GhANK-C3H15 gene and / or interaction gene of cotton and application of GhANK-C3H15 gene and / or interaction gene in regulating cold resistance of crops

Abstract

The invention provides a cotton GhANK-C3H15 gene and / or an interaction gene and application of the gene and / or the interaction gene in regulating the cold resistance of crops, and belongs to the technical field of gene engineering. The invention provides a GhANK-C3H15 gene for regulating and controlling the cold resistance of crops, the genome sequence of the GhANK-C3H15 gene is as shown in SEQ ID No. 1, and the GhANK-C3H15 gene encodes a protein as shown in SEQ ID No. 3. The expression level of the GhANK-C3H15 gene under the stress of low temperature is obviously up-regulated. According to the present invention, the GhANK-C3H15 gene and the GhbHLH30 are subjected to interaction to respectively silence the GhANK-C3H15 gene and the GhbHLH30 gene, such that the cold resistance is significantly reduced; and after the GhANK-C3H15 gene is over-expressed, the cold resistance can be obviously enhanced.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to cotton. GhANK-C3H15 Genes and / or interacting genes and their use in regulating crop cold resistance. Background Technology

[0002] cotton( Gossypium Cotton, belonging to the genus Gossypium of the Malvaceae family, is an important global economic crop and source of natural fiber. However, cotton is a warm-loving crop and is not cold-hardy. my country's main cotton-producing areas are concentrated in Xinjiang, located in the northwest inland region. Its unique climate and geographical location make it highly susceptible to cold air intrusion. During the cotton sowing season, frequent low-temperature meteorological disasters such as prolonged rainfall, cold waves, and late spring frosts cause ground temperatures to fall far below the minimum requirements for cotton seed germination and seedling growth, resulting in varying degrees of seed rot, bud rot, root rot, and even seedling death. Low-temperature stress has become one of the important factors limiting cotton yield.

[0003] CCCH (C3H) proteins belong to the zinc finger protein subfamily. Their defining characteristic is the presence of a CCCH motif composed of three cysteine ​​(C) residues and one histidine (H) arranged in a specific sequence. The core amino acid sequence is CX. 7 / 8 -C-X5-C-X3-H. This motif can bind one zinc ion (Zn). 2+ This is the structural basis for their binding to nucleic acids or proteins. As key regulators of gene expression, CCCH-type zinc finger proteins function mainly through three mechanisms: directly binding to DNA to participate in transcriptional regulation, binding to RNA to participate in post-transcriptional regulation, and mediating protein-protein interactions. The CCCH gene family is widely involved in multiple processes of plant growth and development, including regulating cell wall development, influencing cell elongation, participating in abiotic stress responses, and playing important roles in RNA metabolism and hormone metabolism.

[0004] Currently, the functions of the C3H gene family have been demonstrated in Arabidopsis thaliana and rice. Oryza sativa ),broccoli( Brassica oleracea var italica This has been verified in various plants, including rice. (The C3H gene family members are also mentioned.) OsDOS Overexpression of jasmonic acid can delay leaf senescence by negatively regulating the jasmonic acid signaling pathway. Under drought conditions, root elongation and leaf expansion in rice seeds are inhibited, but overexpression of jasmonic acid can help delay these effects. OsC3H10 Transgenic rice can promote root development and alleviate the inhibitory effect of drought on plant growth. In soybeans, the GmZF351 protein can enhance the transcriptional activation of stress-responsive genes through a histone demethylation-mediated mechanism, improving cell water retention and antioxidant capacity, thereby enabling plants to grow normally under saline-alkali and drought conditions. Arabidopsis thaliana... C3HGene family members are involved not only in abiotic stress responses and hormone regulation processes such as abscisic acid, AtC3H14 and AtC3H15 It has also been shown to regulate the biosynthesis of the secondary cell wall in Arabidopsis thaliana. Overexpression AaZFP3 Arabidopsis thaliana with certain genes exhibits an early flowering phenotype. Broccoli BoC3H4 Genes can increase a plant's salt tolerance, but at the same time reduce its disease resistance. Overexpression CpC3H3 The genetically modified Arabidopsis thaliana flowered earlier and exhibited enhanced drought resistance. However, in cotton... GhANK-C3H15 There is limited research on gene function. Summary of the Invention

[0005] This invention provides cotton GhANK-C3H15 Genes and / or interacting genes and their use in regulating crop cold resistance, wherein the genes and / or interacting genes can positively regulate crop cold resistance.

[0006] This invention provides a method for regulating the low-temperature stress resistance of cotton. GhANK-C3H15 Genes, the ones mentioned GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

[0007] In one specific embodiment of the present invention, the... GhANK-C3H15 The CDS sequence of the gene is shown in SEQ ID No. 2.

