Use of ghnfxl1 gene in improving salt tolerance and / or cold tolerance of gossypium plants

Abstract

The present application relates to the technical field of plant genetic engineering, and particularly relates to application of GhNFXL1 gene in improving salt tolerance and / or cold tolerance of cotton plants. Through various tests, it is found that silencing of the GhNFXL1 gene can reduce the salt tolerance and cold tolerance of the plants; overexpression of the GhNFXL1 gene can enhance the salt tolerance and cold tolerance of the plants, and the results show that the GhNFXL1 positively regulates the salt tolerance and cold tolerance of the cotton plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and in particular, relates to application of GhNFXL1 gene in improving salt tolerance and / or cold tolerance of Gossypium. BACKGROUND

[0002] Saline-alkali soils are widely distributed in the world, especially in the arid and semi-arid regions of the Middle East, North Africa, South Asia, western China and Australia. These saline-alkali soils have a serious impact on crop growth, mainly manifested as inhibition of seed germination, limited root growth and physiological dysfunction, leading to reduced crop yield and quality. For cotton, the impact of saline-alkali soils is particularly significant, as cotton not only has reduced germination rate and emergence rate in high-salt environments, but also has poor root development, leaf yellowing and slow growth, ultimately affecting fiber quality and yield. In addition, cotton is a warm-loving plant, and low temperature can cause slow growth, affect the efficiency of photosynthesis, and lead to leaf yellowing and shedding. The impact of low temperature is particularly evident during the flowering and boll setting period, which can cause flower bud abscission and reduced boll setting rate, ultimately leading to significant reduction in cotton yield.

[0003] NFXL (Nuclear Factor X-Like) proteins are a class of transcription factors belonging to the NFX family. They play an important role in various biological processes, including cell proliferation, differentiation and signal transduction. AtNFXL1 can be induced to be up-regulated in flowers by heat stress. At 22℃, there is no significant difference in pod length between wild type and AtNFXL1 mutant plants. However, after the plants in the reproductive stage are treated at 38℃ and then restored to normal temperature, the AtNFXL1 mutant plants are more sensitive to high temperature stress compared with the wild type, with increased proportion of shortened pods and reduced fertility. TaNFXL1 gene is induced to express by Fusarium graminearum, and the disease resistance of wheat is reduced after silencing TaNFXL1 gene.

[0004] In order to improve the salt tolerance and cold tolerance of cotton, protect cotton from low temperature stress during the growth and development period, ensure the yield and quality of cotton, and facilitate the promotion of cotton varieties and the breeding of resistant varieties, it is of great significance for adapting to different planting areas, promoting agricultural science and technology, and ensuring national cotton safety. SUMMARY

[0005] The present application encompasses the following technical solutions:

[0006] According to an aspect of the present application, the application relates to the use of a nucleic acid corresponding to GhNFXL1 gene in improving salt tolerance and / or cold tolerance of Gossypium.

[0007] According to still another aspect of the present application, there is provided a method for producing a Gossypium plant with improved salt tolerance and / or cold tolerance, comprising introducing a nucleic acid corresponding to the GhNFXL1 gene into the Gossypium plant.

[0008] According to still another aspect of the present application, there is provided a use of the Gossypium plant produced by the method as described above for producing cotton propagation material, wherein the propagation material is suitable for sexual reproduction, vegetative reproduction or tissue culture of regenerable cells.

[0009] According to still another aspect of the present application, there is provided a breeding method for a salt-tolerant and / or cold-tolerant Gossypium plant, comprising:

[0010] detecting the expression level of the GhNFXL1 gene in the Gossypium plant using a detection agent, and selecting a cotton plant or variety with a high expression level for breeding.

[0011] Advantages:

[0012] (1) The present application first explicitly defines the functional role of the gene GhNFXL1 in cotton salt tolerance and cold tolerance, which provides a target gene for enhancing cotton salt tolerance and cold tolerance.

[0013] (2) The present application finds through various tests that silencing of the GhNFXL1 gene reduces the salt tolerance and cold tolerance of the plant; overexpression of the GhNFXL1 gene enhances the salt tolerance and cold tolerance of the plant, indicating that GhNFXL1 plays a positive regulatory role in cotton salt tolerance and cold tolerance.

