway.

[0032] In one or more embodiments, the NO synthesis pathway includes (but is not limited to) NO production by coNOS, NO production by other metabolic pathways.

[0033] In one or more embodiments, the Gossypium plant cell, tissue or organ does not directly produce a living Gossypium plant, or does not serve as Gossypium plant propagation material.

[0034] In another aspect of the application, a method for identifying the cotton fiber quality or yield trait of a Gossypium plant (e.g., for its seeds) is provided, comprising: identifying the expression or activity of coNOS in a test Gossypium plant; if the test Gossypium plant has high coNOS expression or activity (e.g., higher than the average of the species), it has high cotton fiber quality or yield (including high fiber density, high number of mature fibers); if the test Gossypium plant has low coNOS expression or activity (e.g., lower than the average of the species), it has low cotton fiber quality or yield (including low fiber density, low number of mature fibers); preferably, for a test Gossypium plant with low coNOS expression or activity, it is identified as a Gossypium plant in need of coNOS or its up-regulator to regulate so as to restore normal coNOS expression or activity.

[0035] In one or more embodiments, it further comprises identifying the presence of other nitric oxide synthesis pathways in the test Gossypium plant, if there is other nitric oxide synthesis pathway (e.g., NO production with arginine as substrate), it has high cotton fiber quality or yield (including high fiber density, high number of mature fibers).

[0036] In another aspect of the application, a method for screening potential substances for improving the cotton fiber quality or yield trait is provided, comprising:

[0037] (1) treating a Gossypium plant cell or tissue (e.g., fiber cell or fiber tissue) system, which expresses coNOS and / or has other nitric oxide synthesis system, with a candidate substance;

[0038] (2) detecting the coNOS and / or other nitric oxide synthesis system in the system; if the candidate substance statistically increases (e.g., increases by 5%, 10%, 15%, 20%, 30%, 50% or 80% or more) the expression or activity of coNOS and / or increases the amount of nitric oxide, it indicates that the candidate substance is a potential substance for improving the cotton fiber quality or yield trait.

[0039] In one or more embodiments, the screening method further comprises setting up a control group, so as to clearly distinguish the increase or decrease of the expression or activity of coNOS.

[0040] In another preferred embodiment, the method further comprises: performing further cell experiments and / or transgenic experiments on the obtained potential substances, so as to further determine the substances that are excellent in effect of improving cotton fiber quality or yield traits from the candidate substances.

[0041] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as modulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)) directed against coNOS protein or its encoding gene, or its upstream or downstream protein or gene, CRISPR constructs, small molecule compounds, etc.; or donors or reagents for NO.

[0042] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 coNOS construct vector information. 35S: :2301 vector information, the target fragment is inserted between XbaI and KpnI. The vector map is generated by SnapGene.

[0044] Figure 2 Function of coNOS in cotton.

[0045] (A) NO signal intensity in fiber cells. Green fluorescence represents NO signal intensity, and red fluorescence represents cell outline. TM-1 is wild type, and FENG is a coNOS transgenic line.

[0046] (B) Statistics of NO signal intensity in fiber cells. The error bars represent standard deviation (STDEV). “***” indicates that the experimental group and the control group are analyzed by two-sample equal variance hypothesis student’s t test, P < 0.005, n≥10.

[0047] Figure 3 Phenotype of coNOS transgenic line.

[0048] (A) Mature fiber phenotype in FENG. Fiber seeds become larger in number. Bar = 1 cm.

[0049] (B) Scanning electron microscope observation of fiber cell number on the day of flowering. Compared with wild type TM-1, the number of fiber cells in FENG is significantly increased, with an increase of about 18%. Bar = 100 μm.

[0050] (C) FENG transgenic lines surface fiber number statistics results.

[0051] (D) Transmission electron microscopy observation of fiber cell wall thickness on the day of flowering. Compared with wild type TM-1, the FENG fiber cell wall thickness is significantly reduced. Bar = 5 μm.

[0052] (E) FENG transgenic lines surface fiber number statistics results. Error bars indicate standard deviation (STDEV); "**" indicates that the experimental group and the control group are analyzed by double sample equal variance hypothesis student's t test, P < 0.01, "***" indicates that the experimental group and the control group are analyzed by double sample equal variance hypothesis student's t test, P < 0.005, n≥20. DETAILED DESCRIPTION

[0053] The present inventors have disclosed a new target that can be applied to improve fiber traits and / or yield traits of Gossypium plants: coNOS, which is a nitric oxide (NO) synthase. The present application has functionally identified coNOS gene, and obtained Gossypium plants with significantly increased fiber density, significantly increased fiber number, and significantly increased yield through transgenic technology. The present application also discloses that coNOS increases the content of nitric oxide in fibers and controls the synthesis of fiber cell wall.