[0008] The present invention also provides the above. GhANK-C3H15 Proteins encoded by genes.

[0009] The present invention also provides the above. GhANK-C3H15 Application of genes, the aforementioned proteins, and / or interacting proteins of the aforementioned proteins in regulating cotton's resistance to low-temperature stress.

[0010] In one specific embodiment of the present invention, the interacting protein includes GhbHLH30.

[0011] In one specific embodiment of the present invention, the regulation includes overexpression of the GhANK-C3H15 Enhancing cotton's resistance to low-temperature stress by incorporating genes encoding genes and / or interacting proteins; The silence GhANK-C3H15 After the genes encoding genes and / or interacting proteins are removed, the resistance of cotton to low temperature stress is reduced; Knock out the GhANK-C3H15 After the genes encoding the genes and / or interacting proteins are removed, the cotton's resistance to low-temperature stress is reduced.

[0012] This invention also provides a method for improving the low-temperature stress resistance of cotton, including increasing the concentration of certain components in the target cotton genome. GhANK-C3H15 The expression levels of genes encoding genes and / or interacting proteins, the GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

[0013] In one specific embodiment of the present invention, the improvement of the target cotton genome GhANK-C3H15 Methods for measuring the expression levels of genes encoding genes and / or interacting proteins, including transforming the target cotton using an overexpression vector; The base vector for the overexpression vector includes pCAMBIA3301.

[0014] The present invention also provides the above. GhANK-C3H15 Application of genes, the aforementioned proteins, and / or interacting proteins in the breeding of cotton varieties or lines with target resistance to low-temperature stress.

[0015] This invention also provides a method for cultivating cold-resistant crops, including overexpression in the crop genome. GhANK- C3H15 The genes encoding genes and / or interacting proteins, said GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

[0016] Beneficial effects: This invention uses bioinformatics to identify a regulator of cold resistance in the cotton genome. GhANK-C3H15 Genes, the ones mentioned GhANK-C3H15 The genomic sequence of the gene is shown in SEQ ID No. 1 and encodes the protein shown in SEQ ID No. 3. Transcriptomic analysis indicates that the gene... GhANK-C3H15 The gene expression level was significantly upregulated under low temperature stress. This invention describes... GhANK-C3H15 The gene interacts with GhbHLH30, and then the gene is silenced and overexpressed, respectively. GhANK-C3H15 Genes and GhbHLH30 The two cotton plants obtained by silencing the gene showed significantly reduced cold resistance; while those overexpressing the gene showed significantly reduced cold resistance. GhANK-C3H15 The gene in Arabidopsis thaliana can significantly enhance cold resistance, proving the above. GhANK-C3H15 Genes and interacting genes help enhance the cold resistance of plants, providing genetic resources for breeding cold-resistant cotton varieties. Attached Figure Description

[0017] Figure 1 Figure 1 shows the phylogenetic analysis results of the GhANK-C3H protein from upland cotton. In the figure, a: domain analysis diagram, b: phylogenetic tree; Figure 2 A schematic diagram of the gene structure characteristics of the GhANK-C3H protein in upland cotton; Figure 3 Chromosomal distribution map of the GhANK-C3H gene in upland cotton; Figure 4 A schematic diagram showing the distribution of cis-acting elements in the promoter of the GhANK-C3H gene in upland cotton; Figure 5 Under low temperature stress GhANK-C3H15 Figure 1 shows the results of gene expression and function analysis. Figure a: Low temperature stress based on transcriptome data. GhANK-C3H15 b: Gene expression levels; b: qRT-PCR analysis under low temperature stress GhANK-C3H15 Gene expression trends; c: plant phenotype; d: qRT-PCR detection GhANK-C3H15 Gene silencing efficiency; e: silencing GhANK- C3H15 Effects of genes on MDA content in cotton under low-temperature stress; f: silence GhANK-C3H15 Effects of genes on T-AOC content in cotton under low temperature stress; Figure 6 Overexpression under low temperature stress GhANK-C3H15 The figure shows the phenotypic and physiological indicators of Arabidopsis thaliana with gene expression. Figure a: qRT-PCR detection of overexpressing Arabidopsis thaliana. GhANK-C3H15 a: Gene expression level; b: Plant phenotype; c: MDA content in Arabidopsis thaliana overexpressing under low temperature stress; d: T-AOC content in Arabidopsis thaliana overexpressing under low temperature stress; Figure 7 The figure shows the functional analysis results of GhANK-C3H15 protein. In the figure, a: subcellular localization results of GhANK-C3H15 protein; bc: interaction verification between GhANK-C3H15 protein and GhbHLH30 protein. Figure 8 Under low temperature stress GhbHLH30 Figure 1 shows the results of gene expression and function analysis. In the figure, a: qRT-PCR analysis under low temperature stress. GhbHLH30 a: Gene expression trends; b: qRT-PCR detection GhbHLH30 Gene silencing efficiency (n=3); c: plant phenotype; d: silencing GhbHLH30 Effect of genes on MDA content in cotton under low temperature stress (n=3); e: silence GhbHLH30 Effects of genes on T-AOC content in cotton under low temperature stress (n=3). Detailed Implementation

