Mechanism and application of gossypium hirsutum ghEIL7 gene mediated by ethylene and jasmonic acid double-pathway network synergistically regulating fusarium wilt resistance

Abstract

The present application relates to the field of plant genetic engineering, and specifically provides a mechanism for mediating the resistance of verticillium wilt by the GhEIL7 gene of Gossypium hirsutum through the synergistic regulation of the ethylene and jasmonic acid dual-pathway network, and application thereof. The present application significantly reduces the resistance of cotton to L. theoma through VIGS silencing of GhEIL7; and significantly enhances the resistance to verticillium wilt in Arabidopsis thaliana through heterologous overexpression of GhEIL7. Mechanism research shows that GhEIL7 directly binds to the promoter of the downstream target gene GhGELPs and activates its transcription, and GhGELPs is involved in the jasmonic acid synthesis pathway; at the same time, GhEIL7 activates the key gene GhERF1 of the ethylene signaling pathway, and then induces the expression of GhPDF1.2. GhEIL7 enhances the resistance of cotton to verticillium wilt by synergistically regulating the ethylene pathway defense response and jasmonic acid biosynthesis, forming a dual-branch cross network. The present application provides a key gene resource for molecular breeding against verticillium wilt, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to upland cotton. GhEIL7 Mechanism and application of gene-mediated synergistic regulation of Verticillium wilt resistance by ethylene and jasmonic acid dual pathway networks. Background Technology

[0002] Cotton is an indispensable economic crop globally and a core source of natural fiber. With the continued promotion of large-scale, intensive cotton cultivation, the problem of soil microecological imbalance caused by long-term continuous cropping has become increasingly prominent, leading to a year-on-year increase in the frequency and severity of various soil-borne diseases. Among these, diseases caused by *Verticillium dahliae* (…) are particularly prevalent. Verticillium dahliae Verticillium wilt, caused by infection, has become the "number one killer" restricting the improvement of cotton yield and quality in my country, and is known in the industry as the "cancer" of cotton. Currently, Verticillium wilt is widespread in major cotton-producing areas such as Xinjiang, the Yellow River Basin, and the Yangtze River Basin in my country, and the physiological races of the pathogen are constantly mutating, resulting in a continuous increase in pathogenicity, posing unprecedented challenges to traditional control methods.

[0003] Breeding and promoting cotton varieties highly resistant to Verticillium wilt is the most fundamental, economical, and effective way to control this disease. However, there is currently a lack of stable, high-quality germplasm resources with high resistance to Verticillium wilt in cotton production. Traditional breeding methods suffer from problems such as long cycles, low efficiency, and insufficient precision in targeted improvement, making it difficult to quickly cultivate disease-resistant varieties that meet industry needs. Bio-breeding technology, with molecular design breeding at its core, has become an important direction for enhancing the core competitiveness of the agricultural industry due to its significant advantages in rapidly, accurately, and efficiently improving crop disease resistance, and is also an inevitable trend in the breeding of Verticillium wilt-resistant cotton varieties. Therefore, identifying the key genes regulating Verticillium wilt resistance in cotton, analyzing their molecular mechanisms of resistance, and then using molecular breeding technology to target and improve cotton disease resistance traits is a crucial breakthrough for current innovation in Verticillium wilt-resistant cotton germplasm.

[0004] Ethylene is a core signaling molecule regulating plant growth, development, and stress responses. EIN3 / EIL family transcription factors, as key regulatory nodes in its signaling pathway, play a crucial role in plant responses to biotic and abiotic stresses. Currently, members of the EIN3 / EIL family have been shown to participate in disease resistance regulation in various plants; however, their specific functions in regulating cotton Verticillium wilt resistance, particularly the molecular mechanism by which they mediate resistance responses through the synergistic regulation of the ethylene and jasmonic acid signaling pathways, remain fully elucidated. Summary of the Invention

[0005] The purpose of this invention is to clone upland cotton. GhEIL7The gene was systematically analyzed to elucidate its role in the synergistic regulation of cotton Verticillium wilt resistance through the ethylene-jasmonic acid dual signaling pathway. This provides new key gene resources and theoretical support for molecular breeding of cotton to resist Verticillium wilt, and has significant theoretical value and application prospects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, this invention cloned an EIN3 / EIL transcription factor family gene from upland cotton, named GhEIL7, whose coding region (CDS) is 1845 bp in length and whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] SEQ ID NO. 1: The second aspect of the present invention provides overexpression GhEIL7 Genes enhance Arabidopsis thaliana's resistance to Verticillium dahliae ( Verticillium dahliae Applications of resistance to ).

[0008] Furthermore, the application involves simultaneously activating downstream genes of the ethylene signaling pathway through the GhEIL7 transcription factor. GhERF1, GhPDF1.2 Key genes in the jasmonic acid synthesis pathway GhGELPs It synergistically enhances the resistance of Arabidopsis thaliana to Verticillium dahliae.

