Application of GhNFXL1 gene in improving salt tolerance and / or cold tolerance of cotton plants

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of a GhNFXL1 gene to improvement of salt tolerance and / or cold tolerance of gossypium plants. Various experiments show that the salt tolerance and cold tolerance of the plant can be reduced by silencing of the GhNFXL1 gene; the overexpression of the GhNFXL1 gene enhances the salt tolerance and cold tolerance of the plant, and the result shows that the GhNFXL1 plays a positive regulation role in the salt tolerance and cold tolerance of cotton.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to application of a GhNFXL1 gene in improving salt tolerance and / or cold tolerance of cotton plants. Background Art

[0002] Salt-alkali lands are widely distributed around the world, especially in arid and semi-arid regions such as the Middle East, North Africa, South Asia, western China and Australia. These salt-alkali lands cause serious damage to the growth of crops, mainly manifested in the inhibition of seed germination, restricted root growth and physiological dysfunction, resulting in reduced crop yield and quality. For cotton, the impact of salt-alkali land is particularly significant. In a high-salt environment, cotton not only has reduced germination and emergence rates, but also poor root development, and may also cause leaf yellowing and slow growth, ultimately affecting the quality and yield of the fiber. In addition, cotton is a thermophilic plant, and low temperatures will cause its growth to slow down, affect the efficiency of photosynthesis, and cause leaf yellowing and shedding. Especially during the flowering and boll-setting periods, the impact of low temperatures is particularly obvious, which may cause buds to fall off and reduce the boll-setting rate, ultimately resulting in a significant decrease in cotton yield.

[0003] NFXL (Nuclear Factor X-Like) proteins are a type of transcription factor that belongs to the NFX family. They play an important role in a variety of biological processes, including cell proliferation, differentiation, and response signal transduction. AtNFXL1 can be induced to be upregulated in flowers by heat stress. At 22°C, there is no significant difference in the length of pods between wild-type and AtNFXL1 mutant plants. When plants in the reproductive stage were treated at 38°C and then restored to normal temperature, AtNFXL1 mutant plants were more sensitive to high temperature stress than wild-type plants, with an increased proportion of shortened pods and reduced fertility. The TaNFXL1 gene is induced to express by Fusarium graminearum. After silencing the TaNFXL1 gene, the disease resistance of wheat is reduced.

[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, it is beneficial to the promotion of cotton varieties and the breeding of resistant varieties, which is of great significance for adapting to different planting areas, promoting agricultural scientific and technological progress, and ensuring national cotton safety. Summary of the invention

[0005] The present invention covers the following technical solutions:

[0006] According to one aspect of the present invention, it relates to the use of a nucleic acid corresponding to the GhNFXL1 gene in improving the salt tolerance and / or cold tolerance of cotton plants.

[0007] According to another aspect of the present invention, a method for producing a cotton plant with improved salt tolerance and / or cold tolerance comprises introducing a nucleic acid corresponding to the GhNFXL1 gene into the cotton plant.

[0008] According to another aspect of the present invention, it relates to a use of a cotton 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 another aspect of the present invention, it relates to a breeding method for salt-tolerant and / or cold-tolerant cotton plants, comprising:

[0010] The detection agent is used to detect the expression level of the GhNFXL1 gene in cotton plants, and cotton plants or varieties with high expression levels are selected for breeding.

[0011] Beneficial effects:

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

[0013] (2) Through various experiments, the present invention found that silencing of the GhNFXL1 gene would reduce the salt tolerance and cold tolerance of the plant; overexpression of the GhNFXL1 gene would enhance the salt tolerance and cold tolerance of the plant. The results showed that GhNFXL1 plays a positive regulatory role in the salt tolerance and cold tolerance of cotton.