[0018] This invention provides a method for regulating the low-temperature stress resistance of cotton. GhANK-C3H15 Genes, the ones mentioned GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

[0019] The present invention GhANK-C3H15The gene was screened from cotton, especially upland cotton, and is located on chromosome A13. Its genome sequence is shown in SEQ ID No. 1; its CDS sequence is shown in SEQ ID No. 2.

[0020] In this embodiment of the invention, qRT-PCR was used to analyze... GhANK-C3H15 The expression patterns of genes under low-temperature stress showed that the expression levels of the genes generally increased after low-temperature treatment, indicating that... GhANK-C3H15 Genes may be involved in cotton's defense response to low-temperature stress. This is then described through VIGS silencing. GhANK-C3H15 Following gene expression, cotton's tolerance to low-temperature stress was significantly reduced, specifically manifested as a decrease in total antioxidant capacity (T-AOC) and an increase in malondialdehyde (MDA) content; overexpression of the aforementioned gene in Arabidopsis thaliana... GhANK-C3H15 After gene modification, the plant's cold resistance can be significantly enhanced; therefore, the present invention... GhANK- C3H15 The gene is a cold resistance regulator in cotton.

[0021] The present invention also provides the above. GhANK-C3H15 Proteins encoded by genes.

[0022] The amino acid sequence of the protein described in this invention is shown in SEQ ID No. 3.

[0023] The protein of this invention is located in the cell nucleus and specifically interacts with downstream GhbHLH30. The genomic sequence of GhbHLH30 is shown in SEQ ID No. 4, the CDS sequence is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6.

[0024] The present invention also provides the above. GhANK-C3H15 Application of genes, the aforementioned proteins, and / or interacting proteins of the aforementioned proteins in regulating cotton's resistance to low-temperature stress.

[0025] The interacting protein described in this invention includes GhbHLH30, and the regulation described in this invention includes: overexpression of the [specific protein name missing]. GhANK- C3H15 Enhancing cotton's resistance to low-temperature stress by incorporating genes encoding genes and / or interacting proteins; The silence GhANK-C3H15 After the genes encoding genes and / or interacting proteins are removed, the resistance of cotton to low temperature stress is reduced; Knock out the GhANK-C3H15 After the genes encoding the genes and / or interacting proteins are removed, the cotton's resistance to low-temperature stress is reduced.

[0026] The present invention does not specifically limit the method of regulation, and any method in the art that induces changes in gene expression can be used, such as mutagenesis, gene mutation, silencing, gene editing, knock-in, knockout, or overexpression.

[0027] This invention also provides a method for improving the low-temperature stress resistance of cotton, including increasing the concentration of certain components in the target cotton genome. GhANK-C3H15 The expression levels of genes encoding genes and / or interacting proteins, the GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

[0028] This invention can increase the expression level of the gene by constructing an overexpression vector. In one embodiment, the gene is... GhANK-C3H15 A recombinant overexpression vector was constructed by inserting a gene into a base vector, the base vector including pCAMBIA3301.

[0029] The present invention also provides the above. GhANK-C3H15 Application of genes, the aforementioned proteins, and / or interacting proteins in the breeding of cotton varieties or lines with target resistance to low-temperature stress.

[0030] The present invention GhANK-C3H15 Genes and GhbHLH30 Genes can regulate plant cold hardiness on their own. That is, reducing the expression level of any gene or knocking it out can reduce the plant's cold hardiness. In a single plant, reducing the expression level of two genes together or knocking them out sequentially can also reduce the plant's cold hardiness. Conversely, increasing the expression level of any gene or overexpressing it can significantly improve the plant's cold hardiness. Increasing the expression level of two genes simultaneously can significantly improve the plant's cold hardiness.

[0031] In this invention, it can be achieved by reducing GhANK-C3H15 Genes and GhbHLH30 The expression levels of any one or two genes in the genome are used to construct low-hardiness crops as models of cold-intolerant crops.

[0032] This invention also provides a method for cultivating cold-resistant crops, including overexpression in the crop genome. GhANK- C3H15 The genes encoding genes and / or interacting proteins, said GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

[0033] This invention does not specifically limit the type of crop. In one embodiment, Arabidopsis thaliana is used as an example for illustrative purposes. GhANK-C3H15 A recombinant overexpression vector was constructed by inserting a gene into a base vector, the base vector including pCAMBIA3301.

