Application of AOP-like gene in improving disease resistance of cotton verticillium wilt

Abstract

The invention discloses an application of an AOP-like gene in improving the disease resistance of cotton verticillium wilt, and belongs to the field of biotechnology application. The AOP-like gene provided by the invention is used for coding AOP protease. The invention provides a full-length ORF nucleotide sequence and an amino acid sequence of AOP-like in a sea island cotton genetic standard line Hai7124 and an upland cotton genetic standard line TM-1. The gene is induced by verticillium dahlia to realize obvious up-regulation expression. Silent AOP-like in cotton enhances the resistance to verticillium wilt. The research of the invention provides a new cotton verticillium wilt resistance thought.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to the AOP-like gene and its application in improving cotton resistance to Verticillium wilt. The gene's ORF is 972 bp in length and encodes an AOP protease, a member of the dioxygenase family, encoding 324 amino acids. Transcriptional profiling analysis revealed that this gene's expression was significantly upregulated after cotton roots were induced by Verticillium wilt. Plant AOP proteins are key enzymes in plant aliphatic glucosinolate metabolism, responsible for the hydroxylation or alkenylation of substrates (methylsulfinyl alkyl glucosides). The abundant and diverse glucosinolate metabolites (thioglucosides) in plants possess defensive activities against lepidopteran insects, herbivores, and fungi. Current research on plant glucosinolate metabolism is concentrated in the Brassicaceae family. This invention reveals a novel disease resistance function involving cotton AOP-like proteins: inhibiting the expression of the AOP-like gene encoding cotton AOP-like proteins enhances cotton resistance to Verticillium wilt. Biotechnology has clarified that this gene has a negative regulatory role in plant resistance to Verticillium wilt, expanding our understanding of the role of AOP-like proteins in plant resistance to Verticillium wilt. Background Technology

[0002] The AOP (Alkenyl hydroxalkyl producing) subfamily is a key enzyme in thioglycoside metabolism, belonging to the dioxygenase (2OGD, 2-oxoglutarate-dependent dioxygenas) superfamily. AOP proteins are characterized by the domains 2OG-FeII_Oxy and DIOX_N. In plants, the 2OGD family is divided into three classes: DOXA, DOXB, and DOXC. The AOP family is structurally characterized as DOXC. In Arabidopsis, the AOP family includes AOP1, AOP2, and AOP3. AOP1 is the ancestral gene, while AOP2 and AOP3 originate from two different gene duplication events. Thioglycoside synthesis involves three steps: main chain elongation, core structure formation, and side chain modification. AOP proteases participate in aliphatic thioglycosides, with methylsulfinyl alkyl glucoside as the substrate. Specifically, AOP2 catalyzes the substrate alkenylation reaction, and AOP3 catalyzes the substrate hydroxylation reaction. In plants, glucosinolates participate in various biological stress processes, including defense against lepidopteran insects, herbivores, and fungi. Currently, there are numerous reports on how the accumulation of AOP protease substrates in cruciferous plants enhances disease resistance. For example, 3-methylsulfinylpropyl glucoside (GIB) in broccoli, Indian mustard, and cabbage can resist white rust (Albugo candida), black mold (Mycosphaerella brassicicola), and black shank (Leptosphaeria maculans); while 4-methylsulfinylbutyl glucoside (GRA) in cabbage can resist black shank.

[0003] Cotton is an important economic crop, providing textile fibers and seed oil. *Verticillium dahliae* Kleb. (V. dahliae) is a soil-borne fungal disease that poses a serious threat to the growth of over 200 plant species, including important economic crops such as rapeseed, tomato, and cotton. The function of the AOP protease in cotton against Verticillium wilt has not been reported. Therefore, the role of AOP proteins in cotton resistance to Verticillium wilt is unclear. In this study, we screened a cotton AOP-like negative regulator of cotton resistance to Verticillium wilt.

[0004] By analyzing the root transcriptome of cotton plants induced by Verticillium wilt, an AOP-like protein, a member of the 2OGD family, was identified. Compared with control plants, the AOP-like silenced group showed enhanced resistance to Verticillium wilt. These results highlight the crucial role of AOP-like proteins in plant resistance to Verticillium wilt and broaden our understanding of their function in cotton resistance to the disease. Summary of the Invention

[0005] The purpose of this invention is to provide a cotton AOP (AOP-like) gene and its application in improving cotton Verticillium wilt resistance and breeding new germplasm with improved Verticillium wilt resistance. The full-length cDNA ORF nucleotide sequence and the amino acid sequence of the encoded protein of this gene in Sea Island cotton (Hai7124) and Upland cotton (TM-1) are provided. Using this gene as a target gene, AOP-like gene expression was suppressed by virus-induced gene silencing (VIGS) to clarify its role in cotton Verticillium wilt resistance and to cultivate new germplasm for production application. Furthermore, molecular biology techniques revealed that the AOP-like gene is positively regulated by the transcription factor MYB15, thereby conferring cotton Verticillium wilt resistance.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention seeks protection for the use of an AOP-like gene with a nucleotide sequence such as SEQ ID NO:1 or SEQ ID NO:2 in at least one of the following (a1)-(a3):

[0008] (a1) Improve cotton's resistance to Verticillium wilt;

[0009] (a2) Prepare products to improve cotton resistance to Verticillium wilt;

[0010] (a3) Develop new cotton germplasm with improved resistance to Verticillium wilt.

