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

Abstract

1 Abstract The present disclosure relates to the technical field of plant genetic engineering, and in particular, to the use of GhNFXL1 gene in improving the salt tolerance and / or cold tolerance of a Gossypium plant. According to the present disclosure, through various experiments, silencing the GhNFXL1 gene will reduce the salt tolerance and cold tolerance of plants; the overexpression of the GhNFXL1 gene enhances the salt tolerance and cold tolerance of plants. The results show that GhNFXL1 plays a positive regulatory role in the salt tolerance and cold tolerance of cotton. Abstract The present disclosure relates to the technical field of plant genetic engineering, and in particular, to the use of GhNFXLI gene in improving the salt tolerance and / or cold tolerance of a Gossypium plant. According to the present disclosure, through various experiments, silencing the GhNFXLI gene will reduce the salt tolerance and cold tolerance of plants; the overexpression of the GhNFXL1 gene enhances the salt tolerance and cold tolerance of plants. The results show that GhNFXL1 plays a positive regulatory role in the salt tolerance and cold tolerance of cotton. 1 20 25 20 11 21 17 F eb 2 02 5 1 7 F e b 2 0 2 5 2 0 2 5 2 0 1 1 2 1 4 4 / 5 FIG. 4 4 / 5 A 0.20 0.15 I 0.10 ** 0.05 0.00 WT B OE-1 OE-2 OE-3 C Mock GhNFXL1-OE Mock GhNFXL1-OE FIG. 4 4 20 25 20 11 21 17 F eb 2 02 5 2 0 2 5 2 0 1 1 2 1 1 7 F e b 2 0 2 5 CBA M o c kM o c k G h N F X L 1 - O E 4 / 5

Description

SEQUENCE LISTING The sequence listing xml file submitted herewith, named “Sequence Listing WI-AU24-22132-P.xml”, created on Jan. 27, 2025, and having a file size of 22,932 bytes, is incorporated by reference herein. TECHNICAL FIELD

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

[0002] Salt-alkali land is 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 have caused serious damage to crop growth mainly by inhibiting seed germination, limiting root growth and causing physiological dysfunction, resulting in reduced crop yield and quality. The impact of saline-alkali land on cottons is particularly significant. In high-salt environments, the germination and emergence rates of cotton are reduced, the root development is poor, and leaf yellowing and slow growth are caused, ultimately affecting fiber quality and yield. In addition, cotton is a thermophilic plant. 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 period, the impact of low temperatures is particularly obvious, which may cause bud shedding 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 factors that belong to the NFX family. They play important roles in a variety of biological processes, including cell proliferation, 2025201121   17 Feb 2025 differentiation and response signal transduction. AtNFXL1 can be up-regulated 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 returned to normal temperature, AtNFXL1 mutant plants were more sensitive to high temperature stress than wild-type plants, with an increased proportion of shorter 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, and facilitate the promotion of cotton varieties and the selection of resistant varieties, it is of great significance for adapting to different planting areas, promoting the progress of agricultural science and technology, and ensuring the safety of national cotton. SUMMARY

[0005] The present disclosure uses the following technical solution:

[0006] In one aspect, the present disclosure relates to use of a nucleic acid corresponding to GhNFXL1 gene in improving salt tolerance and / or cold tolerance of a Gossypium plant.

[0007] In another aspect, the present disclosure relates to a method for producing a Gossypium plant with improved salt tolerance and / or cold tolerance, including introducing a nucleic acid corresponding to GhNFXL1 gene into a Gossypium plant.

[0008] In still another aspect, the present disclosure relates to use of the Gossypium plant produced by the aforementioned method for producing a cotton propagation material, where the propagation material is applicable to sexual propagation, vegetative propagation or tissue culture of regenerative cells.