[0034] To further illustrate the present invention, the following embodiments are used to describe the cotton provided by the present invention. GhANK-C3H15 The genes and / or interacting genes and their uses in regulating crop cold resistance are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0035] Unless otherwise specified, the materials and methods used in the embodiments of this invention are derived from conventional commercially available products or methods described in the literature.

[0036] 1. Materials and Methods 1.1 Plant materials, RNA extraction and cDNA synthesis Upland cotton TM-1 seeds were preserved by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and have been published in an article (Dai, Y., Zhou, J., Zhang, B., Zheng, D., Wang, K., & Han, J. (2025). Time-course transcriptome analysis reveals gene co-expression networks and transposable element responses to cold stress in cotton. BMC Genomics, 26(1),235. https: / / doi.org / 10.1186 / s12864-025-11433-z). Cotton plants were grown in a greenhouse with a photoperiod of 16h light / 8h dark. Under the same growth conditions, one true leaf was collected from each plant, and three biological replicates were set up for each sample. Total RNA was extracted using a commercial RNA extraction kit, and RNA quality was detected using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized by reverse transcription according to the kit instructions (Nanjing Novizan Biotechnology Co., Ltd.).

[0037] 1.2 Upland Cotton GhANK-C3H Gene family identification and bioinformatics analysis The upland cotton (AD1) 'TM-1' T2T genome dataset JZU_v1.0 was downloaded from the cotton genome database (https: / / www.cottongen.org / ), and the Arabidopsis genome dataset (Araport11) was obtained from the Arabidopsis Information Resource Database (https: / / www.arabidopsis.org / ). Based on Arabidopsis protein sequences, cotton protein sequences containing both ankyrin repeat sequences and C3H domains were screened. Potential GhANK-C3H family member sequences were compared with those from the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / #! / home / contact) and the Protein Family Database (https: / / www.ebi.ac.uk / interpro / entry / pfam / ), and genes identified by both methods were used as candidate genes. The physicochemical properties of GhANK-C3H proteins, including amino acid number, molecular weight (MW), and isoelectric point (pI), were analyzed and predicted using the ExPASy online tool (https: / / www.expasy.org / ). Subcellular localization of the GhANK-C3H protein was predicted using the WoLF PSORT online tool (https: / / wolfpsort.hgc.jp / ). The GhANK-C3H protein sequence of *Gossypium hives* was aligned using MUSCLE, and a rootless phylogenetic tree was constructed using FastTree with maximum likelihood (ML). The phylogenetic tree was visualized using the interactive Tree of Life online tool (https: / / itol.embl.de / ). Promoter cis-regulatory elements were analyzed using the Plant Cis-Regulatory Elements Database (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ), and the results were visualized using a gene structure display server (https: / / gsds.gao-lab.org / ).

[0038] 1.3 GhANK-C3 H gene expression analysis Low temperature stress GhANK-C3HGene expression data were obtained from a public transcriptome dataset (Lin, Z.,Wang, Z., Zhang, Y., Tan, S., Masangano, M., Kang, M., Cao, X., Huang, P.,Gao, Y., Pei, X., Ren, X., He, K., Liang, Y., Ji, G., Tian, ​​Z., Wang, X.,&Ma,X. (2025). Gene expression modules during the emergence stage of uplandcotton under low-temperature stress and identification of the GhSPX9 cold-tolerance gene. Plant Physiology and Biochemistry, 218, 109320. https: / / doi.org / https: / / doi.org / 10.1016 / j.plaphy.2024.109320), and the specific data are shown in Table 1.

[0039] Table 1. TPM represents the transcriptional abundance of genes.

[0040] 1.4 qRT-PCR analysis Specific primers for the target gene were designed, and qRT-PCR amplification was performed using the SYBR Green quantitative PCR kit. All primer sequences used in this invention are shown in Table 2. After pre-denaturation, the reaction system underwent multiple amplification cycles, with real-time monitoring of the fluorescence signal in each cycle to quantify the accumulation of PCR products. A 2... -ΔΔCt The relative transcriptional level of genes was calculated using a method with three biological replicates and three technical replicates per experiment. GhUBQ7 The gene is an internal reference gene.

[0041] Table 2 qRT-PCR primer information

[0042] 1.5 Virus-induced gene silencing (VIGS) Augmentation based on the VIGS sequence design website (https: / / vigs.solgenomics.net / ) GhANK-C3H15 and GhbHLH30The 300bp specific coding sequence of the gene is inserted between the EcoR1 and BamH1 sites in the VIGS vector pTRV2.