[0009] In the ethylene signaling branch, GhEIL7 directly binds to and activates. GhERF1 promoter, GhERF1 Further inducement GhPDF1.2 This expression ultimately enhances the basic resistance of cotton to Verticillium dahliae. In the jasmonic acid synthesis pathway, GhEIL7 is activated. GhGELPs Transcription involves GhGELPs in fatty acid metabolism, promoting the production of α-linolenic acid. α-Linolenic acid is catalyzed by GhLOX and GhAOS1 to generate 12-OPDA, which is then converted into jasmonic acid (JA) and active methyl jasmonic acid (MeJA) via β-oxidation, activating the defense response mediated by the JA signaling pathway.

[0010] The third aspect of the present invention provides overexpression GhEIL7 Applications of gene-related biomaterials, wherein the application is any of the following: A1) Application in enhancing Arabidopsis thaliana's resistance to Verticillium dahliae; A2) Application in the preparation of Arabidopsis thaliana with enhanced resistance to Verticillium dahliae; The biomaterial is any one of B1) to B3) below: B1) An expression cassette containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1; B2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 1; B3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2), wherein the microorganism is Agrobacterium; GhEIL7 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0011] Furthermore, the application involves simultaneously activating downstream genes of the ethylene signaling pathway through the GhEIL7 transcription factor. GhERF1, GhPDF1.2 Key genes in the jasmonic acid synthesis pathway GhGELPs It synergistically enhances the resistance of Arabidopsis thaliana to Verticillium dahliae.

[0012] A fourth aspect of the present invention provides a method for cultivating Arabidopsis thaliana with enhanced resistance to Verticillium dahliae, the method comprising overexpressing in Arabidopsis thaliana... GhEIL7 Genes were used to obtain Arabidopsis thaliana with enhanced resistance to Verticillium dahliae.

[0013] In the method described, overexpression is performed in Arabidopsis thaliana. GhEIL7 The gene was created using transgenic technology in Arabidopsis thaliana. GhEIL7 Gene expression levels increase.

[0014] In the method described above, transgenic technology is used to induce transgenic cells in Arabidopsis thaliana. GhEIL7 The increased gene expression was achieved by introducing a plant expression vector into Arabidopsis thaliana that integrates the nucleic acid molecule shown in SEQ ID NO. 1.

[0015] The beneficial effects of this invention are: (1) The first cloning and functional identification of cotton GhEIL7 The gene was confirmed to positively regulate plant resistance to Verticillium dahliae, providing a key gene resource for molecular breeding of cotton wilt resistance.

[0016] (2) First revealed GhEIL7 The disease resistance mechanism, which forms a dual-branch synergistic network by simultaneously activating defense genes in the ethylene signaling pathway and key genes in the jasmonic acid synthesis pathway, enriches the theory of cross-regulation of plant hormones in disease resistance and provides a new strategy for multi-pathway synergistic breeding.

[0017] (3) The heterologous overexpression experiment verified that GhEIL7 The cross-species conservation of the function indicates that this gene can be applied to resistance improvement in other plants (such as Arabidopsis thaliana), demonstrating its broad applicability.

[0018] (4) Provides containing GhEIL7 Gene expression cassettes, recombinant vectors, and recombinant microorganisms, as well as methods for cultivating disease-resistant plants, have the potential to be directly transformed into breeding tools and can be used to create new cotton germplasm resistant to Verticillium wilt or disease-resistant varieties of other crops. Attached Figure Description

[0019] Figure 1 for GhEIL7 Gene and protein structure. (A) Chromosomal location of GhEIL7 gene; (B) Gene structure; (C) Spatial topological characteristics of GhEIL7 protein; (D) Ramachandran diagram analysis of GhEIL7 protein structure prediction.

[0020] Figure 2 Phylogenetic tree of GhEIL7 protein.

[0021] Figure 3 Subcellular localization of GhEIL7 protein.

[0022] Figure 4 for GhEIL7 The transcriptional expression pattern of genes. (A) GhEIL7 Transcriptional expression patterns of genes in different tissues; (B) GhEIL7 Transcriptional expression patterns of genes during early ovule development (day 0, flowering day; -3 days, -1 day, 0 days, 1 day, 3 days); (C) GhEIL7 Transcriptional expression patterns of genes during ovule development (5, 15, 20, and 25 days after flowering); (D) GhEIL7 Transcriptional expression patterns of genes during fiber development (5, 15, 20, and 25 days after flowering); (E) GhEIL7 Transcriptional expression patterns of genes during seed germination; (F) GhEIL7 Transcriptional expression patterns of genes during abiotic stress processes; (G) GhEIL7 Transcriptional expression patterns of genes during Verticillium dahliae stress; (H)RT-qPCR verification GhEIL7 Transcriptional expression patterns of genes during Verticillium dahliae stress.