[0009] In still another aspect, the present disclosure relates to a method for breeding a salt-tolerant and / or cold-tolerant Gossypium plant, including:

[0010] Using a detection agent to detect the expression level of GhNFXL1 gene in a Gossypium plant, and selecting cotton plants or varieties with high expression levels for breeding. 2025201121   17 Feb 2025

[0011] Beneficial effects

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

[0013] (2) According to the present disclosure, through various experiments, silencing the GhNFXL1 gene will reduce the salt tolerance and cold tolerance of plants; the overexpression of the GhNFXL1 gene enhances the salt tolerance and cold tolerance of plants. The results show 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 disclosure can be used to prevent and control cold damage and salt damage in cottons, specifically to enhance the genetic improvement or molecular breeding of cotton's salt tolerance and cold tolerance, such as cultivating salt-tolerant and cold-tolerant cotton varieties. BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 shows the expression analysis of GhNFXL1 gene under salt stress and cold stress;

[0016] FIG. 2 shows that GhNFXL1 gene silencing reduces salt tolerance and cold tolerance;

[0017] FIG. 3 shows the detection of physiological indicators of salt stress and cold stress under GhNFXL1 gene silencing;

[0018] FIG. 4 shows that the overexpression of GhNFXL1 gene in cotton enhances salt tolerance and cold tolerance; and

[0019] FIG. 5 shows the detection of physiological indicators of salt stress and cold stress in cotton with GhNFXL1 gene overexpression. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In the present disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in 2025201121   17 Feb 2025 the art. Meanwhile, in order to better understand the present disclosure, definitions and explanations of relevant terms are provided below.

[0021] The terms “comprise / comprising” and “include / including” used herein are synonymous, inclusive or open-ended and do not exclude additional, unquoted members, elements or methods and steps. Numerical ranges expressed by endpoints herein include all numerical values and fractions within the range, as well as the quoted endpoints.

[0022] In the present disclosure, “Gossypium” is a genus of the angiosperm family Malvaceae. Its preferred varieties include Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium herbaceum.

[0023] In the present disclosure, “nucleic acid”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence” and “nucleic acid fragment” are used interchangeably and refer to polymers of single- or double-stranded RNA and / or DNA, which optionally contain synthetic, non-natural or altered nucleotide bases. Nucleotides (usually in their 5'-monophosphate form) are designated by the following one-letter codes: “A” represents adenylate or deoxyadenylate, “C” represents cytidylate or deoxycytidylate, and “G” represents guanylate or deoxyguanylate, corresponding to RNA or DNA, respectively; “U” represents uridylate; “T” represents deoxythymidylate; “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, and refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, and apply 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 linkage and sulfation, Y-carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues.

[0025] “Recombinant DNA construct” refers to a combination of nucleic acid fragments that do not normally exist together in nature. Thus, a recombinant DNA construct may contain 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 found in nature. 2025201121   17 Feb 2025 In some specific embodiments, the recombinant DNA construct is a plasmid or a virus. In some embodiments, the recombinant DNA construct of the present disclosure contains 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, and the like).

[0026] In the present disclosure, 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” means mating of two parent plants.

[0028] “Agronomically superior (trait)”, as used herein, means that a genotype has better or optimal performance of a number of discernible traits that allows a producer to harvest a product of commercial importance. The traits include but not limited to seed yield, germination potential, nutritional potential, 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, number of tillers, panicle size, early seedling vigor and seedling emergence under low temperature stress, and the like.

[0029] All documents mentioned herein are incorporated by reference to the same extent as if each individual document is individually incorporated by reference. The references related to herein are cited in their entirety and for all purposes unless they conflict with the purpose and / or technical solutions of the present disclosure.

[0030] Description of the technical solution

[0031] A first aspect of the present disclosure relates to use of nucleic acids corresponding to the GhNFXL1 gene in improving the salt tolerance and / or cold tolerance of Gossypium plant.

[0032] The GhNFXL1 (Gossypium hirsutum NF-X1-type zinc finger protein NFXL1) gene is well 2025201121   17 Feb 2025 known in the art, and its NCBI Reference Sequence is XM 016812982.2 (defined as SEQ ID NO.1). The nucleic acid corresponding to the GhNFXL1 gene referred to in the present disclosure also includes its functionally equivalent sequence, such as its CDS sequence (defined as SEQ ID NO.2). A more preferred nucleic acid sequence is the sequence corresponding to positions 422 to 3661 of the ORIGIN sequence of XM 016812982.2 (ATGAGCTTT...TTATGAATGA, defined as SEQ ID NO.3). When the nucleic acid corresponding to the GhNFXL1 gene has the nucleotide sequence shown in SEQ ID NO.3, the effect of enhancing the salt tolerance and cold tolerance of cotton is better.