[0014] (3) The gene GhNFXL1 discovered by the present application can be applied to prevent and treat cotton cold damage and salt damage, and can be specifically used for genetic improvement or molecular breeding of cotton salt tolerance and cold tolerance, such as breeding of salt-tolerant and cold-tolerant cotton varieties. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 : Salt stress and cold stress expression analysis of the GhNFXL1 gene;

[0016] Figure 2 : Silencing of the GhNFXL1 gene reduces the salt tolerance and cold tolerance of cotton;

[0017] Figure 3 : Salt stress and cold stress physiological index detection of the GhNFXL1 gene silencing;

[0018] Figure 4 : Overexpression of the GhNFXL1 gene enhances the salt tolerance and cold tolerance of cotton;

[0019] Figure 5 : Salt stress and cold stress physiological index detection of the GhNFXL1 gene overexpression cotton. DETAILED DESCRIPTION

[0020] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and phrases related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and molecular biology, and laboratory procedures used herein are terms and procedures that are well understood by one of ordinary skill in the art. Also, for better understanding of the present application, the definitions and explanations of the related terms are provided below.

[0021] The terms "comprise", "contain", and "include" used in the present application are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps. Numerical ranges expressed in the form "from x to y" are understood to include all numbers and fractions subsumed within the given range, as well as the recited endpoints.

[0022] In the present application, "cotton plants" are a genus (Gossypium) of the family Malvaceae of dicotyledonous plants, and preferred species include Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium herbaceum.

[0023] In the present application, "nucleic acid", "polynucleotide", "nucleic acid sequence", "nucleotide sequence", and "nucleic acid fragment" are used interchangeably and refer to polymers of RNA and / or DNA that are single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually in the form of their 5'-monophosphates) are referred to by single-letter codes as follows: "A" for adenosine or deoxyadenosine, "C" for cytosine or deoxycytosine, and "G" for guanosine or deoxyguanosine, corresponding to RNA or DNA, respectively; "U" for uridine; "T" for deoxythymidine; "R" for purine (A or G); "Y" for pyrimidine (C or T); "K" for G or T; "H" for A or C or T; "I" for inosine; and "N" for any nucleotide.

[0024] "Polypeptide", "peptide", "amino acid sequence", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" can also include modifications, including but not limited to, glycosylation, lipid attachment, and sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0025] A "recombinant DNA construct" refers to a combination of nucleic acid segments that are not normally found together in nature. Accordingly, a recombinant DNA construct can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged to function in a manner that is not naturally found. In some embodiments, the recombinant DNA construct is a plasmid or a virus. In some embodiments, the recombinant DNA construct of the present application comprises regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly U sequences, etc.).

[0026] In the present application, the terms "overexpression of a gene" and "inhibition / reduction of gene expression" or their similar expressions refer to higher and lower levels of expression than in wild-type plants, respectively.

[0027] "Crossing" of two parent plants is the mating of two parent plants.

[0028] "Agronomically elite (trait)", as used herein, refers to a genotype that has superior or optimal performance in a number of distinguishable traits that allow the producer to harvest a product of commercial importance. This includes, but is not limited to, seed yield, vigor, nutrient potential, disease resistance, greenness, growth rate, total biomass or rate of accumulation, fresh weight at maturity, dry weight at maturity, fruit yield, grain yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, tiller number, panicle size, early seedling vigor, and emergence under low temperature stress, etc.

[0029] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is individually incorporated by reference. Unless and to the extent that it conflicts with the spirit and / or technical scope of the present application, the documents mentioned in the present application are incorporated by reference in their entirety and for all purposes.

[0030] Scheme Description

[0031] The first aspect of the present application relates to the use of a nucleic acid corresponding to the GhNFXL1 gene in improving salt tolerance and / or cold tolerance of a Gossypium plant.

[0032] The GhNFXL1 gene is known in the art, and its NCBI Reference Sequence is XM_016812982.2 (defined as Sequence 1). The nucleic acid corresponding to the GhNFXL1 gene referred to in the present application also includes functional equivalent sequences thereof, such as the CDS sequence thereof (defined as Sequence 2), and more preferably the sequence corresponding to nucleotides 422-3661 of the ORIGIN sequence of XM_016812982.2 (ATGAGCTTT... TTATGAATGA, defined as Sequence 3). When the nucleic acid corresponding to the GhNFXL1 gene has a nucleotide sequence as shown in Sequence 3, the effect of applying it to enhance the salt tolerance and cold tolerance of cotton is better.