[0023] Figure 5 To verify the interaction between the GhEIL7 transcription factor and the target gene promoter. (A) Molecular interaction docking between GhEIL7 transcription factor and the GhGELPs gene promoter binding site; (B) Yeast single-hybrid verification of the interaction between GhEIL7 transcription factor and the GhGELPs gene promoter; (C) Phenotypic of tobacco dual-luciferase experiment with the interaction between GhEIL7 transcription factor and the GhGELPs gene promoter; (D) Fluorescence values ​​of tobacco dual-luciferase experiment with the interaction between GhEIL7 transcription factor and the GhGELPs gene promoter, where I is PGreenII 62-SK + pGreenII-0800LUC; II is PGreenII 62-SK-GhGELP20D + pGreenII-0800LUC; III is PGreenII 62-SK + pGreenII-0800LUC-GhEIL7; IV is PGreenII 62-SK-GhGELP20D + pGreenII-0800LUC-GhEIL7.

[0024] Figure 6The effect of GhEIL7 gene silencing on cotton Verticillium wilt resistance. (A) Phenotypic and stem vascular bundle browning of pTRV2::00 control and pTRV2::GhEIL7 silenced plants 20 days after inoculation with Verticillium dahliae (Vd991). (B) VIGS silencing efficiency detection: qPCR analysis of GhEIL7 gene expression level in pTRV2::GhEIL7 silenced plants. (C) Disease index statistics of pTRV2::00 and pTRV2::GhEIL7 plants after inoculation with Vd991. (D) qPCR analysis of Vd991 biomass level in the two groups of plants. (E~I) Expression levels of Verticillium wilt defense-related genes GhERF1, GhPDF1.2, GhLOX, GhAOS1 and GhGELPs in different treatments. Data are the mean ± standard error of three biological replicates, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA).

[0025] Figure 7 To enhance the resistance of Arabidopsis thaliana to Verticillium dahliae by overexpression of the GhEIL7 gene. (A) GhEIL7 PCR identification of Arabidopsis thaliana overexpression lines. M: DNA marker; WT: wild type; Mock: blank control; 1–5: independent GhEIL7-OE lines. (B) qPCR detection of GhEIL7 expression levels in different GhEIL7-OE lines, with the highest expression level in line 3 (GhEIL7-3-OE) selected for subsequent experiments. (C) Disease symptoms in WT and GhEIL7-3-OE plants 20 days after inoculation with Verticillium dahliae (V991). Mock is the uninoculated control. (D) Statistical analysis of disease index in WT and GhEIL7-3-OE plants after inoculation. (E) qPCR analysis of pathogen biomass levels in plants. (F~J) Expression levels of defense-related genes AtERF1, AtPDF1.2, AtLOX, AtAOS1, and AtGELPs in WT and GhEIL7-OE plants after V991 inoculation. Data are the mean ± standard error of three biological replicates, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA).

[0026] Figure 8 A diagram illustrating the mechanism by which the GhEIL7 transcription factor regulates cotton resistance to Verticillium wilt. Detailed Implementation

[0027] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0028] Example 1: Upland Cotton GhEIL7 Gene cloning and functional identification 1. Materials and Methods 1.1 Experimental Materials and Strains Plant materials: Nicotiana benthamiana, upland cotton Zhongmian 49, and Arabidopsis thaliana ecotype from Colombia.

[0029] Strain: Verticillium dahliae ( Verticillium dahliae The highly pathogenic bacterial strain Vd991, Escherichia coli DH5α strain, Agrobacterium GV3101 competent cells, and yeast EGY48 were all provided by our laboratory.

[0030] Vectors: pCAMBIA2300-eGFP, pCAMBIA2300-mCherry, pTRV1, pTRV2, pCAMBIA1300-eGFP, pJG4-5, pLacZi, pGreenII 0800-LUC, pGreenII 62-SK.

[0031] 1.2 Test Reagents Nucleic acid extraction kits: RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit and rapid plasmid mini-preparation kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Nucleic acid synthesis and dyes: nucleic acid dyes and cDNA first-strand synthesis kit were purchased from Beijing Dingguo Biotechnology Co., Ltd.; PCR and real-time PCR reagents: DNA Mark, 2×PhantaMaster Mix (Dye Plus), and 2×Cham Q Universal SYBR qPCR Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Restriction endonucleases were purchased from New England Biolabs (NEB).

[0032] 1.3 Test Methods 1.3.1 GhEIL7 Gene cloning and vector construction Total RNA was extracted from cotton leaves according to the instructions of the polysaccharide and polyphenol plant total RNA extraction kit, and the extracted RNA was stored at -80℃. Using the extracted total RNA as a template, cDNA was obtained by reverse transcription. Primers for the target gene fragment were designed using Primer Premier 6 software (Table 1). PCR amplification was performed using the aforementioned primers with the cDNA first strand as a template. Different expression vectors were combined with the PCR-amplified and recovered target fragment, digested with restriction endonucleases, purified by gel electrophoresis, and then ligated. The ligation product was transformed into competent *E. coli* DH5α cells, plated on antibiotic-resistant plates, and positive single colonies were picked. After verification by bacterial PCR, the colonies were sent to a sequencing company for sequencing identification.