[0033] A functionally equivalent sequence also includes a gene sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity with any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.3, and are capable of expressing similar or identical NFXL transcription factor activity, which can be isolated from any plant. Among them, 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), Needleman-Wunsch global alignment method (J.Mol.Biol., 48 (3): 443-53, 1970), Smith-Waterman local alignment method (J.Mol.Biol., 147: 195-197, 1981), Pearson and Lipman similarity search method (PNAS, 85 (8): 2444-2448, 1988), Karlin and Altschul algorithm (Altschul et al., J.Mol.Biol., 215 (3): 403-410, 1990; PNAS, 90: 58735877, 1993). This is familiar to those skilled in the art.

[0034] Obviously, a functionally equivalent sequence also includes a DNA sequence capable of hybridizing to DNA of the aforementioned sequences under stringent conditions. The “stringent conditions” used in the present disclosure are well known, including hybridizing at 60°C for 12 to 16 hours in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA, and then washing at 65°C with a washing solution containing 0.1% SDS and 0.1% SSC for 15 to 60 minutes.

[0035] Obviously, a functionally equivalent sequence also includes a sequence in which the aforementioned sequences have been codon-optimized or subjected to general nucleic acid modifications.

[0036] A second aspect of the present disclosure relates to a method for producing Gossypium plant with improved salt tolerance and / or cold tolerance, including introducing a nucleic acid corresponding to GhNFXL1 gene into a Gossypium plant. 2025201121   17 Feb 2025

[0037] The method enables the overexpression of 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 contains the nucleic acid and other nucleotide sequences corresponding to the GhNFXL1 gene, such as nucleotide sequences encoding functional units such as tags for protein purification, fluorescent protein markers, and DNA binding sites, or encoding elements that have regulatory effects on gene transcription and expression, including but not limited to promoters, strong promoters, enhancers or transcription factor binding sites.

[0040] According to the method of the present disclosure, the nucleic acid / recombinant DNA construct may be directly introduced into a plant to produce a Gossypium plant using a transformation method 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 the Gossypium plant of the present disclosure. The transformation method may include direct and indirect transformation methods. In some embodiments, the recombinant DNA construct is introduced into the Gossypium plant by a gene gun-mediated transformation method, a pollen tube pathway method or a liposome transformation method.

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

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

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

[0044] A third aspect of the present disclosure relates to use of the Gossypium plant produced by 2025201121   17 Feb 2025 the method as described above for producing a cotton propagation material, wherein the propagation material is applicable to sexual propagation, vegetative propagation or tissue culture of regenerative cells.

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

[0046] In some embodiments, the propagation material applicable to tissue culture of regenerative cells is selected from a group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, anthers, flowers, seeds and stems.

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

[0048] using a detection agent to detect the expression level of the GhNFXL1 gene in a Gossypium plant, and selecting cotton plants or varieties with high expression levels for breeding.

[0049] In some embodiments, the detection agent includes a reagent for detecting changes in mRNA expressed by the GhNFXL1 gene, which is applicable to 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] 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 signature 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, microfluidic Sanger sequencing, microscopy-based technology, RNAP sequencing and in vitro viral high-throughput sequencing.

[0051] qRT-PCR and digital PCR are preferred for PCR.

[0052] The generalized isothermal technology can be further divided into the following methods: 2025201121   17 Feb 2025 methods that rely on primer displacement to initiate repeated template copies (hereinafter, for example, 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 continuous reuse or de novo synthesis of single primer molecules, SDA (strand displacement amplification and nucleic acid-based amplification (NASBA and TMA)).