[0054] In a preferred embodiment of the present application, a nitric oxide synthase NOS gene and its codon-optimized gene coNOS and their application in improving Gossypium plants are provided. In the embodiment, the NOS gene is isolated and cloned, the NOS gene sequence is improved by codon preference and coNOS is synthesized, and the expression amount of coNOS gene is regulated by molecular biology and transgenic technology, thereby realizing the regulation of plant growth and development and obtaining high-quality transgenic cotton with significantly increased fiber number. The functions and uses of coNOS protein and nitric oxide small molecule (Nitric Oxide, hereinafter referred to as NO) signal are also provided, which have a positive effect on promoting fiber cell differentiation and improving cotton fiber density quality traits.

[0055] As used herein, the "plant" includes Gossypium plants expressing coNOS or its homologs, preferably it also has a nitric oxide synthesis / metabolism pathway. According to the knowledge in the art, there are plants with coNOS and the signal pathways in which it is involved, which inherently have the mechanism of action as claimed in the present application and can achieve the technical effects as claimed in the present application. In some preferred ways, the plant is an economic crop, preferably a Gossypium crop. More specifically, for example, Gossypium hirsutum and the like.

[0056] As used herein, "high expression or high activity" with respect to a target gene / protein refers to statistically significant increase in expression or activity in a particular plant (e.g., a modified plant) as compared to the average expression or activity in a similar or identical plant, such as 10%, 20%, 60%, 80%, 100%, 200%, 300%, 500%, 800% or more.

[0057] As used herein, "up-regulation" includes promotion, overexpression, increase, enhancement, etc., which is statistically significant or significant up-regulation, promotion, increase or enhancement, such as 5%, 10%, 20%, 60%, 80%, 100%, 200%, 300%, 500%, 800% or more.

[0058] As used herein, "pathway," "pathway," "signaling pathway / pathway" and "regulatory pathway / pathway" are used interchangeably.

[0059] As used herein, a suitable "control plant" is an integral part of the experimental design and can include a corresponding wild-type plant or a corresponding transgenic plant without the gene of interest. The control plant is generally of the same plant species or even the same or a similar variety as the plant to be evaluated. The control plant can also be an individual that has been isolated from the transgenic plant. As used herein, a control plant refers not only to a whole plant, but also to a plant part, including a leaf, an ovule, a fiber cell, an ovule, a seed or a part thereof.

[0060] In the present application, the coNOS is a protein having the amino acid sequence shown in SEQ ID NO: 2, or a sequence variant having the same function as the coNOS protein.

[0061] The variant includes, but is not limited to, deletion, insertion and / or substitution of several (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, still more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (e.g., 30 or less, or 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and / or N-terminus. Any protein that is highly homologous to the coNOS protein (e.g., 70% or more homologous to the polypeptide sequence shown in SEQ ID NO: 2; preferably 80% or more homologous; more preferably 90% or more homologous, such as 95%, 98% or 99% homologous) and has the same function as the coNOS protein is also included in the present application.

[0062] In the present invention, the "coNOS protein" also includes their homologues. It should be understood that, although the coNOS protein obtained from a specific species is preferably studied in the present invention, other polypeptides or genes highly homologous (e.g. having more than 70%, more particularly 80%, 85%, 90%, 95%, even more than 98% sequence identity) to the coNOS protein obtained from the same species or other species, particularly from a Malvaceae plant, are also within the scope of the present invention.

[0063] The present invention also relates to polynucleotide sequences encoding the coNOS protein of the present invention or sequence variants thereof. The polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO: 2 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a polypeptide having the sequence of SEQ ID NO: 2, but differs from the gene sequence shown in SEQ ID NO: 1. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogues and derivatives of the polypeptides having the same amino acid sequence as the present invention.

[0064] The present invention also relates to constructs (vectors) comprising the polynucleotides, and host cells genetically engineered with the constructs (vectors) or polypeptide-encoding nucleic acids.

[0065] In the present invention, the polynucleotide sequence encoding the polypeptide of the present invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of the expression vector is that it usually contains an origin of replication, a promoter, a marker gene and a translation control element. Preferably, the expression vector can also selectively add resistance elements, screening (selection) elements or reporter gene elements, such as Bar, GUS.

[0066] Based on the new findings of the present inventors, the use of a coNOS protein or a modulating molecule (upregulator) thereof is provided for improving the quality or yield traits of cotton fibers; preparing a preparation for improving the quality or yield traits of cotton fibers; or, as a molecular marker for identifying the quality or yield traits of cotton fibers of a Gossypium plant.