[0043] Table 3 Primer information used for VIGS amplification

[0044] The recombinant vectors pTRV2::GhANK-C3H15 and pTRV2::GhbHLH30 were transformed into Agrobacterium GV3101 strain. Silencing the phytoene dehydrogenase (PDS) gene induced an albino phenotype in the plants. The CDS fragment of PDS (GH_A10G1058) was inserted between the EcoR1 and BamH1 sites of the pTRV2 vector to construct the recombinant vector; therefore, pTRV2::PDS was used as the phenotypic marker. Cotton plants were grown in a greenhouse with a photoperiod of 16 h light / 8 h dark. Agrobacterium infection was performed when the cotton cotyledons were fully expanded, followed by 24 h of dark incubation. Phenotypic identification was then performed at the two-leaf-one-heart stage.

[0045] 1.6 Arabidopsis thaliana overexpression experiment Gene-specific primers were designed using the primer design website (https: / / crm.vazyme.com / cetool / simple.html) to insert the amplified fragment between the EcoR1 and BamH1 sites of pCAMBIA3301, constructing the overexpression vector pCAMBIA3301-GhANK-C3H15, which was then transformed into Agrobacterium GV3101 strain. Agrobacterium was used to infect Arabidopsis thaliana Col-0 ecotype inflorescences, and the infection was repeated once after one week to obtain... GhANK-C3H15 Arabidopsis plants with overexpressed genes.

[0046] GhANK-C3H15 -F (SEQ ID No.15): acgggggactcttgaggatccATGTGCCGTGGTTCAAAATTCG; GhANK-C3H15 -R (SEQ ID No. 16): ccgggtaccgagctcgaattcTGCCACCATCTGCTCCTGC.

[0047] 1.7 Measurement of physiological and biochemical indicators After treatment at 4℃ for 24 h, true leaf samples were collected from pTRV2::GhANK-C3H15, pTRV2::GhbHLH30, pTRV2::00 (empty vector control), and wild-type (WT) plants. Malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) were determined according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd.). One true leaf was collected from each plant, and each sample consisted of leaves from three plants grown under identical conditions. All experiments were performed in triplicate.

[0048] 1.8 Subcellular localization Gene-specific primers were designed using a primer design website, and the amplified fragment was inserted into pSGFP (Gao JS, WuN, Shen ZL, et al. Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypiumhirsutum L.). Gene To construct the GhANK-C3H15-GFP fusion expression vector, a gene was created by removing the KpnI restriction site from the gene (d. 2016;576(2 Pt 2):763-769. doi:10.1016 / j.gene.2015.11.002). The recombinant vector was transformed into Agrobacterium GV3101 strain and used to infect tobacco plants at the appropriate growth stage. Subcellular localization of the GhANK-C3H15 protein was observed using laser confocal microscopy.

[0049] GhANK-C3H15 -F (SEQ ID No.17): atactagtggatccggtaccATGTGCCGTGGTTCAAAATTCG; GhANK-C3H15 -R (SEQ ID No. 18): cccttgctcaccatggtaccTGCCACCATCTGCTCCTGC.

[0050] 1.9 Yeast Two-Hybrid (Y2H) Experiment Gene-specific primers were designed using a primer design website, and the amplified fragment was inserted between the EcoRI and Pstl restriction sites of pGBKT7 to construct the bait vector pGBKT7- GhANK-C3H15 The pGADT7 empty vector was co-transformed into Y2HGold yeast competent cells, and self-activation was verified according to the kit instructions (Beijing Coolplay Technology Co., Ltd.). Subsequently, the prey vector pGADT7- was constructed. GhbHLH30The amplified fragment was inserted between the EcoRI and BamHI restriction sites of pGADT7 to construct the prey vector pGADT7- GhbHLH30 Compare it with pGBKT7- GhANK-C3H15 The cells were co-transformed into Y2HGold competent cells to verify protein-protein interactions. The experimental system included negative controls (pGBKT7-Lam, pGADT7-T), positive controls (pGBKT7-53, pGADT7-T), and a self-activation experimental group (pGBKT7-...). GhANK-C3H15 pGADT7), experimental group (pGBKT7- GhANK-C3H15 pGADT7- GhbHLH30 The vectors required for the yeast two-hybrid experiment were purchased from Coollab Corporation.

[0051] GhANK-C3H15 -F (SEQ ID No.19): atggccatggaggccgaattcATGTGCCGTGGTTCAAAATTCG; GhANK-C3H15 -R (SEQ ID No.20): ctagttatgcggccgctgcagTTATGCCACCATCTGCTCCTG; GhbHLH30 -F (SEQ ID No.21):gccatggaggccagtgaattcATGGCTGCTTACTCTTTCAACAAC; GhbHLH30 -R (SEQ ID No. 22): cagctcgagctcgatggatccTCAAGATGAGCTTGAGGTATCAATG.