[0014] (3) The gene GhNFXL1 discovered in the present invention can be used to prevent and treat cotton chilling and salt damage, and can be used to enhance cotton salt tolerance and cold tolerance in genetic improvement or molecular breeding, such as breeding salt-tolerant and cold-tolerant cotton varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :Expression analysis of GhNFXL1 gene under salt stress and cold stress;

[0016] Figure 2 :GhNFXL1 gene silencing reduces salt and cold tolerance in cotton;

[0017] Figure 3 :Detection of physiological indicators of salt stress and cold stress after GhNFXL1 gene silencing;

[0018] Figure 4 :GhNFXL1 gene overexpression in cotton enhances salt tolerance and cold tolerance;

[0019] Figure 5 :Detection of physiological indicators of cotton under salt stress and cold stress with overexpression of GhNFXL1 gene. DETAILED DESCRIPTION

[0020] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0021] The terms "comprising", "including" and "comprising" used in the present invention are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members, elements or method steps. The numerical ranges expressed as endpoints in the present invention include all values ​​and fractions contained in the range, as well as the cited endpoints.

[0022] In the present invention, "Gossypium" refers to a genus (Gossypium) of the angiosperm Malvaceae family, and its preferred species include upland cotton (Gossypium hirsutum), sea island cotton (Gossypium barbadense), tree cotton (Gossypium arboreum), and grass cotton (Gossypium herbaceum).

[0023] In the present invention, "nucleic acid", "polynucleotide", "nucleic acid sequence", "nucleotide sequence" and "nucleic acid fragment" are used interchangeably and refer to single-stranded or double-stranded RNA and / or DNA polymers, which optionally contain synthetic, non-natural or altered nucleotide bases. - Nucleotides (usually in their 5'-monophosphate form) are referred to by the following single-letter codes: "A" represents adenylic acid or deoxyadenylic acid, "C" represents cytidylic acid or deoxycytidylic acid, and "G" represents guanylic acid or deoxyguanylic acid, corresponding to RNA or DNA, respectively; "U" represents uridylic acid; "T" represents deoxythymidylic acid; "R" represents purine (A or G); "Y" represents pyrimidine (C or T); "K" represents G or T; "H" represents A or C or T; "I" represents inosine; and "N" represents any nucleotide.

[0024] "Polypeptide", "peptide", "amino acid sequence" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues 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" may also include modifications including, but not limited to, glycosylation, lipid attachment and sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0025] "Recombinant DNA construct" refers to a combination of nucleic acid fragments that do not usually exist together in nature. Therefore, the recombinant DNA construct can include regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that existing in nature. In some specific embodiments, the recombinant DNA construct is a plasmid or a virus. In some embodiments, the recombinant DNA construct of the present invention includes regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

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

[0027] "Hybridization" The mating of two parent plants.

[0028] "Agronomically superior (trait)", as used herein, refers to a genotype having better or optimal performance of a number of distinguishable traits that allow producers to harvest products of commercial importance. Including but not limited to seed yield, germination vigor, vegetative vigor, disease resistance, greenness, growth rate, total biomass or accumulation rate, 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 tissues, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in vegetative tissues, total plant protein content, fruit protein content, seed protein content, protein content in vegetative tissues, drought tolerance, nitrogen absorption, root lodging, harvest index, stem 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 documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as references individually. Unless they conflict with the purpose and / or technical solution of the present invention, the cited documents involved in the present invention are cited with all contents and purposes.

[0030] Description of the program

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

[0032] The GhNFXL1 gene is well 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 invention also includes its functional equivalent sequence, such as its CDS sequence (defined as Sequence 2), and a more preferred nucleic acid sequence is the sequence corresponding to the 422nd to 3661st nucleotides 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, it is preferably used to enhance the salt tolerance and cold tolerance of cotton.

[0033] Functionally equivalent sequences also include sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity with any of the sequences shown in SEQ ID NOs. 1 to 3, and are capable of expressing gene sequences having similar or identical NFXL transcription factor activity, and can be isolated from any plant. The percentage of sequence identity can be obtained by well-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 algorithm of Karlin and Altschul (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] Functional equivalent sequences obviously also include DNA sequences that can hybridize with the DNA of the above sequence under stringent conditions. The "stringent conditions" used in the present invention are well known, including hybridization at 60°C for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA, followed by washing at 65°C with a washing solution containing 0.1% SDS and 0.1% SSC for 15 to 60 minutes.

[0035] Functionally equivalent sequences obviously also include sequences in which the above sequences have been codon-optimized or generally modified.

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

[0037] The method can overexpress the GhNFXL1 gene in cotton plants.