[0067] Once the function of the coNOS is known, various methods known to those skilled in the art can be used to overexpress the coNOS to improve the fiber quality or yield traits of cotton. For example, an expression unit (e.g., an expression vector or a virus) carrying the coNOS gene can be delivered to the target and expressed to produce active coNOS by methods known to those skilled in the art.

[0068] Other methods for increasing the expression of the coNOS gene or its homologs are known in the art. For example, the expression of the coNOS gene or its homologs can be enhanced by driving with a strong promoter. Alternatively, the expression of the coNOS can be enhanced by an enhancer (e.g., the first intron of the waxy gene of rice, the first intron of the Actin gene, etc.). Suitable strong promoters include, but are not limited to, the 35s promoter, the Ubi promoter of rice and corn, etc.

[0069] In the present application, the upregulator of the polypeptide of the coNOS or its encoding gene includes a promoter, an agonist, an activator. The "upregulation" or "promotion" includes the "upregulation" or "promotion" of the activity of the polypeptide or the "upregulation" or "promotion" of the expression of the polypeptide. Any substance that can increase the activity of the coNOS, increase the stability of the coNOS, upregulate the expression of the coNOS, or increase the effective action time of the coNOS can be used in the present application as a substance useful for upregulating the coNOS. They can be compounds, chemical small molecules, biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0070] The coNOS or its upregulator is particularly suitable for use in a class of plants whose expression of the coNOS is lower than the average of the class of plants or whose coNOS is not expressed; thus, the use of the coNOS protein or its upregulator can restore the phenotype of the class of plants to the wild type or a better phenotype.

[0071] The transformation of host cells with recombinant DNA can be performed using conventional techniques known to those skilled in the art. The transformation of plants can be performed using methods such as Agrobacterium transformation or biolistic transformation, etc.

[0072] The present application also includes plants or plant progeny obtained by any of the foregoing methods, which plants include transgenic plants into which a nucleic acid encoding the polypeptide has been introduced.

[0073] Nitric oxide is a gaseous signaling molecule that plays a very important role in the growth and development of animals and plants. Nitric oxide has important applications in the medical field, especially in the treatment of cardiovascular diseases. In plants, nitric oxide plays a certain role in resisting adverse environments such as drought and cold. However, the effect of nitric oxide on cotton fibers in cotton plants is not well understood in the art.

[0074] The inventors' research results show that nitric oxide and its regulatory gene coNOS have important regulatory effects on fiber cell density, and have very strong application value in improving fiber yield and fiber quality.

[0075] Any substance that can promote the effect of coNOS, participate in the synthesis pathway of nitric oxide, and increase the amount of nitric oxide in cotton plants can be applied in the present application.

[0076] Based on the inventors' new findings, the present application provides molecular markers suitable for analyzing the fiber quality or yield traits of cotton plants, including the coNOS gene. The present application also relates to specific detection schemes for the coNOS or the synthesis pathway of nitric oxide, which can achieve early determination of the fiber quality or yield traits of cotton plants and determination of plants with excellent traits. The method comprises: identifying the expression or activity of coNOS in a test cotton plant; if the test cotton plant has high coNOS expression or activity (e.g., higher than the average value of the cotton plant species), it has high fiber quality or yield (including high fiber density and large number of mature fibers); if the test cotton plant has low coNOS expression or activity (e.g., lower than the average value of the cotton plant species), it has low fiber quality or yield (including low fiber density and small number of mature fibers); preferably, for a test cotton plant with low coNOS expression or activity, it is identified as a cotton plant that needs to be regulated with coNOS or its upregulator to restore normal coNOS expression or activity.

[0077] According to the inventors' new findings, those skilled in the art can use any of a variety of techniques known in the art or under development to analyze nucleic acid sequences, which can be included in the present application. The methods include, but are not limited to, PCR amplification, probe methods, sequencing methods, hybridization methods, restriction enzyme analysis methods, and allele polymorphism analysis methods (such as melting curve methods) for identifying nucleic acid sequences.

[0078] The identification method described in the present application only needs to perform PCR reaction and / or agarose gel electrophoresis, and by judging the length of the corresponding PCR product, the phenotype of the sample to be tested can be accurately and quickly determined, which is low in cost, suitable for large-scale identification, and requires very little sample amount. If necessary, those skilled in the art can design primers for identifying the molecular markers.

[0079] Methods for obtaining DNA from a sample to be tested are well known to those skilled in the art, for example the traditional phenol / chloroform / isoamyl alcohol method can be used, or a commercially available DNA extraction kit can be used, such kits being well known to those skilled in the art. Polymerase chain reaction (PCR) technology is well known to those skilled in the art, and is based on the principle of in vitro enzymatic synthesis of specific DNA fragments. The method of the present application can be carried out using conventional PCR technology.