[0052] 1.10 Luciferase Complementation Imaging (LCI) Experiment Gene-specific primers were designed using a primer design website. The amplified fragments were inserted between the Kpn I and Sal I restriction sites of the n / cLUC vector, respectively, to construct the cLUC-GhANK-C3H15 and nLUC-GhbHLH30 recombinant vectors. These vectors, along with the empty vectors pCambia1300-cLUC and pCambia1300-nLUC (negative control), were transformed into Agrobacterium and used to infect tobacco plants at the appropriate growth stage. Fluorescence signals were detected using a multifunctional imaging system. The experimental system included negative control 1 (cLUC-GhANK-C3H15, nLUC), negative control 2 (cLUC, nLUC-GhbHLH30), negative control 3 (cLUC, nLUC), and the experimental group (cLUC-GhANK-C3H15, nLUC-GhbHLH30). The empty vectors were previously preserved by the research group.

[0053] GhANK-C3H15 -F (SEQ ID No.23): gtacgcgtcccggggcggtaccATGTGCCGTGGTTCAAAATTCG; GhANK-C3H15 -R (SEQ ID No. 24): gaacgaaagctctgcaggtcgacTTATGCCACCATCTGCTCCTG; GhbHLH30 -F (SEQ ID No.25): cggggggacgagctcggtaccATGGCTGCTTACTCTTTCAACAAC; GhbHLH30 -R (SEQ ID No. 26): gtcccggggcgtcgacAGATGAGCTTGAGGTATCAATGTAGG.

[0054] 1.11 Statistical Analysis Statistical analysis was performed using t-tests, and bar charts were generated using Graphpad Prism 9.5 software.

[0055] Example 1 1.1 Identification and Evolutionary Analysis of the GhANK-C3H Gene Family in Upland Cotton Thirty GhANK-C3H genes were identified from the upland cotton genome using HMMER and BLASTP analyses. Based on their chromosomal locations, these 30 upland cotton GhANK-C3H proteins were named GhANK-C3H1 to GhANK-C3H30. The physicochemical properties of all members of the upland cotton GhANK-C3H family are shown in Table 4. The amino acid lengths of GhANK-C3H proteins range from 642 to 1011 aa, with predicted molecular weights of 70174.29 to 114236.55 kDa and theoretical isoelectric points of 5.68 to 8.35. Subcellular localization predictions showed that 22 GhANK-C3H proteins were located in the nucleus, 3 in the endoplasmic reticulum, 2 in the cell membrane, 2 in the extracellular space, and 1 in the chloroplast.

[0056] Table 4. Physicochemical properties of all members of the GhANK-C3H family of upland cotton.

[0057] 1.2 Phylogenetic analysis and classification of the GhANK-C3H gene family in upland cotton Phylogenetic analysis was performed on members of the GhANK-C3H gene family in upland cotton. Results are as follows: Figure 1 As shown, GhANK-C3H proteins all contain typical C-X7-C-X5-C-X3-H domains and their variants.

[0058] Based on the phylogenetic structure and domain characteristics of the GhANK-C3H gene in upland cotton, a phylogenetic tree was constructed, and its gene structural characteristics were analyzed. The results are as follows: Figure 2 As shown, the N-terminus of the GhANK-C3H protein contains an ankyrin repeat sequence, and the C-terminus of this repeat sequence is connected in series with a CCCH domain.

[0059] 1.3 Chromosomal localization analysis of the GhANK-C3H gene family in upland cotton Based on the genomic and annotation data of tetraploid cotton, this invention provides a detailed analysis of the chromosome distribution of the identified GhANK-C3H gene. The results are as follows: Figure 3 As shown, the 30 GhANK-C3H genes are randomly distributed across 18 chromosomes. The number of genes on the homologous chromosomes of the At and Dt subgenomes is completely identical.

[0060] 1.4 Analysis of cis-regulatory elements of the promoter of the GhANK-C3H gene in upland cotton To further explore the potential regulatory mechanisms of the GhANK-C3H gene family, this invention analyzed the cis-regulatory elements in the upstream 2000 bp promoter regions of 30 GhANK-C3H genes. The results are as follows: Figure 4 As shown, these promoter regions contain a total of 77 different types of cis-acting elements, which participate in 40 different biological functions in multiple species, and it is speculated that they may have similar functions in cotton.

[0061] The promoter regions of all 30 GhANK-C3H genes contain at least one light-responsive element (such as Box4, G-Box, MRE, and GATA motifs), with Box4 and G-Box being the two most abundant element types. The upstream regions of most GhANK-C3H genes contain plant hormone response elements such as ABRE, P-box, and TCA-element, which are involved in gibberellin, abscisic acid, salicylic acid, and jasmonic acid. Furthermore, this invention identified cis-regulatory elements involved in plant growth and development, including CAT-box, A-box, and GCN4 motifs. The GhANK-C3H gene promoters also contain one or more stress-responsive elements: ARE and GC motifs involved in gene expression regulation under anaerobic conditions, the MYB binding site MBS involved in drought-induced expression, and LTR and TC-rich repetitive sequences in response to low temperatures. These results indicate that GhANK-C3H genes may participate in multiple stress response processes, providing a basis for further analysis of the regulatory mechanisms of the upland cotton GhANK-C3H gene family.