[0033] The functional equivalent sequence also includes a gene sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to any one of the sequences shown in Sequences 1-3, and capable of expressing a gene having similar or identical NFXL transcription factor activity, which can be isolated from any plant. The percentage of sequence identity can be obtained by known bioinformatics algorithms, including the Myers and Miller algorithm (Bioinformatics, 4(1): 11-17, 1988), the Needleman-Wunsch global alignment method (J. Mol. Biol., 48(3): 443-53, 1970), the Smith-Waterman local alignment method (J. Mol. Biol., 147: 195-197, 1981), the Pearson and Lipman similarity search method (PNAS, 85(8): 2444-2448, 1988), and the Karlin and Altschul algorithm (Altschul et al., J. Mol. Biol., 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.

[0034] The functional equivalent sequence obviously also includes a DNA sequence that can hybridize to the above-mentioned sequences under stringent conditions. The "stringent conditions" used in the present application are known in the art, including, for example, hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA at 60°C for 12-16 hours, followed by washing with a washing solution containing 0.1% SDS and 0.1% SSC at 65°C for 15-60 minutes.

[0035] The functional equivalent sequence obviously also includes a sequence obtained by codon optimization or general nucleic acid modification of the above-mentioned sequences.

[0036] The second aspect of the present application relates to a method for producing a Gossypium plant with improved salt tolerance and / or cold tolerance, comprising introducing a nucleic acid corresponding to the GhNFXL1 gene into the Gossypium plant.

[0037] The method can allow overexpression of the GhNFXL1 gene in the Gossypium plant.

[0038] In some embodiments, the nucleic acid is introduced into the Gossypium plant in the form of a recombinant DNA construct.

[0039] The recombinant DNA construct comprises the nucleic acid corresponding to the GhNFXL1 gene and other nucleotide sequences, such as nucleotide sequences encoding functional units for protein purification, fluorescent protein markers, and binding sites for DNA, or nucleotide sequences encoding elements having a regulatory effect on gene transcription and expression, including but not limited to promoters, strong promoters, enhancers, or transcription factor binding sites, etc.

[0040] The method of the present application can also directly introduce the nucleic acid / recombinant DNA construct into the plant to produce the Gossypium plant, which is prepared using transformation methods known to those skilled in the art of plant biotechnology. Any method can be used to transform the recombinant DNA construct into a plant cell to produce the Gossypium plant of the present application. The transformation method can include direct and indirect transformation methods. In some embodiments, the recombinant DNA construct is introduced into the Gossypium plant by gene gun-mediated transformation, pollen tube pathway, or liposome transformation.

[0041] The recombinant DNA construct provided by the present application can be inserted into a plasmid, cosmid, yeast artificial chromosome, bacterial artificial chromosome, or any other vector suitable for transformation into a host cell. The preferred host cell is a bacterial cell, especially a bacterial cell used for cloning or storing polynucleotides, or for transforming plant cells, such as Escherichia coli, Agrobacterium, Rhizobium radiobacter, and Rhizobium rhizogenes. In some preferred embodiments, the recombinant DNA construct is introduced into the Gossypium plant by Agrobacterium.

[0042] In some embodiments, the method further comprises:

[0043] Crossbreeding the Gossypium plant introduced with the nucleic acid corresponding to the GhNFXL1 gene with a second Gossypium plant to produce a progeny plant; the second Gossypium plant is an agronomically elite variety.

[0044] The third aspect of the present application relates to the use of the Gossypium plant produced by the method described above for producing cotton propagation material, wherein the propagation material is suitable for sexual reproduction, vegetative reproduction, or tissue culture of regenerable cells.

[0045] In some embodiments, the propagation material suitable for sexual reproduction is selected from the group consisting of microspores, pollen, ovary, ovule, embryo sac and egg cell.

[0046] In some embodiments, the propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, anther, flower, seed and stem.

[0047] The fourth aspect of the present application relates to a method for breeding a salt-tolerant and / or cold-tolerant Gossypium plant, comprising:

[0048] The expression level of the GhNFXL1 gene in the Gossypium plant is detected using a detection agent, and a cotton plant or variety with a high expression level is selected for breeding.