[0080] In the light of the function of coNOS or nitric oxide, in addition to using it as a molecular marker for directed screening of plants, it is also possible to screen for substances or potential substances which can be used to direct regulation of cotton fibre quality or yield traits by modulating this mechanism.

[0081] The present application provides a method for screening for potential substances which can be used to improve cotton fibre quality or yield traits, comprising: (1) treating a Gossypium cell or tissue system which expresses coNOS and / or has other nitric oxide synthesis systems with a candidate substance; (2) detecting coNOS and / or other nitric oxide synthesis systems in the system; if the candidate substance statistically increases the expression or activity of coNOS and / or increases the amount of nitric oxide, then it is indicated that the candidate substance is a potential substance which can be used to improve cotton fibre quality or yield traits.

[0082] Methods for screening for substances which act on a target, using the target as a target, are well known to those skilled in the art, and these methods can be used in the present application. The candidate substance can be selected from the group consisting of peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0083] Methods for detecting the interaction between proteins and the strength of the interaction are well known to those skilled in the art, for example GST pull-down, bi-molecular fluorescence complementation, yeast two-hybrid system or co-immunoprecipitation.

[0084] Through large-scale screening, a class of substances which specifically act on coNOS protein or its encoding gene, or which modulate the NO synthesis pathway, modulate the level of NO in a plant and have a regulatory effect on cotton fibre quality or yield can be obtained.

[0085] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not specified, are generally according to the conventional conditions as described in J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Science Press, or according to the conditions suggested by the manufacturer.

[0086] Example 1, coNOS full-length gene synthesis

[0087] The cotton codon-optimized NOS gene was synthesized by Shanghai Xinzhuo Biotechnology Co., Ltd.

[0088]

[0089] coNOS encoding amino acid sequence (SEQ ID NO: 2):

[0090] MEDHMFGVQQIQPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLIRGGAAEQSGLIQAGDIILA

[0091] VNGRPLVDLSYDSALEVLRGIASETHVVLILRGPEGFTTHLETTFTGDGTPKTIRVTQPLGPPTKA

[0092] VDLSHQPPAGKEQPLAVDGASGPGNGPQHAYDDGQEAGSLPHANGLAPRPPGQDPAKKATRVS

[0093] LQGRGENNELLKEIEPVLSLLTSGSRGVKGGAPAKAEMKDMGIQVDRDLDGKSHKPLPLGVEN

[0094] DRVFNDLWGKGNVPVVLNNPYSEKEQPPTSGKQSPTKNGSPSKCPRFLKVKNWETEVVLTDTL

[0095] HLKSTLETGCTEYICMGSIMHPSQHARRPEDVRTKGQLFPLAKEFIDQYYSSIKRFGSKAHMERL

[0096] EEVNKEIDTTSTYQLKDTELIYGAKHAWRNASRCVGRIQWSKLQVFDARDCTTAHGMFNYICN

[0097] HVKYATNKGNLRSAITIFPQRTDGKHDFRVWNSQLIRYAGYKQPDGSTLGDPANVQFTEICIQQ

[0098] GWKPPRGRFDVLPLLLQANGNDPELFQIPPELVLEVPIRHPKFEWFKDLGLKWYGLPAVSNMLL

[0099] EIGGLEFSACPFSGWYMGTEIGVRDYCDNSRYNILEEVAKKMNLDMRKTSSLWKDQALVEINIA

[0100] VLYSFQSDKVTIVDHHSATESFIKHMENEYRCRGGCPADWVWIVPPMSGSITPVFHQEMLNYRL

[0101] TPSFEYQPDPWNTHVWKGTNGTPTKRRAIGFKKLAEAVKFSAKLMGQAMAKRVKATILYATET

[0102] GKSQAYAKTLCEIFKHAFDAKVMSMEEYDIVHLEHETLVLVVTSTFGNGDPPENGEKFGCALM

[0103] EMRHPNSVQEERKSYKVRFNSVSSYSDSQKSSGDGPDLRDNFESAGPLANVRFSVFGLGSRAYP

[0104] HFCAFGHAVDTLLEELGGERILKMREGDELCGQEEAFRTWAKKVFKAACDVFCVGDDVNIEKA

[0105] NNSLISNDRSWKRNKFRLTFVAEAPELTQGLSNVHKKRVSAARLLSRQNLQSPKSSRSTIFVRLH

[0106] TNGSQELQYQPGDHLGVFPGNHEDLVNALIERLEDAPPVNQMVKVELLEERNTALGVISNWTD

[0107] ELRLPPCTIFQAFKYYLDITTPPTPLQLQQFASLATSEKEKQRLLVLSKGLQEYEEWKWGKNPTI

[0108] VEVLEEFPSIQMPATLLLTQLSLLQPRYYSISSSPDMYPDEVHLTVAIVSYRTRDGEGPIHHGVCS

[0109] SWLNRIQADELVPCFVRGAPSFHLPRNPQVPCILVGPGTGIAPFRSFWQQRQFDIQHKGMNPCPM

[0110] VLVFGCRQSKIDHIYREETLQAKNKGVFRELYTAYSREPDKPKKYVQDILQEQLAESVYRALKE

[0111] QGGHIYVCGDVTMAADVLKAIQRIMTQQGKLSAEDAGVFISRMRDDNRYHEDIFGVTLRTYEV

[0112] TNRLRSESIAFIEESKKDTDEVFSS

[0113] Example 2, Construction of 35S::coNOS1 expression vector and Agrobacterium transformation

[0114] 1. Construction of 35S::coNOS1 expression vector

[0115] The sequencing correct T vector (containing the complete coNOS gene nucleotide sequence in the T vector) was used as a template, 25 μL high-fidelity enzyme MIX (purchased from Yixing Bio) was added, the homologous arms at both ends of the 35S::2301 enzyme cutting site were introduced at the 5' end of the primer, after adding the upstream and downstream primers, dd H2O was added to complete the system to 50 μL, and PCR reaction was performed. PCR reaction conditions: 94°C pre-denaturation for 5 min; then 94°C denaturation for 30 s, 58°C recombination for 30 s, 72°C extension for 1.5 min, a total of 38 cycles; finally 72°C extension for 10 min. The PCR product was electrophoretically purified and recovered to obtain the cloning fragment.

[0116] The 35S::2301 vector was double digested with restriction enzymes XbaI and KpnI, and the vector fragment was obtained after electrophoretic purification and recovery. The cloning fragment and the vector fragment were recombined with homologous recombination enzyme, and the 35S::coNOS vector was obtained after sequencing confirmation. Figure 1 )。

[0117] Primer sequence:

[0118] coNOS1-2301-F: 5'-GAGAGAACAGTCGACTCTAGAATGGAAGATCATAT GTTTGGAGTTCA-3'(SEQ ID NO: 3);

[0119] coNOS1-2301-R: 5'-GAACGATCGAGCTCGGAGAAGAAAACACTTCATC AG-3'(SEQ ID NO: 4).

[0120] 2. Agrobacterium tumefaciens transformation

[0121] Agrobacterium tumefaciens was transformed by freeze-thaw method. One single colony of LBA4404 (Invitrogen) was inoculated into 3 ml LB medium (25 μg / ml rifampicin (Rif) and 50 μg / ml kanamycin (Kan) or gentamycin (Gen)) and incubated at 28°C, 220 rpm overnight. 2 ml bacterial solution was inoculated into 50 ml LB medium (25 μg / ml Rif and 50 μg / ml Gen) and incubated at 28°C, 220 rpm until OD 600 = 0.5 (about 6 h). The solution was placed on ice for 30 min and centrifuged at 5000 g, 4°C for 5 min. The pellet was resuspended in 10 ml 0.15 M NaCl. The solution was centrifuged at 5000 g, 4°C for 5 min. The pellet was resuspended in 1 ml 20 mM CaCl2, 50 μl / tube, snap-frozen in liquid nitrogen and stored at -70°C. The binary vector containing the gene of interest (35S::coNOS vector constructed as described above) and 50 μl / tube competent cells were mixed and placed on ice for 30 min, snap-frozen in liquid nitrogen for 1 min. The bacterial solution was thawed at 37°C water bath for 5 min, 1 ml LB medium was added and the solution was incubated at 28°C, 220 rpm for 2-4 h. 50-100 μl of the solution was spread on LB medium (25 μg / ml Rif, 50 μg / ml Gen and 50 μg / ml kanamycin (Kan) or hygromycin (Hyg)) and single colonies were picked after 2 days for PCR identification. Agrobacterium clones with the vector were obtained.

[0122] Example 3, Plant Transformation and Screening of Transgenic Progeny

[0123] Agrobacterium containing 35S::coNOS vector plasmid was cultured on YEB medium containing kanamycin 50 mg / L, rifampicin 100 mg / L and streptomycin 300 mg / L for 2-3 days, and single colonies were inoculated into YEB liquid medium containing the same antibiotics and incubated at 28°C, 200 rpm / min overnight. The bacterial solution was centrifuged at 4000 rpm / min for 10 min, the pellet was resuspended in 1 / 2MS liquid medium containing glucose 30 g / L and acetosyringone 100 μmol / L, and the OD 600 value was adjusted to 0.4-0.6. The solution was used as infection solution.