[0062] Example 2 2.1 Silence GhANK-C3H15 Effects of genes on cotton's tolerance to low temperature stress This invention, based on transcriptome data, detected the expression pattern of the GhANK-C3H gene under low-temperature treatment (4℃), such as... Figure 1 As shown, by analyzing the expression characteristics of the GhANK-C3H gene in upland cotton under different low-temperature treatment durations, it was found that some genes (GhANK-C3H4, GhANK-C3H5, GhANK-C3H14, GhANK-C3H15, GhANK-C3H19, GhANK-C3H20, GhANK-C3H29, GhANK-C3H30) were continuously induced and their expression levels were significantly upregulated during the treatment period. This indicates that these genes are regulated by low-temperature stress and may play a key role in the long-term low-temperature acclimatization process of the plant.

[0063] The VIGS gene was used to silence the above 8 genes to verify the cold resistance of cotton. After 24 hours of low temperature treatment (4℃), the phenotype of the GhANK-C3H15-silenced plants was the most obvious.

[0064] This invention analyzes GhANK-C3H15Gene expression patterns under low temperature stress, qRT-PCR results as follows Figure 5 As shown, the expression levels of the genes generally increased after low-temperature treatment. Figure 5 (a and b) indicate GhANK-C3H15 Genes may be involved in cotton's defense response to low-temperature stress.

[0065] To further clarify GhANK-C3H15 To investigate the function of genes in response to low-temperature stress, this invention employs virus-induced gene silencing (VIGS) technology. The experimental system includes wild-type (WT), an empty vector control group (pTRV2::00), and a carrier group. GhANK-C3H15 The effectiveness of the VIGS system was verified by using a vector group containing a 300bp specific fragment of the gene (pTRV2::GhANK-C3H15) and a vector group carrying the PDS gene (pTRV2::PDS) as a positive control.

[0066] The newly emerged true leaves exhibited an albino phenotype, indicating successful PDS gene silencing. Subsequently, true leaf samples were collected from plants in both the experimental group (pTRV2::GhANK-C3H15) and the control group (pTRV2::00). One true leaf was collected from each plant, and each biological replicate contained leaf samples from three plants. The technical replication was validated three times using qRT-PCR, and the results are as follows: Figure 5 As shown in Figure c, the leaves and overall growth of the control group plants under low-temperature stress were significantly better than those under normal conditions. GhANK-C3H15 Gene-silenced plants. Gene silencing efficiency detected by qRT-PCR is as follows: Figure 5 As shown in d, compared with the control group, the silent plants had... GhANK-C3H15 Gene expression levels were significantly downregulated. Furthermore, compared to the control group, the total antioxidant capacity (T-AOC) of silent plants decreased, and the malondialdehyde (MDA) content increased. Figure 5 (e and f). The above results indicate that silence... GhANK-C3H15 Genes can reduce the tolerance of cotton plants to low-temperature stress.

[0067] 2.2 Overexpression GhANK-C3H15 Cold resistance of genetically enhanced transgenic Arabidopsis thaliana To further explore GhANK-C3H15 To investigate the function of genes under low-temperature stress, this invention transforms the overexpression vector pCAMBIA3301-GhANK-C3H15 into wild-type Arabidopsis thaliana Col-0 ecotype, obtaining three genes. GhANK-C3H15 The homozygous overexpression lines with the highest gene expression levels (OE1, OE7, and OE8) were used for subsequent cold hardiness testing. Figure 6(a) Transgenic homozygous lines and wild-type lines were treated at 4°C for 48 h, followed by recovery culture at room temperature for 2 days. Plant phenotypes were observed and relevant physiological indicators (MDA and T-AOC) were measured. Results are as follows: Figure 6 As shown in figures b and c, the overexpression lines exhibited better leaf chlorosis, milder wilting symptoms, and less leaf damage due to low-temperature stress. These results indicate that overexpression in Arabidopsis thaliana... GhANK- C3H15 Genes can enhance a plant's cold resistance.