[0049] In some embodiments, the detection agent comprises a reagent for detecting the change of mRNA expressed by the GhNFXL1 gene, which is suitable for at least one of the following methods: sequencing, polymerase chain reaction (PCR), isothermal amplification reaction, resonance light scattering method, biological mass spectrometry, electrochemical analysis, gel electrophoresis, capillary electrophoresis, microarray, CRISPR-Cas detection system.

[0050] wherein the nucleic acid sequencing can be Maxam-Gilbert sequencing, chain termination method, shotgun sequencing, bridge PCR, single molecule real-time sequencing, ion semiconductor (ion torrent sequencing), sequencing by synthesis, sequencing by ligation (SOLiD sequencing), chain termination (Sanger sequencing), massively parallel signature sequencing (MPSS), polymerase cloning sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunneling current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidic Sanger sequencing, microscopy-based techniques, RNAP sequencing and in vitro viral high-throughput sequencing.

[0051] wherein the PCR is preferably qRT-PCR and digital PCR.

[0052] wherein the generalized isothermal techniques can be subdivided into methods that rely on the displacement of primers to initiate repeated template copies (illustrated below, HDA (helicase-dependent amplification), exonuclease-dependent amplification (EP1866434), recombinase polymerase amplification (RPA), recombinase-mediated amplification (RAA), loop-mediated amplification (LAMP), rolling circle amplification (RCA), multiple displacement amplification (MDA), cross priming amplification (CPA)) and methods that rely on the continued reuse or de novo synthesis of single primer molecules, SDA (strand displacement amplification and nucleic acid-based amplification (NASBA and TMA)).

[0053] Among them, according to the type of nucleic acid molecule detected, the CRISPR / Cas technology nucleic acid detection can be divided into DNA (CRISPR / Cas9, CRISPR / Cas12 and CRISPR / Cas12 recognize DNA sequence) and RNA (CRISPR / Cas13 recognizes RNA sequence) detection.

[0054] In some embodiments, the detection agent includes a reagent for detecting changes in the protein expressed by the GhNFXL1 gene, which is suitable for at least one of the following detection methods: immunoassay, biological mass spectrometry, lectin-based detection method, and nucleic acid aptamer-based detection method.

[0055] As the immunoassay, various enzyme immunoassay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), double monoclonal antibody sandwich immunoassay, monoclonal polyclonal antibody sandwich immunoassay, immunostaining method, immunofluorescence method, Western blotting method, biotin-avidin method, immunoprecipitation method, colloidal gold agglutination method, immunochromatography, latex agglutination method (LA) and immunoturbidimetry (TIA) and the like can be mentioned without particular limitation.

[0056] As the reagent used in the immunoassay, an anti-GhNFXL1 antibody already on the market can be used, or an antibody can be prepared by a conventional method based on the known amino acid sequence of GhNFXL1.

[0057] As the mass spectrometry, there is no particular limitation, and a mass spectrometer can be used by combining an ion source using electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser desorption ionization (SELDI) and the like with a time-of-flight analyzer (TOF), an ion trap analyzer (IT), a Fourier transform analyzer (FT) and the like. LC-MS and CE-MS and the like can be used, which are formed by connecting a mass spectrometer with a separation device such as high-speed liquid chromatography (HPLC) or capillary electrophoresis (CE). In addition, as a method of obtaining mass spectrometry data, data-independent analysis (DIA), data-dependent analysis (DDA), multiple reaction monitoring method (MRM) and the like can be mentioned. In the mass spectrometry, cases where the sample is stably isotopically labeled with iTRAQ reagent (SCIEX Corporation) and the like are also included.

[0058] Embodiments of the present application will be described in detail below with reference to Examples. It should be understood that these Examples are only intended to illustrate the present application and are not intended to limit the scope of the present application. In the following Examples, the experimental methods not specified in the specific conditions can be preferably referred to the guidelines given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or can be referred to other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0059] In the following specific examples, the amount of raw material components are measured parameters, and slight deviations within the range of weighing accuracy are possible without special instructions. Involving temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0060] The transgenic cotton recipient material used in this example is Gossypium hirsutum "ZM113".

[0061] The vectors used in this example are as follows: pTRV2: gene silencing vector, used for silencing material transformation; pCambia2300-GFP: overexpression vector, used for overexpression material transformation.

[0062] The strains used in this example are as follows: E. coli competent DH5α, Agrobacterium competent GV3101.