[0033]

[0034] 1.3.2 Physicochemical properties and structural analysis of GhEIL7 protein First, those that have been successfully sequenced GhEIL7 The full-length gene sequence was translated into an amino acid sequence, and then a series of analyses were performed using the following online tools: the ExPASy ProtParam online tool (https: / / web.expasy.org / protparam / ) was used to predict the protein's amino acid composition, molecular weight, and total average hydrophilicity and other physicochemical properties; the AlphaFold protein structure database (https: / / alphafold.ebi.ac.uk / ) and the SWISS-MODEL online tool and database (https: / / swissmodel.expasy.org / ) were combined to predict and model the protein's three-dimensional spatial structure.

[0035] 1.3.3 Phylogenetic analysis of GhEIL7 protein Using MEGA-X software, the ClustalW tool was called to perform sequence alignment (parameter settings: Gap opening penalty=10, Gap extension penalty=0.2). After alignment, a phylogenetic tree was constructed using the neighbor-joining (NJ) method, with the Poisson correction model selected as the distance model. 1000 bootstrap tests were set to evaluate the reliability of the tree, and other parameters were set to the software default values.

[0036] 1.3.4 Subcellular localization of GhEIL7 protein Will GhEIL7The gene CDS sequence was cloned into the expression vector pCAMBIA2300-eGFP to construct the pCAMBIA2300-GhEIL7-eGFP fusion expression vector. Agrobacterium-containing bacterial suspension (GV3101) of pCAMBIA2300-GhEIL7-eGFP and Agrobacterium-containing nuclear localization marker vector pCAMBIA2300-mCherry were mixed at a 1:1 ratio and injected into leaves of 3-4 week-old *Nicotiana benthamiana* for transient expression. Fluorescence signals were observed using a laser confocal microscopy system (Zeiss LSM900) 36-48 h after infection.

[0037] 1.3.5 Molecular docking analysis of downstream target genes of GhEIL7 Using a three-dimensional structural model of the GhEIL7 protein amino acid sequence, EIN3 / EIL family transcription factor binding motifs were selected from the JASPAR database (https: / / jaspar.elixir.no / ) for analysis. GhGELPs Transcription factor binding sites were predicted from the promoter sequence 2000 bp upstream of the gene transcription start site, yielding the high-confidence binding site TAGATTCATTT. The corresponding DNA double-stranded 3D structure was then constructed using the online Sequence to Structure tool (https: / / scfbio-iitd.res.in / software / drugdesign / bdna.jsp). The GhEIL7 protein model and the DNA sequence containing this site were submitted to HDOCK SERVER (http: / / hdock.phys.hust.edu.cn / ) for protein-nucleic acid molecular docking. The conformation with the best docking score was selected as the final interaction model. The complex structure was visualized and analyzed using PyMOL 2.5 software.

[0038] 1.3.6 Yeast single-hybrid spot-to-spot experiment (Y1H) Will GhEIL7 The CDS sequence of the gene was cloned into the yeast expression vector pJG4-5 to construct a prey plasmid. PCR amplification. GhGELPsThe promoter sequence 2000 bp upstream of the ATG gene was cloned into the reporter vector pLacZi to construct the bait plasmid. The linearized bait plasmid pLacZi-GhGELPs was transformed into yeast strain EGY48 using lithium acetate (LiAc) transformation. The transformation products were plated on SD / -Ura-deficient medium for screening, and positive clones were obtained after incubation at 30°C for 2–3 days. Subsequently, the prey plasmid pJG4-5-GhEIL7 and the empty vector pJG4-5 (negative control) were transformed into the above positive yeast strains, respectively. The transformation products were plated on SD / -Trp / -Ura-deficient medium and incubated at 30°C for 2–3 days to obtain transformants. The obtained transformants were inoculated onto X-gal induction medium plates and incubated at 30°C in the dark for 2–3 days, and the colony color development was observed.

[0039] 1.3.7 Dual-luciferase reporter gene assay (DLRA) Will GhGELPs The promoter sequence 2000 bp upstream of the ATG gene was cloned into the pGreenII 0800-LUC vector to construct a reporter plasmid to drive the expression of firefly luciferase (LUC); GhEIL7 The complete CDS sequence was cloned into the pGreenII 62-SK vector to construct the effect plasmid, with the empty vector pGreenII 62-SK serving as a negative control. The reporter plasmid and effect plasmid were mixed at a 1:1 ratio and injected into leaves of *Nicotiana benthamiana* using Agrobacterium GV3101-mediated transient transformation. Four groups were established: pGreenII-0800-LUC+pGreenII 62SK (Ⅰ), pGreenII-0800-LUC+pGreenII 62SK-GhEIL7 (Ⅱ), pGreenII-0800-LUC-GhGELPs+pGreenII 62SK (Ⅲ), and pGreenII-0800LUC-GhGELPs+pGreenII 62SK-GhEIL7 (Ⅳ), with three biological replicates in each group. 48-72 h post-infection, total protein was extracted from tobacco leaves using a dual-luciferase reporter gene assay kit (Beijing Cooler Master Technology Co., Ltd.). The activities of firefly luciferase (LUC) and kidney luciferase (REN) were measured using a chemiluminescence analyzer, and the LUC / REN ratio was calculated. One-way ANOVA and Dunnett's test were performed using GraphPadPrism 9 software for statistical analysis.