[0053] According to the type of nucleic acid molecule detected, CRISPR-Cas-based nucleic acid detection can be divided into DNA detection (DNA sequences recognized by CRISPR / Cas9, CRISPR / Cas12 and CRISPR / Cas12) and RNA detection (RNA sequences recognized by CRISPR / Cas13).

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

[0055] The immunoassay 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, Western blot, biotin-avidin method, immunoprecipitation, colloidal gold agglutination, immunochromatography, latex agglutination (LA), and immunoturbidimetry (TIA), and the like.

[0056] As reagents 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] The mass spectrometry method is not particularly limited, and a mass spectrometer can be used that combines an ion source using electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser desorption ionization (SELDI), or the like with a time-of-flight analyzer (TOF), an ion trap analyzer (IT), a Fourier transform analyzer (FT), or the like. In addition, examples of methods for obtaining mass spectrum data include data independent analysis (DIA), data dependent analysis (DDA), and multiple reaction monitoring (MRM), and the like. The 2025201121   17 Feb 2025 mass spectrometry also includes cases where the sample is labeled with a stable isotope such as iTRAQ reagent (SCIEX).

[0058] Embodiments of the present disclosure will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. For experimental methods that do not specify specific conditions in the following examples, reference is made to the instructions given in the present disclosure in priority, or the experimental manuals or conventional conditions in the art are followed, or reference is made to other experimental methods known in the art, or conditions recommended by the manufacturer are followed.

[0059] In the following specific examples, the measurement parameters of the 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 receptor material used in this example is Gossypium hirsutum “ZM113”.

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

[0062] The strains used in this example are as follows: Escherichia coli competent DH5a, Agrobacterium competent GV3101.

[0063] For the remaining materials or reagents, unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0064] Example 1 Cotton planting and treatment

[0065] Cotton planting: ZM13 seeds with full grains were chosen and washed with sterile water, then soaked in a container, placed in an artificial climate incubator at 28OC overnight to promote germination. When turning white, the seeds were transplanted into moist nutrient soil, covered with mulch, and placed in an artificial climate incubator at 28 °C with alternating natural light for 16 hours and darkness for 8 hours. 2025201121   17 Feb 2025

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

[0067] Cotton salt treatment: When the cotton seedlings grew to have two leaves and one central bud, 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 group and the control group were placed in an artificial climate incubator with alternating natural light of 16 hours and darkness of 8 hours.

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

[0069] Root tissues of Gossypium hirsutum ZM113 were sampled at 0h, 1h, 3h, 6h, and 12h after stress treatment at 4°C, and RT-qPCR was used to analyze the expression of the GhNFXL1 gene, using GhUBQ7 gene as the internal reference gene.

[0070] The RT-qPCR reaction system was shown in the table below. Component Volume 2xChamQ Universal SYBR qPCR Master Mix 10 uL cDNA 1 uL Forward Primer 0.4 uL Reverse Primer 0.4 uL ddH2O Up to 20 gL

[0071] The RT-qPCR reaction program was as follows: Program Cycle number Temperature (°C) Time (sec) Predenaturation 1 95 30 Cycle reaction 40 95 10 60 30 Dissolution curve 1 95 15 2025201121   17 Feb 2025 60 60 95 15

[0072] The primers used in RT-qPCR were as follows: Primer name                         Primer sequence RT-GhNFXL1-F    5’- TGGCGGTAAGGAAGGAGGTTGA-3’ (SEQ ID No. 4) RT-GhNFXL1-R     5’- AACAGAAGTGGGTGCCCTCG-3’ (SEQ ID No. 5) GhUBQ7-F     5’- GAAGGCATTCCACCTGACCAAC-3’ (SEQ ID No. 6) GhUBQ7-R    5’- CTTGACCTTCTTCTTCTTGTGCTTG-3’ (SEQ ID No. 7)

[0073] Data analysis was performed by the 2-AACT method, with three parallel replicates, t-test, ** indicated P<0.01.The results showed that as the salt stress time increased, the expression of GhNFXL1 gene significantly increased (A in FIG. 1). During 0-3 h, the expression of GhNFXL1 gene continued to increase and reached a peak at 3h. At 6-24 h, the expression of GhNFXL1 gene began to decrease. These results proved that the GhNFXL1 gene responded to salt stress. As the cold stress time increased, the expression of the GhNFXL1 gene increased significantly (B in FIG. 1). During 0-12 h, the expression of the GhNFXL1 gene continued to increase and reached a peak at 12h. At 24 h, the expression of the GhNFXL1 gene began to decrease. These results proved that the GhNFXL1 gene responded to cold stress.