[0063] The remaining materials or reagents, unless otherwise specified, are prepared according to the existing method or directly purchased from the market in the example.

[0064] Example 1 Cotton planting and treatment

[0065] Cotton planting: Select ZM13 seeds with full grains, clean them with sterile water, soak them in a container, and place them in a 28℃ artificial climate incubator overnight to promote germination. After the seeds are white, transplant them into moist nutrient soil, cover them with mulch, and place them in a 28℃ artificial climate incubator with natural light for 16h / darkness for 8h alternately.

[0066] Cotton cold treatment: When the cotton seedlings grow to two leaves and one heart, the cotton in the treatment group is placed in a 4℃ artificial climate incubator, and the control group is placed in a 28℃ artificial climate incubator, with natural light for 16h / darkness for 8h alternately.

[0067] Cotton salt treatment: When the cotton seedlings grow to two leaves and one heart, the treatment group is added with 200mM NaCl solution, and the control group is added with equal volume of H2O. The treatment group and the control group are placed in an artificial climate incubator with natural light for 16h / darkness for 8h alternately.

[0068] Example 2 Expression analysis of GhNFXL1 gene under salt stress and cold stress

[0069] The root tissues of Gossypium hirsutum ZM113 were sampled at 0h, 1h, 3h, 6h, and 12h after 4℃ stress treatment, and the expression of GhNFXL1 gene was analyzed by RT-qPCR, with GhUBQ7 gene as the reference gene.

[0070] The RT-qPCR reaction system is as follows:

[0071]

[0072] The RT-qPCR reaction program is as follows:

[0073]

[0074] The primers used for RT-qPCR are as follows:

[0075]

[0076] Data analysis utilizes 2 -ΔΔCT Method, three parallel repeats, t-test, ** indicates P < 0.01. The results show that with the increase of salt stress time, the expression amount of GhNFXL1 gene increases significantly Figure 1 A) During 0-3h, the expression amount of GhNFXL1 gene continuously increases and reaches the peak at 3h. During 6-24h, the expression amount of GhNFXL1 gene begins to decrease, which proves that GhNFXL1 gene responds to salt stress. With the increase of cold stress time, the expression amount of GhNFXL1 gene increases significantly Figure 1 B) During 0-12h, the expression amount of GhNFXL1 gene continuously increases and reaches the peak at 12h. At 24h, the expression amount of GhNFXL1 gene begins to decrease, which proves that GhNFXL1 gene responds to cold stress.

[0077] Example 3 Construction of GhNFXL1 gene expression vector

[0078] 1. Recovery of target fragment and linearized vector

[0079] (1) Linearization of expression vector: incubate gene silencing vector pTRV2 and overexpression vector pCambia2300-GFP in the following reaction system at 37℃ for 1h double enzyme digestion to obtain linearized vector:

[0080]

[0081] (2) Obtain target fragment: use cotton cDNA as template, amplify sequence using Novozyme's high-fidelity enzyme 2xPhantaMax MasterMix(Dye Plus), and the PCR reaction system is as follows:

[0082]

[0083] PCR amplification system is as follows:

[0084]

[0085] Primer information

[0086]

[0087] (3) The above PCR products and linearized expression vector were purified using the EasyPure Quick Gel Extraction Kit from Beijing TransGen Biotech Co., Ltd. The specific steps are as follows:

[0088] (a) Quickly cut the agarose gel containing the target band under UV light to avoid prolonged exposure to UV light that could damage the DNA. Cut the gel into small pieces and place them into a clean 1.5 mL centrifuge tube for subsequent gel dissolution.

[0089] (b) Weigh the gel using an electronic balance. Add 3 times the volume of BufferGDP according to the formula 100mg equals 100μL. Incubate in a 55°C water bath for 15 minutes, inverting and mixing 2-3 times during the process to completely dissolve the gel.

[0090] (c) Place the FastPure DNAMini Columns-G adsorption column in a 2 mL collection tube, carefully transfer the sol liquid cooled to room temperature into the adsorption column, and centrifuge at 12000 rpm for 1 min.

[0091] (d) Discard the filtrate and then return the adsorption column to the collection tube. Add 300 μL of Buffer GDP to the adsorption column, let it stand at room temperature for 3 min, and then centrifuge at 12000 rpm for 1 min.

[0092] (e) Discard the filtrate and then return the adsorption column to the collection tube. Add 700 μL of Buffer GW and centrifuge at 12,000 rpm for 1 min.