[0124] Cotton TM-1 seeds (Gossypium hirsutum TM-1 variety) were sterilized and placed on 1 / 2MS0 (1 / 2MS salts + 5 g / L glucose + 7 g / L agar powder, pH 6.0) medium and incubated in the dark. Hypocotyls of the aseptic seedlings were cut into 1.0 cm sections after 5-7 days and used as transformation explants.

[0125] The explants were soaked in Agrobacterium solution for 15-20 min, and then transferred to co-cultivation 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 dark at 22°C for 2 days. Then the explants were transferred to medium MSB2 (MSB1 + 500 mg / L cephalosporin + 80 mg / L kanamycin) for induction of resistant callus. The explants were subjected to induction of resistant callus, proliferation of callus and induction of embryogenic callus (medium MSB3: MS salt + B5 organic + 30 g / L glucose + 2.5 g / L Gelrite, pH 6.0), 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; double KNO3 in MS salt and remove NH4NO3), and regeneration of resistant plantlets. When the plantlets grew to 3-4 true leaves, they were transplanted into flower pots and grown in an artificial climate room.

[0126] More than 5 coNOS expression lines (FENG) were obtained.

[0127] Example 4, Molecular biological identification of transgenic plants

[0128] 1. RNA extraction from Gossypium hirsutum

[0129] The ovules of Gossypium hirsutum TM-1 (Gossypium hirsutum variety "TM-1", purchased from the Cotton Institute of the Chinese Academy of Agricultural Sciences) were collected on the day of flowering and ground into powder in liquid nitrogen.

[0130] The polysaccharide and polyphenol total RNA extraction kit (purchased from Tiangen Bioscience Co., Ltd.) was used for RNA extraction.

[0131] The process is briefly described as follows:

[0132] (1) Take an appropriate amount of sample and add 700 μL of lysis solution SL (add 1580 μL of β-mercaptoethanol before use);

[0133] (2) Centrifuge at 10000 x g for 2 minutes at low temperature;

[0134] (3) Carefully pipette the supernatant into the filter column CS and centrifuge at 10000 x g for 1 minute at low temperature;

[0135] (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;

[0136] (5) Centrifuge at 10000 x g for 30 s at low temperature, and discard the waste liquid;

[0137] (6) Add 350 μL of RNase-free water RW1 to the adsorption column CR3;

[0138] (7) 10000xg low temperature centrifugation for 30s, discard the waste liquid;

[0139] (8) Add at least 80 μL of DNAes I to the adsorption column CR3 to remove residual DNA and make the RNA purer, and stand at room temperature for 15 minutes;

[0140] (9) Add 350 μL of RNase-free water RW1 to the adsorption column CR3;

[0141] (10) 10000xg low temperature centrifugation for 30s, discard the waste liquid;

[0142] (11) Add 500 μL of rinse liquid RW to the adsorption column CR3;

[0143] (12) 10000xg low temperature centrifugation for 30s, discard the waste liquid;

[0144] (13) Repeat steps (12) and (13);

[0145] (14) 10000xg low temperature centrifugation for 2 minutes;

[0146] (15) Add appropriate amount of RNAse-free H2O, 10000xg low temperature centrifugation for 1 minute;

[0147] (16) Use instruments such as NanoDrop to detect the concentration and state of RNA extraction.

[0148] 2、PCR

[0149] DNA extraction was performed by the method of cold phenol. 2g material was ground into powder in liquid nitrogen, transferred into 50ml centrifuge tube, 8ml extraction buffer (1M Tris-HCl, 50mM EDTA, 1% SDS, pH 9.0) and equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24:1) were added, mixed well, and placed on ice for 1h, mixed every 10min. Centrifuged at 13000g for 20min at 4°C. The phenol: chloroform: isoamyl alcohol extraction was repeated 2-4 times, and finally extracted once with chloroform: isoamyl alcohol (24:1). The supernatant was taken, 1 / 2 volume of high-salt solution (0.8M sodium citrate, 1.2M NaCl) and 1 / 2 volume of isopropanol were added, mixed well, and placed at -70°C for 1h. Centrifuged at 13000g for 20min at 4°C, the supernatant was removed, the precipitate was washed with 1ml 70% ethanol twice, blown at room temperature for 20min, and dissolved in 1ml sterile water. Centrifuged at 13000g for 10min at 4°C. The supernatant was taken, 5-10μl RNase (10mg / ml) was added, and digested at 37°C for 30min.