[0074] Example 3 Construction of GhNFXL1 gene expression vector

[0075] 1. Recovery of target fragment and linearized vector

[0076] (1) Linearization of expression vector: The gene silencing vector pTRV2 and the overexpression vector pCambia2300-GFP were incubated at 37°C for 1 h for double digestion according to the following reaction system to obtain the linearized vector: Component Volume Plasmid 4 pL BamHAp 2 pL 2025201121   17 Feb 2025 EcorR I 2 pL rCutSmart 10 pL ddH2O Up to 50 gL

[0077] (2) Acquisition of target fragment: With cDNA as a template, sequences were amplified using Vazyme’s high-fidelity enzyme 2*Phanta Max Master Mix (Dye Plus). The PCR reaction system was as follows: Component Volume 2*Phanta Max Master Mix (Dye Plus ) 25 pL cDNA 2 pL Forward Primer 2 pL Reverse Primer 2 pL ddH2O Up to 50 gL

[0078] PCR amplification system: Temperature Time — 98 C 3 min 98 C 30 s 56 C 30 s 35 cycles 72 C 30 s 72 C 5 min

[0079] Primer information Primer name                                 Primer sequence pTRV2-GhNFXL1-F         5’-aaggttaccgaattctctagaTGGCGGTAAGGAAGGAGGTT-3’ (SEQ ID No. 8) pTRV2-GhNFXL1-R     5’-cgtgagctcggtaccggatccAAAAGCAAACATACCGTATCTCCTTA-3’ 2025201121   17 Feb 2025 (SEQ ID No. 9) pCambia2300-GhNFXL1-F   5’-gactcttgaggatccgaattcATGAGCTTTCAAGGCCGAAATAGAT-3’ (SEQ ID No. 10) pCambia2300-GhNFXL1-R    5’-tagctttgtatatcactgcagTTCATAAGCCTTTTCCCAATCATCT-3’ (SEQ ID No. 11)

[0080] (3) Recovery and purification of the above PCR product and linearized expression vector using the EasyPure Quick Gel Extraction Kits of TransGen Biotech Co., Ltd. The specific steps were as follows:

[0081] (a) Quickly cut the agarose gel containing the target band under UV light to avoid damage to DNA caused by long-term exposure to UV light. Cut the gel block into small pieces and put them into a 1.5-mL clean centrifuge tube for subsequent gel dissolution.

[0082] (b) Weigh the gel on an electronic balance. Calculate the volume based on 100 mg equivalent to 100 uL. Add 3 times the volume of Buffer GDP and place in a 55°C water bath for 15 min. During this period, invert and mix 2-3 times to completely dissolve the gel block.

[0083] (c) Place the FastPure DNA Mini Columns-G adsorption column in a 2-mL collection tube, carefully transfer the gel liquid cooled to room temperature to the adsorption column, and centrifuge at 12,000 rpm for 1 min.

[0084] (d) Discard the filtrate and put the adsorption column back into the collection tube. Add 300 gL of Buffer GDP to the adsorption column, allow to stand at room temperature for 3 min, and then centrifuge at 12,000 rpm for 1 min.

[0085] (e) Discard the filtrate and put the adsorption column back into the collection tube. Add 700 gL of Buffer GW and centrifuge at 12000 rpm for 1 min.

[0086] (f) Repeat step (5).

[0087] (g) Discard the filtrate, put the adsorption column back into the collection tube, and centrifuge at 12,000 rpm for 2 min.