[0093] (f) Repeat step (5).

[0094] (g) After discarding the filtrate, put the adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 min.

[0095] (h) Place the adsorption column in a sterile 1.5 ml centrifuge tube, add 40 μL of Elution Buffer (preheated to 55 °C in a water bath) to the center of the adsorption column, and incubate at room temperature for 5 min. Elute the DNA by centrifugation at 12000 rpm for 1 min, and store at -20 °C for later use.

[0096] 2. Vector construction and transformation of E. coli: using vectors from Nanjing Novizan Biotechnology Co., Ltd. The UltraOne Step Cloning Kit performs homologous recombination of linearized vectors and target fragments to construct vectors, which are then prepared in the following liquid systems on ice:

[0097]

[0098] The reaction was carried out at 50°C for 10 min, and then immediately cooled on ice. The recombinant product was then transformed into competent E. coli cells.

[0099] (1) Add all 10 μL of the recombinant ligation product to 100 μL of freshly thawed DH5α competent cells and let stand on ice for 30 min.

[0100] (2) Heat shock in a 42℃ water bath for 45 seconds, then immediately place on ice for 2 minutes.

[0101] (3) Add 500 μL of LB liquid medium without antibiotics and place it on a shaker at 37°C for 1 h.

[0102] (4) Centrifuge at 5,000 r / min for 2 min, discard 400 μL of supernatant in a clean bench, leave 100 μL of bacterial culture and spread it on LB solid medium containing the corresponding antibiotic, blow dry and invert it in a 37℃ incubator overnight.

[0103] (5) On the second day, pick single clones from the plate and shake them in 500 μL of LB liquid medium containing the corresponding antibiotic until the bacterial culture is turbid. Perform bacterial culture PCR and send positive clones to Shanghai Sangon Biotech Co., Ltd. for sequencing. Compare the sequencing results returned by the company with the target sequence. If the comparison results are completely correct, preserve the bacteria.

[0104] 3. Plasmid extraction: Using Beijing Total Gold plasmid extraction equipment... The Plasmid MiniPrep Kit is used for the extraction of E. coli plasmids. The specific steps are as follows:

[0105] (1) Take 5 mL of Escherichia coli culture that has been cultured overnight at 37℃ in a shaker and put it into a 10 mL centrifuge tube. Centrifuge at 12,000 r / min for 5 min and discard the supernatant.

[0106] (2) Add 300 μL of colorless solution RB (containing RNase A), and shake vigorously with a vortex mixer to precipitate and suspend the bacteria. No small bacterial clumps should remain.

[0107] (3) Add 300 μL of blue solution LB, gently invert and mix 6-8 times to fully lyse the cells until the solution turns a clear blue color, indicating complete lysis. The operation should be completed within 5 minutes.

[0108] (4) Add 450 μL of yellow solution NB, gently mix by turning it upside down 6-8 times until the solution color changes from blue to yellow and forms a firm yellow aggregate, indicating that the neutralization is complete. Let it stand at room temperature for 2 minutes.

[0109] (5) After centrifugation at 12,000 r / min for 10 min, the supernatant was taken into the centrifugal column, 800 μL at a time, centrifuged at 12,000 r / min for 1 min, and the liquid in the collection tube was discarded.

[0110] (6) 700 μL of rinse solution WB was added, centrifuged at 12,000 r / min for 1 min, the liquid in the collection tube was discarded, the empty tube was centrifuged at 12,000 r / min for 2 min, and placed on the clean bench to dry for 5 min to remove the residual rinse solution.

[0111] (7) The centrifugal column was placed in a clean 1.5 mL centrifugal tube, 40 μL of 65°C preheated Elution Buffer was added to the center of the centrifugal column, and after standing at room temperature for 2 min, the plasmid DNA was eluted by centrifugation at 12,000 r / min for 2 min.

[0112] (8) The plasmid DNA concentration was measured using NanoDrop 2000, and the obtained plasmid was stored at -20°C.

[0113] 4. Agrobacterium transformation: Agrobacterium transformation was performed using GV3101 from Shanghai Weidi Biological Company, and the specific steps were as follows:

[0114] (1) 2 μL of plasmid was added to 100 μL of Agrobacterium competent cells in a frozen state (optimal melting time 5 min), and the tube wall was gently shaken with fingers.