[0040] 1.3.8 GhEIL7 Identification of VIGS gene silencing and disease resistance function Silencing cotton using virus-induced gene silencing technology (VIGS) GhEIL7Gene. PCR amplification GhEIL7 A specific fragment was inserted into the pTRV2 vector to construct the silencing vector pTRV2:: GhEIL7 pTRV2:: GhPDS pTRV1::00 served as a positive control, while pTRV2::00 served as a negative control. After transforming Agrobacterium GV3101 with the recombinant vectors pTRV1 and pTRV2, the mixture was administered at a 1:1 volume ratio and then used to infect cotyledonary cotton seedlings via Agrobacterium injection. The pTRV2::00 vector was then used as a negative control. GhPDS After the plants exhibited a distinct albino phenotype, they were detected by RT-qPCR. GhEIL7 The efficiency of silence. The root soaking method was used to immerse the cotton roots in 10... 7 Plants were soaked in a suspension of Verticillium dahliae spores per mL for 5 min, and the disease phenotype was observed after 20 days. The disease index was statistically analyzed according to GB / T22101.5-2009 standard. The relative biomass of Verticillium dahliae in cotton roots was detected by qRT-PCR using Ve-ITS1-F / ST-VE1-R primers.

[0041] 1.3.9 GhEIL7 Heterologous gene expression and identification of disease resistance function Overexpression in Arabidopsis thaliana using heterologous overexpression technology GhEIL7 Genes. GhEIL7 The CDS sequence of the gene was cloned into the pCAMBIA1300-eGFP vector and transformed into Arabidopsis thaliana via Agrobacterium-mediated flower-dip transformation. Hygromycin resistance screening yielded T3 homozygous transgenic lines, and positive lines and their relative expression levels were identified by PCR and RT-qPCR. The root-dip method was used to transform wild-type Arabidopsis thaliana and... GhEIL7 Heterologous overexpression lines were inoculated with *Verticillium dahliae*, and the disease phenotype of the plants was observed and the disease index was calculated after 20 days. The relative biomass of *Verticillium dahliae* in Arabidopsis roots was detected by qRT-PCR using Ve-ITS1-F / ST-VE1-R primers.

[0042] 1.3.10 qRT-PCR detection of relative expression levels Total RNA was extracted from root tissues of upland cotton 'Zhongmian Institute 49' at different time points (0 h, 3 h, 6 h, 12 h) after treatment with *Verticillium dahliae* (V991) using a plant total RNA purification kit. Reverse transcription was performed using the HiScript IV All-in-One Ultra RT SuperMix for qPCR kit to synthesize the first strand of cDNA for real-time quantitative polymerase chain reaction (qRT-PCR). qRT-PCR detection was performed using the SupRealQ Ultra Hunter SYBR qPCR Master Mix (U+) kit. GhEIL7The relative expression of genes under different treatments was studied. The cotton GhUBQ7 gene was used as an internal control gene, and each sample was tested in triplicate. Relative gene expression levels were analyzed using a 23 ratio. -ΔΔCt The data were calculated using a one-way ANOVA method, and then the significance of differences between groups was tested using Dunnett's multiple comparison method.

[0043] 2 Results and Analysis 2.1 Cloning and Vector Construction of GhEIL7 Gene This study used *Gossypium hirsutum* cv. Zhongmian 49 as experimental material and successfully isolated a candidate gene of the EIN3 / EIL family using homologous cloning technology. The coding region (CDS) of this gene is 1845 bp in length. Based on its location on the cotton chromosome, it was named... GhEIL7 (ID: Gohir.A08G196600 () Figure 1 (A). To systematically verify its function, the expression vectors were constructed as follows: ① Subcellular localization vector pCAMBIA2300- GhEIL7 -eGFP; ② Interaction verification vectors pJG4-5-GhEIL7 (yeast one-hybrid) and pGreenII 62-SK-GhEIL7; ③ VIGS silencing vector pTRV2-GhEIL7 (inserting a 350 bp specific fragment); ④ Overexpression vector pCAMBIA2300- GhEIL7 -eGFP. All vectors were verified to be correctly constructed through enzyme digestion and sequencing, and meet the requirements. GhEIL7 Multi-level functional analysis requirements.