[0150] PCR identification used NPT II-specific primers:

[0151] CRcoNOS-1F: 5'-TAGGAAAGATATGTGTATAATGTTTCAAATTGGAGT

[0152] GAG-3'(SEQ ID NO: 5);

[0153] CRcoNOS-1R: 5'-AAGCGGCTCTTGCGATCA-3'(SEQ ID NO: 6);

[0154] CRcoNOS-2F: 5'-TGGAACACAGCCAAAGTTGAACC-3'(SEQ ID NO: 7);

[0155] CRcoNOS-2R: 5'-TCCGGTTATGCAGTCATTAGTGC-3'(SEQ ID NO: 8).

[0156] The reaction conditions were as follows: pre-denaturation at 94°C for 5min, then 35 cycles of pre-denaturation at 94°C for 30sec, annealing at 56°C for 30sec, and extension at 72°C for 1min, and finally extension at 72°C for 10min. The amplified fragment was 510bp in size.

[0157] After sequencing, the gene expression successful strain FENG was detected and obtained, and subcultured.

[0158] 3. Fluorescent quantitative RT-PCR analysis

[0159] a. RT-PCR analysis

[0160] 1 μg of total RNA, reverse transcription by Oligo (dT) primer, 20 μl reaction. 42 °C for 30 minutes, reverse transcription of first strand cDNA, overnight. Take 0.5 μl of reverse transcription product, 25 μl PCR reaction system to detect the expression of target gene. PCR primer is specific primer of GhTCP4:

[0161] coNOS-qRT-F: 5'-GGTTGTGCTTTGATGGAAATGAGG-3' (SEQ ID NO: 9); coNOS-qRT-R: 5'-CAGCCTTGAACACTTTCTTAGCCC-3' (SEQ ID NO: 10).

[0162] Cotton gene Histone3 (AF024716) as an internal standard, correct the template amount of RT-PCR reaction. PCR reaction conditions: 94 °C pre-denaturation for 5 minutes; then 94 °C denaturation for 30 seconds, 56 °C recombination for 30 seconds, 72 °C extension for 1 minute, a total of 30 cycles; finally 72 °C extension for 10 minutes.

[0163] b. Fluorescence quantitative RT-PCR analysis

[0164] Quantitative RT-PCR detection using chimeric fluorescence method Premix Ex Taq TM II (Perfect RealTime), TaKaRa, DRR041A). Reaction system is shown in Table 1.

[0165] Table 1

[0166]

[0167] Cotton gene Histone3 (AF024716) as an internal standard. Data analysis using Realplex v2.0 (purchased from Eppendorf, Hamburg, Germany, NSW). The experiment was repeated three times, and the average value and variance of each group of data were taken to draw a chart.

[0168] Select transgenic lines with expression higher than wild type TM-1 at least 10 times, called FENG, and subculture.

[0169] Example 5, fiber length and gene expression analysis of transgenic cotton

[0170] a. Gene expression analysis and function verification of coNOS

[0171] The results showed that coNOS could produce NO in cotton, especially in fiber cells.

[0172] The results showed that coNOS could produce NO in cotton, especially in fiber cells. Figure 2 DAF-FM DA is a probe for quantitative detection of nitric oxide (NO). FM4-64 is a lipophilic water-soluble styryl dye that can specifically bind to cell membranes and endomembrane organelles to produce fluorescence.

[0173] The results showed that coNOS could produce NO in cotton, especially in fiber cells.

[0174] b. Trait analysis of transgenic cotton

[0175] The 35S::coNOS transgenic cotton and wild type TM-1 were planted in Shanghai and Hainan farms, and were managed conventionally. At the mature boll stage, mature cotton bolls were collected from single plants, and the consistency of the collection site was maintained as much as possible. A certain number of seeds were randomly taken, the fibers were combed flat, and the fiber length was measured for statistical analysis.

[0176] The results showed that the phenotype of 35S::coNOS transgenic cotton (FENG) was stably inherited. In mature cotton, the fiber length was unchanged, the ovule was enlarged, and the number of fibers was increased Figure 3 A).

[0177] Scanning electron microscopy also showed that the number of fibers on the surface of the ovule of 35S::coNOS transgenic cotton (FENG) on the day of flowering was significantly increased compared to the wild type, with an increase of about 18% Figure 3 B-C).

[0178] Transmission electron microscopy results showed that on the day of flowering, the fiber cell wall was significantly thinned Figure 3 D-E).

[0179] The above results show that coNOS can increase the density of fibers and improve cotton yield by increasing the amount of NO signal.

[0180] In summary, the synthesis of NO by an exogenous gene plays a very important role in fiber development, especially in the early stage of fiber development. It improves the fiber cells while maintaining the fiber length, increases the density of fiber cells, and thus improves the yield of cotton.