[0115] (2) The following operations were performed in sequence: standing on ice for 5 min, 5 min in liquid nitrogen, 5 min in a 37°C water bath, and standing on ice for 5 min.

[0116] (3) In the clean bench, 600 μL of LB liquid medium was added, and the recovery culture was placed in a 28°C 200 rpm shaker for 3 h.

[0117] (4) Centrifugation at 3000 rpm for 3 min, collection of bacterial cells, discarding part of the supernatant in the clean bench, leaving 150 μL of bacterial cells to be resuspended by pipetting, and evenly coating the bacterial liquid on LB solid medium containing Kan and Rif resistance. Incubate in a 28°C incubator for 2 days.

[0118] (5) Positive clone detection: select single clone strain in 600 μL of LB liquid medium containing Kan and Rif resistance, and culture at 28°C and 200 rpm for 12 h.

[0119] (6) PCR verification was performed using bacterial liquid as template.

[0120] Example 4 Silencing expression of GhNFXL1 gene reduces salt tolerance and cold tolerance of cotton

[0121] (1) Select self-pollinated ZM113 seeds preserved in the laboratory, soak the seeds in sterile water overnight at 37°C, select the seeds with white sprouts, plant them with the radicle facing down in a 1:1 mixture of nutrient soil and vermiculite, cover with a transparent lid, and inject Agrobacterium after the cotyledons have fully unfolded.

[0122] (2) Take 40 mL of Agrobacterium tumefaciens culture containing gene silencing vector that has been cultured overnight into a 50 mL clean centrifuge tube, centrifuge at 4000 rpm for 10 min to enrich the bacterial cells, and discard the supernatant.

[0123] (3) Use the prepared resuspension solution to resuspend the bacteria and adjust OD600 = 1.0.

[0124] (4) Incubate at room temperature in the dark for 3 hours.

[0125] (5) Carefully scratch the back of the cotton cotyledon with the needle of a 1mL sterile syringe (do not penetrate the leaf), and inject the bacterial solution into the leaf through the wound until the entire leaf is soaked.

[0126] (6) After standing at room temperature in the dark for 24 hours, it is transferred to a greenhouse for cultivation. After it grows to two leaves and one heart, it is treated with 4℃ or salt.

[0127] RNA was extracted from leaves of TRV:00 and TRV:GhNFXL1 plants, reverse transcribed, and then quantitatively analyzed using fluorescence. The results showed that the expression of the TRV:GhNFXL1 gene was significantly reduced. Figure 2 (A and C). After treatment with 200mM NaCl solution, the results showed that the leaves of TRV:GhNFXL1 plants wilted and the stems lodged. Figure 2 (B). Further analysis of MDA and POD contents revealed that TRV:GhNFXL1 plants had higher MDA contents than TRV:00 plants, while TRV:GhNFXL1 plants had lower POD contents than TRV:00 plants. Figure 3 (A and B). These results indicate that silencing the GhNFXL1 gene reduces cotton salt tolerance. After 3 days of treatment at 4℃, the leaves of TRV:GhNFXL1 plants showed severe wilting. Figure 2 (D). Further analysis of MDA and POD contents revealed that the MDA content of TRV:GhNFXL1 plants was higher than that of TRV:00 plants, while the POD content of TRV:GhNFXL1 plants was lower than that of TRV:00 plants. Figure 3 (C and D). These results indicate that silencing the GhNFXL1 gene reduces the cold tolerance of cotton.

[0128] Example 5: Overexpression of the GhNFXL1 gene enhances the salt and cold tolerance of cotton.

[0129] (1) In a clean bench, cut the sterile seedlings into small segments and put them into an Erlenmeyer flask containing Agrobacterium infection solution containing GhNFXL1 gene overexpression vector for 10 min. After infection, transfer the hypocotyl to a sterile large petri dish and wait for the surface bacterial solution to dry. Then transfer the hypocotyl to a co-culture medium and culture in the dark for 40-46 h. After co-culture, transfer it to sterile water containing CEF and soak for 20 min. Then wash it with sterile water 5-6 times until the residue is washed away and the surface moisture is dried. Then spread the hypocotyl evenly in hypocotyl induction medium containing termethin and kanamycin. Finally, place the hypocotyl in a greenhouse at 28±1℃ for culture. The induced callus tissue was transferred to a callus proliferation medium containing termethin and kanamycin and cultured. The culture was subcultured every 4 weeks. An appropriate amount of callus tissue was then transferred to an embryogenic callus induction medium containing termethin and kanamycin and cultured. The culture was subcultured every 3 weeks until embryogenic callus tissue appeared.