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

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

[0040] The method of the present invention can also directly introduce the nucleic acid / recombinant DNA construct into plants to produce cotton plants, using transformation methods known to those skilled in the field of plant biotechnology. Any method can be used to transform the recombinant DNA construct into plant cells to produce cotton plants of the present invention. The transformation method can include direct and indirect transformation methods. In some embodiments, the recombinant DNA construct is introduced into cotton plants by gene gun-mediated transformation, pollen tube channel method or liposome transformation method.

[0041] Recombinant DNA construct provided by the present invention can be inserted into any carrier in plasmid, clay, yeast artificial chromosome, bacterial artificial chromosome or other suitable transformation into host cell.Preferred host cell is bacterial cell, especially is used for cloning or storing polynucleotide or is used for the bacterial cell of transformed plant cell, for example intestinal bacteria, Agrobacterium, Agrobacterium tumefaciens and Agrobacterium rhizogenes.In some preferred embodiments, described recombinant DNA construct is introduced cotton plant by Agrobacterium.

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

[0043] The cotton plant into which the nucleic acid corresponding to the GhNFXL1 gene is introduced is hybridized with a second cotton plant to produce progeny plants; the second cotton plant is an agronomically superior variety.

[0044] A third aspect of the present invention relates to the use of a cotton plant produced 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.

[0045] In some embodiments, the propagation material suitable for sexual reproduction is selected from the group consisting of microspores, pollen, ovaries, ovules, embryo sacs, and egg cells.

[0046] In some embodiments, the propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristem cells, roots, anthers, flowers, seeds, and stems.

[0047] A fourth aspect of the present invention relates to a method for breeding salt-tolerant and / or cold-tolerant cotton plants, comprising:

[0048] The detection agent is used to detect the expression level of the GhNFXL1 gene in cotton plants, and cotton plants or varieties with high expression levels are selected for breeding.

[0049] In some embodiments, the detection agent includes a reagent for detecting changes in 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, biological mass spectrometry, electrochemical analysis, gel electrophoresis, capillary electrophoresis, microarray, CRISPR-Cas detection system.

[0050] Among them, 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), synthesis sequencing, ligation sequencing (SOLiD sequencing), chain termination (Sanger sequencing), massively parallel tag sequencing (MPSS), polymerase clone sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunnel current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidics Sanger sequencing, microscopy-based technology, RNAP sequencing and in vitro viral high-throughput sequencing.

[0051] Among them, PCR is preferably qRT-PCR and digital PCR.

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

[0053] Among them, according to the type of nucleic acid molecules detected, CRISPR / Cas technology nucleic acid detection can be divided into DNA (CRISPR / Cas9, CRISPR / Cas12 and CRISPR / Cas12 recognize DNA sequences) and RNA (CRISPR / Cas13 recognizes RNA sequences) 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] The immunoassay method is not particularly limited, and examples thereof include various enzyme immunoassays, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), double monoclonal antibody sandwich immunoassay, monoclonal polyclonal antibody sandwich immunoassay, immunostaining, immunofluorescence, protein blotting, biotin-avidin method, immunoprecipitation, colloidal gold agglutination, immunochromatography, latex agglutination (LA) and immunoturbidimetry (TIA).

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

[0057] As mass spectrometry, it is not particularly limited, and the ion source using electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), surface enhanced laser desorption ionization (SELDI) etc. can be used with a mass spectrometer combined with a time-of-flight analyzer (TOF), an ion trap analyzer (IT), a Fourier transform analyzer (FT) etc. It is possible to use LC-MS and CE-MS etc. formed by connecting a mass spectrometer with a separation device such as a high-speed liquid chromatography (HPLC) or capillary electrophoresis (CE). In addition, as a method for obtaining mass spectral data, it is possible to cite: data independent analysis (DIA), data dependent analysis (DDA), multiple reaction monitoring method (MRM) etc. The mass spectrum also includes the case where stable isotope labeling of iTRAQ reagent (SCIEX company) etc. is performed on the sample.

[0058] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0059] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

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

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

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

[0063] As for other materials or reagents, unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0064] Example 1 Cotton Planting and Processing

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

[0066] Cotton cold treatment: When the cotton seedlings grew to two leaves and one heart, the cotton in the treatment group was placed in an artificial climate incubator at 4°C, and the cotton in the control group was placed in an artificial climate incubator at 28°C, with alternating natural light of 16h / darkness of 8h.