[0068] 2.3 Verification of the interaction between GhANK-C3H15 and GhbHLH30 proteins To further clarify GhANK-C3H15 This invention investigates the molecular mechanisms by which genes participate in low-temperature stress responses and employs a yeast two-hybrid (Y2H) assay to screen downstream interacting genes. First, subcellular localization experiments are used to validate the effectiveness of the library screening system. GhANK-C3H15 The coding sequence of the gene was cloned into the transient expression vector hANK-C3H15-GFP. Utilizing the green fluorescence of GFP, the subcellular localization of the GhANK-C3H15 protein was observed using laser confocal microscopy. Results are as follows: Figure 7 As shown in Figure a, the GhANK-C3H15 protein is located in the nucleus of tobacco epidermal cells.

[0069] Subsequently, the effectiveness of the yeast two-hybrid system was verified by a self-activation experiment. The positive control strain grew normally on both SD / -Trp-Leu (two-deficient medium, DDO) and SD / -Trp-Leu-His-Ade (four-deficient medium, QDO), and showed a blue color on QDO medium (blue-white screening); while the negative control and self-activation experimental group strains only grew normally on DDO medium. These results indicate that this yeast two-hybrid system is suitable for screening downstream interacting proteins of the GhANK-C3H15 protein.

[0070] Through library screening, this invention identified a protein, GhbHLH30, that interacts with the GhANK-C3H15 protein. Figure 7 (b) The interaction between the two was further verified in *Nicotiana benthamiana* using luciferase complementation imaging (LCI) experiments. The results showed that when *GhANK-C3H15-nLUC* and *GhbHLH30-cLUC* were co-expressed in *Nicotiana benthamiana*, a significant fluorescence signal was detected on the leaf surface. Figure 7 (c). In summary, this invention confirms that the GhANK-C3H15 protein can specifically interact with the GhbHLH30 protein.

[0071] 2.4 Silence GhbHLH30 Genes reduce cotton's cold resistance To verify GhbHLH30 To investigate whether genes participate in the response to low-temperature stress, this invention employs virus-induced gene silencing (VIGS) technology. The experimental system includes an empty vector control group (pTRV2::00) and a vector carrier... GhbHLH30 Vector genome of a 300bp specific fragment of the gene (pTRV2::GhbHLH30). Experimental procedure and validation. GhANK-C3H15 The process of gene function is completely consistent.

[0072] The results are as follows Figure 8 As shown, under low temperature stress, GhbHLH30 The wilting was more severe in gene-silenced plants. The gene silencing efficiency detected by qRT-PCR was as follows: Figure 8 As shown in Figure b, compared with the control group, the silent plants had... GhbHLH30 Gene expression levels were significantly downregulated, with the difference reaching a statistically significant level. Furthermore, compared to the control group, the malondialdehyde (MDA) content in silent plant seedlings was significantly increased. Figure 8 (d), while the total antioxidant capacity (T-AOC) decreased significantly ( Figure 8 (e). The above results indicate that silence... GhbHLH30 Genes can reduce a plant's cold resistance.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for regulating the low-temperature stress resistance of cotton GhANK-C3H15 Genes, characterized by, The GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No.

1.

2. As described in claim 1 GhANK-C3H15 Genes, characterized by, The GhANK-C3H15 The CDS sequence of the gene is shown in SEQ ID No.

2.

3. The claim 1 or 2 GhANK-C3H15 Proteins encoded by genes.

4. The claim 1 or 2 GhANK-C3H15 The use of genes, the protein of claim 3, and / or the interacting proteins of the protein in regulating crop resistance to low temperature stress.

5. The application according to claim 4, characterized in that, The interacting proteins include GhbHLH30.

6. The application according to claim 4 or 5, characterized in that, The regulation includes overexpression of the aforementioned GhANK-C3H15 After encoding genes and / or interacting proteins, crop resistance to low temperature stress is improved; The silence GhANK-C3H15 After the genes encoding genes and / or interacting proteins are removed, crop resistance to low temperature stress is reduced; Knock out the GhANK-C3H15 After the genes encoding genes and / or interacting proteins are removed, crop resistance to low temperature stress is reduced.

7. A method for improving crop resistance to low-temperature stress, characterized in that, Including improving the genome of target crops GhANK-C3H15 The expression levels of genes encoding genes and / or interacting proteins, the GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No.

1.

8. The method according to claim 7, characterized in that, The improvement of the target crop genome GhANK-C3H15 Methods for measuring the expression levels of genes encoding genes and / or interacting proteins, including transforming the target cotton using an overexpression vector; The base vector for the overexpression vector includes pCAMBIA3301.

9. The claim 1 or 2 GhANK-C3H15 The use of genes, the protein of claim 3, and / or the interacting proteins of the protein in the breeding of crop varieties or lines with target low-temperature stress resistance.

10. A method for cultivating a cold-resistant crop, characterized in that, Including overexpression in crop genomes GhANK-C3H15 The genes encoding genes and / or interacting proteins, said GhANK-C3H15 The genome sequence of the gene is shown in SEQ ID No. 1.

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