[0130] (2) The obtained embryogenic callus was transferred into an embryogenic callus proliferation medium containing termethin and kanamycin for expansion culture. Subculture was carried out every two weeks. When the embryogenic callus proliferated to a certain amount, it could be used for subsequent differentiation into seedlings.

[0131] Leaves of overexpressing plants were collected, DNA was extracted, and RT-qPCR amplification was performed using primers designed to target specific fragments. The results showed that the expression level of the GhNFXL1 gene in the overexpressing lines was significantly higher than that in the Mock lines. Figure 4 (A) This demonstrates the successful construction of GhNFXL1 gene overexpression lines. Mock and T4 generation cotton seeds were hydroponically cultured until they reached the two-leaf-one-heart stage, then transferred to a hydroponic solution containing 200 mM NaCl. The results showed that the leaves of the GhNFXL1 gene overexpression lines exhibited slight wilting and slightly bent stems, while the Mock plants showed more severe wilting and severe stem lodging. Figure 4 (B). Further analysis of MDA and POD levels revealed that the MDA content in GhNFXL1-overexpressing plants was lower than that in Mock plants, while the POD content in GhNFXL1-overexpressing plants was higher than that in Mock plants. Figure 5 (A and B). These results indicate that the GhNFXL1 gene can improve the salt tolerance of cotton. Additionally, cotton seeds from the Mock and T4 generations were selected, and after being soil-grown to the two-leaf stage, they were treated at 4℃ for 2 days. Phenotypic observation revealed that the leaves of the GhNFXL1 gene-overexpressing lines showed slight wilting, while the Mock plants showed more severe wilting, demonstrating that the GhNFXL1 gene-overexpressing lines were more cold-tolerant than the Mock plants. Figure 4MDA and POD contents were further detected, and the results showed that the MDA content of the GhNFXL1 gene overexpression plant was lower than that of the Mock plant, and the POD content of the GhNFXL1 gene overexpression plant was higher than that of the Mock plant Figure 5 C and D). These results show that the GhNFXL1 gene can improve the cold tolerance of cotton.

[0132] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in more detail, but cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of a nucleic acid corresponding to the GhNFXL1 gene in improving cold tolerance in Gossypium plants; the NCBI RefSeq accession number corresponding to the GhNFXL1 gene is XM_016812982.

2.

2. Use of a nucleic acid corresponding to the GhNFXL1 gene in simultaneously improving salt tolerance and cold tolerance in Gossypium plants; the NCBI RefSeq accession number corresponding to the GhNFXL1 gene is XM_016812982.

2.

3. Method for producing Gossypium plants with improved cold tolerance, or Gossypium plants with simultaneously improved salt tolerance and cold tolerance, comprising introducing a nucleic acid corresponding to the GhNFXL1 gene into a Gossypium plant; the NCBI RefSeq accession number corresponding to the GhNFXL1 gene is XM_016812982.

2.

4. The method according to claim 3, wherein the nucleic acid is introduced into the Gossypium plant in the form of a recombinant DNA construct.

5. The method according to claim 4, wherein the recombinant DNA construct is introduced into the Gossypium plant by means of a gene gun-mediated transformation method, a pollen tube pathway method or a liposome transformation method.

6. The method according to claim 4, wherein the recombinant DNA construct is introduced into the Gossypium plant by means of Agrobacterium.

7. The method according to any one of claims 3 to 6, further comprising: crossing the Gossypium plant into which the nucleic acid corresponding to the GhNFXL1 gene has been introduced with a second Gossypium plant to produce progeny plants; the second Gossypium plant being an agronomically elite variety.

8. Use of a Gossypium plant produced by the method according to any one of claims 3 to 7 for producing cotton propagation material, wherein the propagation material is suitable for sexual reproduction, vegetative reproduction or tissue culture of regenerable cells.

9. The use according to claim 8, wherein the propagation material suitable for sexual reproduction is selected from the group consisting of microspores, pollen, ovaries, ovules, embryo sacs and egg cells.

10. The use according to claim 8, wherein the propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, anthers, flowers, seeds and stems.

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