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

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

[0069] Root tissues of Upland Cotton 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 the internal reference gene being GhUBQ7 gene.

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

[0071]

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

[0073]

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

[0075]

[0076] Data analysis and utilization 2 -ΔΔCT Methods, three replicates, t-test, ** indicates P < 0.01. The results showed that with the increase of salt stress time, the expression level of GhNFXL1 gene increased significantly ( Figure 1 (A). During the period of 0-3h, the expression of GhNFXL1 gene continued to increase and reached a peak at 3h. At 6-24h, the expression of GhNFXL1 gene began to decrease. These results prove that GhNFXL1 gene responds to salt stress. With the increase of cold stress time, the expression of GhNFXL1 gene increased significantly ( Figure 1 (B) During the period of 0-12h, the expression of GhNFXL1 gene continued to increase and reached a peak at 12h. At 24h, the expression of GhNFXL1 gene began to decrease. These results prove 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: The gene silencing vector pTRV2 and the overexpression vector pCambia2300-GFP were incubated at 37°C for 1 h and double-digested to obtain linearized vectors according to the following reaction system:

[0080]

[0081] (2) Obtaining the target fragment: Using cotton cDNA as a template, the sequence was amplified using the high-fidelity enzyme 2×PhantaMax MasterMix (Dye Plus) produced by Novazonics. The PCR reaction system was as follows:

[0082]

[0083] The PCR amplification system is as follows:

[0084]

[0085] Primer information

[0086]

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

[0088] (a) Quickly cut the agarose gel containing the target band under ultraviolet light to avoid damage to the DNA caused by prolonged exposure to ultraviolet light. Cut the gel into small pieces as much as possible and place them in a 1.5 mL clean centrifuge tube for subsequent sol preparation.

[0089] (b) Weigh the gel using an electronic balance, assuming that 100 mg is equal to 100 μL volume. Add 3 times the volume of Buffer GDP and incubate at 55°C in a water bath for 15 min. Invert and mix 2-3 times 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 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 minutes, and then centrifuge at 12,000 rpm for 1 minute.

[0092] (e) Discard the filtrate and return the adsorption column to the collection tube. Add 700 μL 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 sterilized 1.5 ml centrifuge tube, add 40 μL Elution Buffer (preheated to 55°C in a water bath) to the center of the adsorption column, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 1 min to elute the DNA, and store at -20°C for later use.

[0096] 2. Construction of vector and transformation of E. coli: The vector was constructed using the UltraOne Step Cloning Kit performs homologous recombination between the linearized vector and the target fragment to construct the vector. Prepare the following liquid system on ice:

[0097]

[0098] React at 50°C for 10 min, cool on ice immediately, and then transform the recombinant product into competent E. coli.

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

[0100] (2) Heat shock at 42°C in a water bath for 45 seconds and immediately place on ice for 2 minutes.

[0101] (3) Add 500 μL of LB liquid culture medium without antibiotics and shake 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, and spread 100 μL of bacterial solution on LB solid medium containing the corresponding antibiotics. After drying, invert and culture in a 37°C incubator overnight.

[0103] (5) On the next day, single clones were picked from the plates and shaken in 500 μL of LB liquid culture medium containing the corresponding antibiotics until the bacterial solution became turbid. PCR was performed on the bacterial solution and the positive clones were sent to Shanghai Biotechnology Co., Ltd. for sequencing. The sequencing results returned by the company were compared with the target sequence. The clones with completely correct results were preserved.

[0104] 3. Plasmid extraction: using Beijing Quanshijin Plasmid MiniPrep Kit was used to extract E. coli plasmids. The specific steps are as follows:

[0105] (1) Take 5 mL of E. coli cultured overnight at 37°C in a shaking incubator and place it in a 10 mL centrifuge tube. Centrifuge at 12,000 rpm for 5 min and discard the supernatant.

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

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

[0108] (4) Add 450 μL of yellow solution NB and gently mix by inverting up and down 6-8 times until the solution changes color from blue to yellow and forms a compact yellow agglomerate, indicating complete neutralization. Let stand at room temperature for 2 min.

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

[0110] (6) Add 700 μL of WB and centrifuge at 12,000 r / min for 1 min. Discard the liquid in the collection tube, centrifuge the empty tube at 12,000 r / min for 2 min, and place it on a clean bench to blow dry for 5 min to remove the residual rinsing solution.