[0181] Example 6, screening method

[0182] Test object: cotton, which normally expresses endogenous coNOS.

[0183] Test group: the above test subjects are treated with candidate substances / primers;

[0184] Control group: no candidate substance is introduced.

[0185] The expression and activity of coNOS in the test group and the control group are detected respectively and compared.

[0186] If the expression or activity of coNOS in the test group is statistically higher (e.g. 50% or more) than that in the control group, it indicates that the candidate substance is a potential substance for improving the quality or yield traits of cotton fibers (including: improving fiber cells, increasing fiber density, increasing the number of mature fibers, and increasing fiber yield).

[0187] Further, the production of NO in the test group and the control group can also be analyzed. If the production of NO increases on the basis of the increased expression or activity of coNOS, it indicates that the candidate substance is a potential substance for improving the quality or yield traits of cotton fibers.

[0188] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Meanwhile, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference individually.

Claims

1. A method for improving Gossypium plants, comprising: Upregulating coNOS in cotton plants 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: improving fiber cells, increasing fiber density, increasing the number of mature fibers, and increasing fiber yield; preferably, the coNOS controls the synthesis of fiber cell walls, reduces fiber cell wall thickness, and regulates fiber cell differentiation or growth; preferably, the reduction of fiber cell wall thickness is to reduce the cell wall thickness in the initial stage of fiber cell development.

3. The method as described in claim 1, characterized in that, The upregulation of coNOS expression or activity includes: Introducing the coding sequence of coNOS into plants; preferably, introducing the coding sequence of coNOS into plants using a construct containing the coding sequence of coNOS. Modifying the endogenous coNOS gene in plants to upregulate coNOS expression or activity; preferably, the modification is carried out through gene editing. Promotes the coNOS gene promoter drive capability; Regulation of coNOS can be achieved by upregulating molecules that interact with coNOS, thereby increasing the expression or activity of coNOS; or Introduce a nitric oxide synthesis pathway in plants to increase nitric oxide content.

4. The application of a coNOS or an upregulator thereof for: improving cotton plants, enhancing cotton fiber quality or yield traits; preparing preparations for enhancing cotton fiber quality or yield traits; or, as a molecular marker for identifying cotton fiber quality or yield traits in cotton plants.

5. The application as described in claim 4, characterized in that, The improvement of cotton fiber quality or yield includes: improving fiber cells, increasing fiber density, increasing the number of mature fibers, and increasing fiber yield; more preferably, the coNOS controls the synthesis of fiber cell walls, reduces the thickness of fiber cell walls, and regulates the differentiation or growth of fiber cells; even more preferably, the reduction of fiber cell wall thickness is to reduce the thickness of the cell wall in the initial stage of fiber cell development.

6. The method as described in any one of claims 1-3 or the application as described in any one of claims 4-5, characterized in that, The upregulators include: polynucleotides or expression constructs encoding coNOS, gene editing reagents that promote coNOS gene expression, upregulators that promote the promoter-driven ability of the coNOS gene, upregulators that interact with the coNOS protein to increase its expression or activity, downregulators of coNOS gene-specific microRNAs, chemical upregulators of coNOS, or combinations thereof.

7. The method as described in any one of claims 1-3 or the application as described in any one of claims 4-5, characterized in that, The coNOS comprises: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO:2; (b) a coNOS 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 coNOS derivative or active fragment having the function of the polypeptide (a) with a sequence having ≥80% homology to the amino acid sequence shown in supplementary b; and (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 supplementary b, 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 exogenous coNOS; preferably, the Gossypium includes a nitric oxide synthesis pathway; more preferably, the NO synthesis pathway includes: NO is generated through coNOS and through other metabolic pathways.

9. A method for identifying cotton fiber quality or yield traits in plants of the genus *Gossypium*, comprising: The expression or activity of coNOS in tested Gossypium species is identified. If the expression or activity of coNOS in the tested Gossypium species is high, the cotton fiber quality or yield is high. If the expression or activity of coNOS in the tested Gossypium species is low, the cotton fiber quality or yield is low. Preferably, for tested Gossypium species with low coNOS expression or activity, they are identified as Gossypium species that need to be regulated with coNOS or its upregulators to restore the coNOS expression or activity to normal.

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 coNOS and / or having other nitric oxide synthesis systems; (2) Detect the presence of CONOS and / or other nitric oxide synthesis systems in the system; If the candidate substance statistically increases the expression or activity of coNOS and / or increases the amount of nitric oxide, it indicates that the candidate substance is a potential substance for improving the quality or yield traits of cotton fibers.

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