[0088] (h) Place the adsorption column in a sterilized 1.5-mL centrifuge tube, add 40 gL of Elution Buffer to the center of the adsorption column (preheated to 55 C in a water bath), and leave it at 2025201121   17 Feb 2025 room temperature for 5 min. Centrifuge at 12,000 rpm for 1 min to elute DNA, and store at -20°C for later use.

[0089] 2. Vector construction and transformation of E. coli: Use the ClonExpress® Ultra One Step Cloning Kit from Nanjing Vazyme Biotech Co., Ltd. to perform homologous recombination between the linearized vector and the target fragment to construct the vector and prepare the following liquid system on ice: Component Volume Linearized vector 2 pL Target fragment 3 pL 2xClonExpress Mix 5 pL ddH2O Up to 10 pL

[0090] React at 50°C for 10 min and immediately cool on ice, then transform the recombinant product into E. coli competent cells.

[0091] (1) Add all 10 pL of the recombinant ligation product to 100 pL of freshly melted DH5a competent cells, and allow to stand on ice for 30 min.

[0092] (2) Put in 42°C water bath for heat shock for 45 sec, then immediately place on ice for 2 min.

[0093] (3) Add 500 gL of LB liquid culture medium without antibiotics and shake on a shaker at 37°C for 1 hour.

[0094] (4) Centrifuge at 5,000 r / min for 2 min, discard 400 gL of the supernatant on the ultra-clean workbench, and spread 100 gL of bacterial liquid on LB solid culture medium containing the corresponding antibiotics, blow dry, and then invert and incubate in an incubator at 37°C overnight.

[0095] (5) The next day, pick the single clones on the plate and shake them in 500 gL of LB liquid culture medium containing the corresponding antibiotics until the bacterial liquid becomes turbid; perform PCR of bacterial liquid, and send the positive clones to Shanghai Sangon Biotech Co., Ltd. for sequencing; compare the sequencing results with the target sequence, and preserve the bacteria with correct results after comparison. 2025201121   17 Feb 2025

[0096] 3. Plasmid extraction: Use the EasyPure® Plasmid MiniPrep Kit to extract E. coli plasmids according to the following steps:

[0097] (1) Transfer 5 mL of E. coli cultured overnight on a 37°C shaker to a 10-mL centrifuge tube, centrifuge at 12,000 r / min for 5 min, and discard the supernatant.

[0098] (2) Add 300 pL of colorless solution RB (containing RNase A), and oscillate vigorously in a vortex mixer to suspend the bacterial sediment, without leaving any small bacterial blocks.

[0099] (3) Add 300 pL of blue solution LB, mix gently by turning up and down for 6 to 8 times, and fully lyse the bacterial cells until the color of the solution changes to bright blue, indicating complete lysis. The operation should be completed within 5 minutes.

[0100] (4) Add 450 pL of yellow solution NB, mix gently by turning up and down for 6 to 8 times, until the color of the solution changes from blue to yellow, forming a tight yellow agglomerate, indicating complete neutralization, and allow to stand at room temperature for 2 min.

[0101] (5) After centrifugation at 12,000 r / min for 10 min, draw the supernatant into the centrifuge column, 800 pL at a time, and centrifuge at 12,000 r / min for 1 min. Discard the liquid in the collection tube.

[0102] (6) Add 700 pL of rinse solution WB, 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 blow dry on a clean workbench for 5 min to remove residual rinse liquid.

[0103] (7) Place the centrifuge column in a 1.5-mL clean centrifuge tube, add 40 pL of 65°C preheated Elution Buffer to the center of the centrifuge column, allow to stand at room temperature for 2 min, and centrifuge at 12,000 r / min for 2 min to elute the plasmid DNA.

[0104] (8) Measure the plasmid DNA concentration using NanoDrop 2000, and store the obtained plasmid at -20°C.

[0105] 4. Agrobacterium transformation: Use GV3101 of Shanghai Weidi Biotechnology Co., Ltd. for Agrobacterium transformation according to the following steps:

[0106] (1) Add 2 pL of plasmid to 100 pL of frozen-thaw Agrobacterium competent cells (preferably thawing for 5 min), and gently shake the tube wall with fingers to mix. 2025201121   17 Feb 2025

[0107] (2) Carry out the following operations in sequence: stand on ice for 5 min, place in liquid nitrogen for 5 min, perform water bath at 37°C for 5 min, and stand on ice for 5 min.