[0111] (7) Place the centrifuge column in a clean 1.5 mL centrifuge tube and add 40 μL of 65°C preheated Elution Buffer to the center of the centrifuge column. After standing at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min to elute the plasmid DNA.

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

[0113] 4. Agrobacterium transformation: GV3101 from Shanghai Weidi Biotechnology Co., Ltd. was used for Agrobacterium transformation. The specific steps are as follows:

[0114] (1) Take 2 μL of plasmid and add it to 100 μL of freeze-thawed Agrobacterium competent cells (5 min of thawing is best), and gently shake the tube wall with your fingers to mix.

[0115] (2) Perform the following steps in sequence: place on ice for 5 min, place in liquid nitrogen for 5 min, place in a 37°C water bath for 5 min, and place on ice for 5 min.

[0116] (3) Add 600 μL of LB liquid culture medium to the clean bench and place in a shaking incubator at 200 rpm at 28°C for 3 h.

[0117] (4) Centrifuge at 3000 rpm for 3 min, collect the cells, discard part of the supernatant in a clean bench, and keep 150 μL of the cells. Use a pipette to resuspend them, and evenly apply the bacterial solution on LB solid medium containing Kan and Rif resistance. Incubate inverted in a 28°C incubator for 2 days.

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

[0119] (6) Perform PCR verification using bacterial culture as template.

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

[0121] (1) Select the seeds of ZM113 of self-fertilization age stored in the laboratory, soak the seeds in sterile water at 37°C overnight, select the white seeds, plant them with the radicle facing downward in a 1:1 mixture of nutrient soil and vermiculite, cover them with a transparent lid, and inject Agrobacterium after the cotyledons are fully expanded.

[0122] (2) Take 40 mL of overnight cultured Agrobacterium containing the gene silencing vector into a 50 mL clean centrifuge tube, centrifuge at 4000 rpm for 10 min to enrich the bacteria, and remove the supernatant.

[0123] (3) Resuspend the cells in the prepared resuspension solution and adjust OD600 to 1.0.

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

[0125] (5) Use the needle of a 1 mL sterile syringe to carefully scratch the back of the cotton cotyledon (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 h, move to a greenhouse for cultivation. After the plants have grown two leaves and one heart, treat them at 4°C or with salt.

[0127] RNA was extracted from leaves of TRV:00 and TRV:GhNFXL1 plants, and then reverse transcribed and analyzed by fluorescence quantitative analysis. The results showed that the expression of TRV:GhNFXL1 gene was significantly decreased ( Figure 2 After treatment with 200 mM NaCl solution, the leaves of TRV:GhNFXL1 plants wilted and the stems fell over ( Figure 2 Further testing of MDA and POD contents showed that the MDA content of TRV:GhNFXL1 plants was higher than that of TRV:00 plants, and the POD content of TRV:GhNFXL1 plants was lower than that of TRV:00 plants ( Figure 3 These results indicate that cotton salt tolerance is reduced after silencing the GhNFXL1 gene. After 3 days of treatment at 4°C, the leaves of TRV:GhNFXL1 plants were severely wilted ( Figure 2 Further testing of MDA and POD contents revealed that the MDA content of TRV:GhNFXL1 plants was higher than that of TRV:00 plants, and 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 reduced the cold tolerance of cotton.

[0128] Example 5 Overexpression of GhNFXL1 gene enhances salt tolerance and cold tolerance in cotton

[0129] (1) In a clean bench, the sterile seedlings were cut into small pieces and placed in a conical flask containing Agrobacterium infection solution containing the GhNFXL1 gene overexpression vector for infection for 10 min. After infection, the hypocotyls were transferred to a sterilized large culture dish and the surface bacterial solution was blown dry. The hypocotyls were then transferred to a co-cultivation medium and cultured in the dark for 40-46 h. After co-cultivation, they were transferred to sterile water containing CEF and soaked for 20 min. After that, they were washed with sterile water only for 5-6 times until the residue was washed off. The surface moisture was blown dry, and the hypocotyls were evenly spread in a hypocotyl induction medium containing timentin and kanamycin. Finally, the hypocotyls were cultured in a greenhouse at 28±1°C. The induced callus tissue was transferred to a callus proliferation medium containing timentin and kanamycin and cultured, subcultured once every 4 weeks, and an appropriate amount of callus tissue was transferred to an embryonic callus induction medium containing timentin and kanamycin and cultured, subcultured every 3 weeks until embryonic callus tissue appeared.