[0044] 2.2 Physicochemical properties and structural analysis of GhEIL7 protein Sequence analysis showed that GhEIL7 Located on the antisense strand of chromosome 8 in the A subgenome of upland cotton (Gossypium hirsutum), the gene structure contains two exons and one intron. The 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), and coding region (CDS) are all located within the exon region. The CDS is 1845 bp in length and is located in the second exon. Figure 1 (A, B); its encoded amino acid sequence contains a conserved EIN3-like DNA-binding domain (IPR047091). Physicochemical property analysis showed that the GhEIL7-encoded protein has a relative molecular mass of 69.52 kDa, a theoretical isoelectric point of 5.53, an instability coefficient of 47.75, and an overall average hydrophilicity of -0.707.

[0045] The three-dimensional structure prediction and homology modeling of the GhEIL7 protein were performed using the AlphaFold and SWISS-MODEL databases, respectively. Figure 1 (C, D). The Laplace diagram conformation test results show that in the dihedral distribution of model amino acid residues, the dominant conformation region accounts for 77.58%, the overall spatial conformation of the residues is stable, and the secondary structure arrangement conforms to the conventional rules; the proportion of outlier conformation residues is 12.77%, the proportion of abnormally twisted side chain residues is 4.80%, and a small number of amino acid side chain conformations deviate from the native optimal conformation.

[0046] 2.3 Phylogenetic analysis of GhEIL7 transcription factor To clarify the evolutionary position of GhEIL7, multiple sequence homology alignment combined with Pfam-HMM domain (PF04873) verification was used to systematically identify and obtain all EIN3 / EIL family members in the genomes of allotetraploid cotton (upland cotton, island cotton) and diploid cotton (Asian cotton, Gossypium ramonde), and a phylogenetic tree was constructed based on the neighbor-joining method (NJ method). The results showed that the EIN3 / EIL family members of the genus Gossypium can be divided into three main evolutionary branches (Clade I, Clade II, Clade III), of which Clade II and Clade III have further differentiated into four subclusters (…). Figure 2 This family exhibits significant subtype differentiation during evolution, with subtypes of different sequence characteristics clustering specifically into particular branches or subclusters, suggesting that functional specialization may have occurred during evolution. GhEIL7 clearly clusters into the CladeⅢc subcluster, indicating a closer evolutionary relationship with members of this subcluster.

[0047] 2.4 Subcellular localization analysis of GhEIL7 protein To identify the transcription factor properties of GhEIL7, subcellular localization validation was performed. The recombinant vector pCAMBIA2300- GhEIL7 -eGFP was transiently expressed in tobacco leaves by co-infection with the nuclear localization marker vector pCAMBIA2300-mCherry. Fluorescence observation results showed that ( Figure 3 The green fluorescence of the GhEIL7 fusion protein completely overlapped with the red fluorescence of the cell nucleus, confirming that the protein is located in the cell nucleus and has typical subcellular distribution characteristics of transcription factors.

[0048] 2.5 Analysis of the transcriptional expression pattern of the GhEIL7 gene For system analysis GhEIL7 The spatiotemporal expression characteristics and stress response patterns of this gene were analyzed, and the transcriptional expression patterns of this gene under different tissues, developmental stages, and adverse treatments were investigated. Figure 4 Tissue expression profile analysis results showed that GhEIL7 It exhibits significant expression specificity in different tissues of upland cotton. Figure 4 The gene (A) was most highly expressed in the stem, and showed varying degrees of expression in the roots, floral organs, and calyx, while its expression was lowest in the stipules, suggesting that this gene may play an important regulatory role in cotton stem development. Expression analysis at the ovule and fiber development stages indicated that… GhEIL7 Significant differences were observed at different stages of ovule development. Figure 4 (B, C): In the early stages of ovule development, the expression level of this gene reaches its peak 1 day after flowering; while during ovule development, its expression level is highest at 15 days. During fiber development ( Figure 4 (D) GhEIL7 The expression of [a specific substance] was significantly upregulated at 20 and 25 days, suggesting its potential involvement in the regulation of cotton fiber elongation and secondary cell wall synthesis. During seed germination, [the following text appears to be incomplete and requires further context: "..."] GhEIL7 The expression exhibits significant temporal dynamic changes ( Figure 4 E): The expression level of this gene continuously increases during the seed water absorption stage (0-24 h), reaching a peak at 5 h; with the appearance of cotyledons, its expression level is upregulated again and maintained at a high level, indicating that this gene may be involved in seed germination and early seedling formation.

[0049] Abiotic stress response analysis showed that GhEIL7 The transcriptional level of [something] responds to a variety of adverse signals. Figure 4 (F): Cold, heat, salinity, and PEG-simulated drought treatments all induced upregulation of this gene expression, with the induction effect under heat stress being particularly significant, suggesting... GhEIL7 It may participate in the regulatory network of cotton's response to various abiotic stresses as a stress response factor. Furthermore, the results of Verticillium dahliae inoculation experiments indicate that... GhEIL7 The expression of [something] changes dynamically under pathogenic stress. Figure 4 (G, H): Expression levels were significantly upregulated 3-6 h after inoculation, followed by a decline at 12 h, exhibiting typical early response characteristics, suggesting that this gene may be involved in regulating the early defense response of cotton against Verticillium wilt.