[0108] (3) Add 600 pL of LB liquid culture medium to the clean bench, place it on a shaker at 200 rpm at 28°C for recovery and culture for 3 hours.

[0109] (4) Centrifuge at 3000 rpm for 3 min, collect the bacterial cells, discard part of the supernatant on a clean bench, leave 150 pL of bacterial cells and resuspend them by pipetting, and spread the bacterial liquid evenly on the solid medium containing Kan and Rif-resistant LB. Incubate upside down in a 28°C incubator for 2 days.

[0110] (5) Positive clone detection: Select single clone strains and culture them in 600 a L of fluid medium containing Kan and Rif-resistant LB at 28 C and 200 rpm for 12 h.

[0111] (6) Perform PCR for verification using the bacterial solution as a template.

[0112] Example 4 Silencing and expressing GhNFXL1 gene reduced salt tolerance and cold tolerance of cotton

[0113] (1) Select self-fertilized ZM113 seeds stored in the laboratory, soak the seeds in sterile water, and leave them at 37°C overnight. Select the white seeds and plant them with the radicle facing down into a 1:1 mixture of nutrient soil and vermiculite, cover with a transparent lid, and perform Agrobacterium injection after full expansion of cotyledons.

[0114] (2) Transfer 40 mL of Agrobacterium liquid cultured overnight and containing the gene silencing vector in a 50-mL clean centrifuge tube, centrifuge at 4000 rpm for 10 min, enrich the bacterial cells, and remove the supernatant.

[0115] (3) Resuspend the bacterial cells with the prepared resuspension solution and adjust OD600 to 1.0.

[0116] (4) Incubate at room temperature for 3 hours under dark conditions.

[0117] (5) Carefully scratch the back of the cotton cotyledon using the needles of a 1-mL sterile syringe (without penetrating the leaf), and inject the bacterial solution into the leaf from the wound until the entire leaf is soaked.

[0118] (6) Allow to stand at room temperature for 24 hours under dark conditions and then move to 2025201121   17 Feb 2025 the greenhouse for culture. When the plant grows to two leaves and one central bud, treat at 4 °C or with salt.

[0119] RNA was extracted from leaves of TRV:00 and TRV: GhNFXL1 plants, and fluorescence quantitative identification was performed after reverse transcription. The results showed that the expression level of TRV:GhNFXL1 gene was significantly reduced (FIG. 2 A and C). After treatment with 200mM NaCl solution, the leaves of TRV:GhNFXL1 plants wilted and the stems became lodging (FIG. 2 B). Further testing of MDA and POD content also 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 (FIG. 3 A and B). These results indicated that silencing the GhNFXLl gene reduced the salt tolerance of cotton. After 3 days of treatment at 4 C, the leaves of TRV:GhNFXL1 plants were severely wilted (FIG. 2 D). Further testing of MDA and POD content 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 (FIG. 3C and D). These results indicate that silencing the GhNFXL1 gene reduced the cold tolerance of cotton.

[0120] Example 5 Overexpression of GhNFXL1 gene enhanced the salt tolerance and cold tolerance of cotton

[0121] (1) On a clean bench, the sterile seedlings were cut into small pieces and placed into a conical flask with Agrobacterium infection solution containing the GhNFXL1 gene overexpression vector for 10 min. After infection, the hypocotyls were transferred to a sterilized large petri dish; when the bacterial solution on the surface was blown dry, the hypocotyls were transferred to a co-cultivation medium and culture them in the dark for 40-46 hours; after co-cultivation, transferred to sterile water containing CEF and soaked for 20 min; then washed with sterile water only 5-6 times until the residue was washed off. The water on surface was blown dry, and the hypocotyls were spread evenly in the hypocotyl induction medium containing timentin and kanamycin. Finally, the hypocotyls were cultured in a greenhouse at 28±1°C. The induced calli were transferred to callus proliferation medium containing timentin and kanamycin for culture, and subcultured once every 4 weeks. An appropriate amount of callus was transferred into an embryonic culture medium containing timentin and kanamycin, and cultured, then subcultured every 3 weeks until embryogenic callus appeared.