[0130] (2) The obtained embryonic callus is transferred to an embryonic callus proliferation medium containing timentin and kanamycin for expansion culture, and subcultured every two weeks. When the embryonic callus proliferates to a certain amount, it can be used for subsequent differentiation into seedlings.

[0131] The leaves of the overexpressed plants were taken, DNA was extracted, and specific fragment primers were designed for RT-qPCR amplification. The results showed that the expression level of the GhNFXL1 gene overexpression strain was much higher than that of the Mock ( Figure 4 A in the middle), proving that the GhNFXL1 gene overexpression strain was successfully constructed. Mock and T4 cotton seeds were selected and hydroponically cultured until they had two leaves and one heart. They were then transferred to a hydroponic solution containing 200 mM NaCl. The results showed that the leaves of the GhNFXL1 gene overexpression strain showed slight wilt and the stems were slightly bent, while the Mock plants showed more severe wilt and severe lodging of the stems ( Figure 4 Further testing of MDA and POD contents showed that the MDA content of GhNFXL1 gene overexpression plants was lower than that of Mock plants, and the POD content of GhNFXL1 gene overexpression plants was higher than that of Mock plants ( Figure 5 A and B in the figure). These results indicate that the GhNFXL1 gene can improve the salt tolerance of cotton. In addition, cotton seeds of the Mock and T4 generations were selected and soil cultured until they had two leaves and one heart. The phenotype was observed after 2 days of treatment at 4°C. The results showed that the leaves of the GhNFXL1 gene overexpression strains showed slight wilt, while the Mock plants showed more severe wilt, indicating that the GhNFXL1 gene overexpression strains have higher cold tolerance than the Mock plants ( Figure 4C). Further testing of MDA and POD contents showed that the MDA content of GhNFXL1 gene overexpression plants was lower than that of Mock plants, and the POD content of GhNFXL1 gene overexpression plants was higher than that of Mock plants ( Figure 5 These results suggest that the GhNFXL1 gene can improve the cold tolerance of cotton.

[0132] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. Use of the nucleic acid corresponding to the GhNFXL1 gene in improving the salt tolerance and / or cold tolerance of cotton plants.

2. A method for producing a cotton plant having improved salt tolerance and / or cold tolerance, comprising introducing a nucleic acid corresponding to the GhNFXL1 gene into the cotton plant.

3. The method according to claim 2, wherein the nucleic acid is introduced into the cotton plant in the form of a recombinant DNA construct.

4. The method according to claim 3, wherein the recombinant DNA construct is introduced into the cotton plant by gene gun-mediated transformation, pollen tube pathway or liposome transformation.

5. The method according to claim 3, wherein the recombinant DNA construct is introduced into cotton plants via Agrobacterium.

6. The method according to any one of claims 2 to 5, further comprising: The cotton plant into which the nucleic acid corresponding to the GhNFXL1 gene is introduced is hybridized with a second cotton plant to produce progeny plants; the second cotton plant is an agronomically superior variety.

7. Use of a cotton plant produced by the method of any one of claims 2 to 6 for producing cotton propagation material, wherein the propagation material is suitable for sexual reproduction, vegetative reproduction or tissue culture of regenerable cells.

8. The use according to claim 7, wherein the propagation material suitable for sexual reproduction is selected from microspores, pollen, ovary, ovule, embryo sac and egg cell; Preferably, said propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristem cells, roots, anthers, flowers, seeds and stems.

9. A method for breeding salt-tolerant and / or cold-tolerant cotton plants, comprising: The detection agent is used to detect the expression level of the GhNFXL1 gene in cotton plants, and cotton plants or varieties with high expression levels are selected for breeding.

10. The breeding method according to claim 9, wherein the detection agent comprises a reagent for detecting changes in the 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, biological mass spectrometry, electrochemical analysis, gel electrophoresis, capillary electrophoresis, microarray, CRISPR-Cas detection system; Alternatively, 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.

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