[0050] The above results indicate that GhEIL7 not only participates in the tissue development and seed germination of cotton, but also plays a potential regulatory role in various abiotic stresses and Verticillium wilt biotic stress responses, making it a transcription factor with multiple regulatory functions.

[0051] 2.6 Downstream target gene mining of GhEIL7 To elucidate the regulatory mechanism of the GhEIL7 transcription factor on downstream target genes, a yeast single-hybrid screening library was used to identify downstream target genes and verify their interactions. The results showed that GhEIL7 interacts with GDSL-type esterases / lipases. GhGELPs ( Gohir.D04G101800 The promoter regions of GhEIL7 and GhEIL7 interact. Molecular docking yields GhEIL7 with... GhGELPs The optimal conformation for promoter DNA fragment interaction was found, with a docking score of -251.97 and a confidence score of 0.8849. This indicates that the conformation has a lower binding free energy and higher confidence, therefore it was selected as the final model for subsequent structural analysis. PyMOL visualization results show ( Figure 5 GhEIL7 (A) may recognize and bind through its DNA-binding domain. GhGELPs The EIN3 / EIL binding motif on the promoter and the multiple hydrogen bond interactions between amino acid residues such as ARG87, GLN83, and ARG413 and DNA bases at the interface provide a structural basis for their specific recognition. Yeast one-hybrid experiments showed that ( Figure 5 Yeast strains co-transformed with pLacZi-GELPs and pJG4-5-GhEIL7 vectors (B) showed a distinct blue color on SD / -Trp / -Ura medium, while the empty vector control showed no color development, indicating that GhEIL7 can interact with... GhGELPs Promoter interaction occurs. Dual-luciferase reporter system detection results of transient transformation in tobacco leaves show ( Figure 5 In the tobacco regions co-expressed with pGreenII 62-SK-GhEIL7 and pGreenII-0800LUC-GhGELPs (C, D), the fluorescence signal was significantly stronger than that in the other three control groups, and the luciferase activity was significantly higher than that in the control groups, further demonstrating that GhEIL7 and... GhGELPs Promoter binding involves interaction.

[0052] 2.7 GhEIL7 Gene silencing reduces cotton's resistance to Verticillium dahliae. To investigate the biological function of the GhEIL7 transcription factor in cotton resistance to Verticillium wilt, we used virus-induced gene silencing (VIGS) technology to silence the gene in upland cotton. GhEIL7 The gene was identified, and its disease resistance phenotype was systematically analyzed. The results showed that, compared with the empty vector control (pTRV2::00), pTRV2:: GhEIL7 Silent plants GhEIL7 The transcriptional level was significantly suppressed ( Figure 6 (B) indicates that the VIGS silencing system is effective. Twenty days after inoculation with *Verticillium dahliae* (Vd991), the silenced plants exhibited more severe Verticillium wilt symptoms, with significantly increased leaf yellowing and wilting, and more pronounced browning of the vascular bundles in the stems. Figure 6 (A). Statistical results of the disease index indicate that... GhEIL7 The disease index of silent plants was significantly higher than that of the control group. Figure 6 (C), and pathogen biomass detection also showed that the relative content of Vd991 in silent plants was significantly increased (C). Figure 6 (D), Explanation GhEIL7 The silence resulted in a significant decrease in cotton's resistance to Verticillium wilt.

[0053] To elucidate its disease resistance mechanism, we further analyzed the expression changes of genes related to Verticillium wilt defense. The results showed that in pTRV2:: GhEIL7 In plants, key genes in the ethylene signaling pathway and key genes in the jasmonic acid synthesis pathway GhERF1 , GhPDF1.2 , GhLOX , GhAOS1 and GhGELPs The expression level of was significantly suppressed ( Figure 6 (E, F, G, H, I). The above results indicate that... GhEIL7 Gene silencing significantly weakens cotton’s resistance to Verticillium dahliae. Its loss of function may lead to increased plant susceptibility to pathogens by disrupting the normal expression of key genes in the ethylene signaling pathway and jasmonic acid synthesis pathway.

[0054] 2.8 Overexpression GhEIL7 Gene-enhanced Arabidopsis resistance to Verticillium dahliae To further verify GhEIL7 We constructed the function of genes in Verticillium wilt resistance. GhEIL7 Five independent Arabidopsis thaliana (Col-0) lines were successfully obtained through gene overexpression, identified by PCR and qPCR expression analysis. GhEIL7 -OE positive strains, among which GhEIL7 The -3-OE strain had the highest expression level and was selected as the material for subsequent functional analysis. Figure 7 (A, B)

[0055] Twenty days after inoculation with Verticillium dahliae (V991), compared with WT plants, GhEIL7 -3-OE plants showed a significant reduction in Verticillium wilt symptoms, with a marked improvement in leaf yellowing and wilting. Figure 7 (C). The statistical results of the disease index show that... GhEIL7 -3-OE plants had a significantly lower disease index than WT ( Figure 7 In the D-cell analysis, quantitative analysis of pathogen biomass also confirmed that the proliferation of V991 in the overexpression lines was significantly inhibited. Figure 7 (E), indicating overexpression GhEIL7 It significantly enhanced the resistance of Arabidopsis thaliana to Verticillium wilt.