[0122] (2) The obtained embryonic callus was transferred to an embryonic callus proliferation 2025201121   17 Feb 2025 medium containing timentin and kanamycin for expansion culture, and subcultured every two weeks. When proliferated to a certain amount, the embryogenic callus could be used for subsequent differentiation into seedlings.

[0123] DNA was extracted from the leaves of the overexpressing plants, and specific fragment primers were designed for RT-qPCR amplification. The results showed that the expression level of the GhNFXL1 gene overexpressing strain was much higher than that of the Mock (FIG. 4 A), proving that the GhNFXL1 gene overexpressing strain was successfully constructed. Cotton seeds of the Mock and T4 generations were selected and cultured in water until two leaves and one central bud appeared, then transferred to a hydroponic solution containing 200mM NaCl. The results showed that the leaves of the GhNFXL1 gene overexpression line showed slight wilting and the stems were slightly bent, while the Mock plants had more severe wilting and serious lodging of stems (FIG. 4 B). Further testing of MDA and POD content showed that the MDA content of GhNFXL1 gene overexpressing plants was lower than that of Mock plants, and the POD content of GhNFXL1 gene overexpressing plants was higher than that of Mock plants (FIG. 5 A and B). These results indicated that the GhNFXL1 gene could improve the salt tolerance of cotton. In addition, cotton seeds of the Mock and T4 generations were selected and cultivated in soil until two leaves and one central bud appeared, and then treated at 4°C for 2 days to observe the phenotype. The results showed that the leaves of the GhNFXL1 gene overexpression line showed weak wilting, while the Mock plants showed more serious wilting, proving that the cold tolerance of GhNFXL1 gene overexpression lines was higher than that of Mock plants (FIG. 4 C). Further testing of MDA and POD content showed that the MDA content of GhNFXL1 gene overexpressing plants was lower than that of Mock plants, and the POD content of GhNFXL1 gene overexpressing plants was higher than that of Mock plants (FIG. 5 C and D). These results indicated that the GhNFXL1 gene could improve the cold tolerance of cotton.

[0124] The foregoing examples only describe several embodiments of the present disclosure, and the description thereof is relatively specific and detailed, but these examples should not be construed as limiting the scope of the present disclosure. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, which all fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the appended claims, and the description and drawings can be used to interpret the content of the claims. 2025201121   17 Feb 2025

Claims

1. Use of a nucleic acid corresponding to GhNFXL1 gene in improving salt tolerance and / or cold tolerance of a Gossypium plant.

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

3. The method according to claim 2, wherein the nucleic acid is introduced into the Gossypium 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 Gossypium plant by a gene gun-mediated transformation method, a pollen tube pathway method or a liposome transformation method.

5. The method according to claim 3, wherein the recombinant DNA construct is introduced into the Gossypium plant by Agrobacterium.

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

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

8. The use according to claim 7, wherein the propagation material applicable to sexual propagation is selected from a group consisting of microspores, pollen, ovaries, ovules, embryo sacs and egg cells; andpreferably the propagation material applicable to tissue culture of regenerative cells is selected from a group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, anthers, flowers, seeds and stems.

9. A method for breeding a salt-tolerant and / or cold-tolerant Gossypium plant, comprising:2025201121   17 Feb 2025using a detection agent to detect the expression level of GhNFXL1 gene in a Gossypium plant, and selecting cotton plants or varieties with high expression levels for breeding.

10. The breeding method according to claim 9, wherein the detection agent comprises a reagent for detecting changes in mRNA expressed by the GhNFXL1 gene, and the reagent is applicable to 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; orthe detection agent comprises a reagent for detecting changes in the protein expressed by the GhNFXL1 gene, and the reagent is applicable to at least one of the following detection methods: immunoassay, biological mass spectrometry, lectin-based assay, and aptamer-based assay.

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