[0056] To investigate its molecular mechanism, we analyzed the expression changes of the ethylene signaling pathway, the jasmonic acid (JA) synthesis pathway, and defense-related genes. The results showed that... GhEIL7 -OE plants AtERF1 , AtPDF1.2 , AtLOX , AtAOS1 and AtGELPs The expression levels of these substances were significantly induced to be upregulated, and the upregulation was significantly higher than that of WT plants ( Figure 7 (F, G, H, I, J). These results indicate that the GhEIL7 transcription factor can enhance Arabidopsis thaliana's resistance to Verticillium dahliae by positively regulating the ethylene signaling pathway, jasmonic acid synthesis pathway, and the expression of downstream defense genes.

[0057] 2.9 Overexpression of the GhEIL7 gene enhances Arabidopsis resistance to Verticillium dahliae. To elucidate the molecular mechanism by which GhEIL7 regulates resistance to Verticillium wilt in cotton, we constructed a dual-branch regulatory network model mediated by GhEIL7. Figure 8 ). In Verticillium dahliae ( Verticillium dahliae After infecting the vascular tissues of cotton roots, the GhEIL7 transcription factor activates downstream defense responses through two cooperating pathways: In the ethylene signaling pathway, GhEIL7 directly binds GhERF1 Cis-acting elements in the promoter region are significantly activated. GhERF1 Transcription; transcription factor GhERF1 further targets and activates GhPDF1.2 The promoter induces its expression. GhPDF1.2 As a defense-related gene, it ultimately enhances the cotton's basic resistance to Verticillium dahliae.

[0058] In the jasmonic acid synthesis pathway, GhEIL7 activates downstream target genes. GhGELPs Transcriptional expression of lipase genes; GhGELPs lipases participate in fatty acid (FA) metabolism, promoting the production of α-linolenic acid. α-Linolenic acid, as a precursor to jasmonic acid (JA) synthesis, is transported to chloroplasts and then catalyzed by GhLOX and GhAOS1 to generate 12-OPDA. 12-OPDA subsequently enters the peroxisome and is converted to JA via β-oxidation, further methylated to form active methyl jasmonic acid (MeJA), activating the JA signaling pathway-mediated defense response.

[0059] In summary, GhEIL7 enhances cotton's resistance to Verticillium wilt by simultaneously activating two pathways: the ERF1-PDF1.2 defense signaling pathway and the JA biosynthesis pathway, revealing a novel mechanism of cross-regulation between the core transcription factor of ethylene signaling and the jasmonic acid metabolic pathway.

Claims

1. Overexpression GhEIL7 Genes enhance Arabidopsis thaliana's resistance to Verticillium dahliae ( Verticillium dahliae Its application in the resistance of ) is characterized by, GhEIL7 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application utilizes the GhEIL7 transcription factor to simultaneously activate downstream genes in the ethylene signaling pathway. GhERF1, GhPDF1.2 Key genes in the jasmonic acid synthesis pathway GhGELPs It synergistically enhances the resistance of Arabidopsis thaliana to Verticillium dahliae.

3. Overexpression GhEIL7 The application of gene-related biomaterials is characterized by, The application is any one of the following: A1) Application in enhancing Arabidopsis thaliana's resistance to Verticillium dahliae; A2) Application in the preparation of Arabidopsis thaliana with enhanced resistance to Verticillium dahliae; The biomaterial is any one of B1) to B3) below: B1) An expression cassette containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1; B2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 1; B3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2), wherein the microorganism is Agrobacterium; GhEIL7 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The application utilizes the GhEIL7 transcription factor to simultaneously activate downstream genes in the ethylene signaling pathway. GhERF1, GhPDF1.2 Key genes in the jasmonic acid synthesis pathway GhGELPs It synergistically enhances the resistance of Arabidopsis thaliana to Verticillium dahliae.

5. A method for cultivating Arabidopsis thaliana with enhanced resistance to Verticillium dahliae, characterized in that, The method involves overexpression in Arabidopsis thaliana. GhEIL7 Genes were used to obtain Arabidopsis thaliana with enhanced resistance to Verticillium dahliae. GhEIL7 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, The overexpression in Arabidopsis GhEIL7 The gene was created using transgenic technology in Arabidopsis thaliana. GhEIL7 Gene expression levels increase.

7. The method according to claim 6, characterized in that, The use of transgenic technology to induce Arabidopsis thaliana GhEIL7 The increased gene expression was achieved by introducing a plant expression vector into Arabidopsis thaliana that integrates the nucleic acid molecule shown in SEQ ID NO. 1.

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