[0011] Secondly, the present invention seeks protection for the use of biological materials associated with AOP-like genes with nucleotide sequences such as SEQ ID NO:1 or SEQ ID NO:2 in at least one of the following (a1)-(a3):

[0012] (a1) Improve cotton's resistance to Verticillium wilt;

[0013] (a2) Prepare products to improve cotton resistance to Verticillium wilt;

[0014] (a3) Develop new cotton germplasm with improved resistance to Verticillium wilt;

[0015] The biomaterials associated with AOP-like genes are the biomaterials described in (b1) or (b2) below:

[0016] (b1) The protein AOP-like encoded by the AOP-like gene;

[0017] (b2) Biological materials for silencing, interfering with, or inhibiting the AOP-like genes;

[0018] The biological material used for silencing, interfering with, or inhibiting the AOP-like gene is at least one of the following (c1) to (c10):

[0019] (c1) Nucleic acid molecules used to silence, interfere with, or inhibit the expression of said AOP-like genes;

[0020] (c2) Primers used to amplify the nucleic acid molecule described in (c1);

[0021] (c3) An expression cassette containing the nucleic acid molecule described in (c1);

[0022] (c4) A recombinant vector containing the nucleic acid molecule described in (c1), or a recombinant vector containing the expression cassette described in (c3);

[0023] (c5) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c3), or a recombinant microorganism containing the recombinant vector described in (c4);

[0024] (c6) A transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c3), or a transgenic plant cell line containing the recombinant vector described in (c4);

[0025] (c7) A transgenic plant tissue containing the nucleic acid molecule described in (c1), or a transgenic plant tissue containing the expression cassette described in (c3), or a transgenic plant tissue containing the recombinant vector described in (c4);

[0026] (c8) A transgenic plant organ containing the nucleic acid molecule described in (c1), or a transgenic plant organ containing the expression cassette described in (c3), or a transgenic plant organ containing the recombinant vector described in (c4);

[0027] (c9) A transgenic plant containing the nucleic acid molecule described in (c1), or a transgenic plant containing the expression cassette described in (c3), or a transgenic plant containing the recombinant vector described in (c4);

[0028] (c10) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c9);

[0029] (c11) Propagation material of the transgenic plant as described in (c9).

[0030] Furthermore, the above application involves using the AOP-like gene as a target gene and employing genetic engineering methods to silence, inhibit, or interfere with the expression of the AOP-like gene or reduce the activity or content of the AOP-like protein encoded by the AOP-like gene in target cotton, thereby improving cotton resistance to Verticillium wilt or cultivating new cotton germplasm with improved resistance to Verticillium wilt.

[0031] Thirdly, the present invention seeks to protect a method for improving cotton resistance to Verticillium wilt, using an AOP-like gene with a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 as a target gene, and through genetic engineering methods, silencing, inhibiting or interfering with the expression of the AOP-like gene or reducing the activity or content of the AOP-like protein encoded by the AOP-like gene in the target cotton, thereby improving cotton resistance to Verticillium wilt.

[0032] Fourthly, this invention claims protection for a method for cultivating new cotton germplasm with enhanced resistance to Verticillium wilt. The method involves using an AOP-like gene with a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 as a target gene, and through genetic engineering methods, silencing, inhibiting, or interfering with the expression of the AOP-like gene in target cotton to obtain transgenic cotton plants. The transgenic cotton plants are then cultivated to obtain new cotton germplasm with enhanced resistance to Verticillium wilt and applied in production.

[0033] Fifthly, the present invention seeks protection for an AOP-like gene associated with resistance to Verticillium wilt in cotton, the gene having a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0034] In a sixth aspect, the present invention seeks protection for the protein AOP-like encoded by the aforementioned AOP-like gene.

[0035] In a seventh aspect, the present invention seeks protection for biological materials containing the AOP-like gene or for silencing, interfering with or inhibiting the AOP-like gene, wherein the biological material is a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

[0036] In the technical solution of this invention, the protein AOP-like encoded by the AOP-like gene is at least one of the proteins described in (d1)-(d3) below:

[0037] (d1) A protein having the amino acid sequence shown in SEQ ID NO:3;

[0038] (d2) A protein having the amino acid sequence shown in SEQ ID NO:4;

[0039] (d3) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (d1) or (d2).

[0040] This invention clones an AOP protease gene, AOP-like, whose cDNA ORF sequence is shown in SEQ ID NO:1 and SEQ ID NO:2. The protein encoded by this gene has the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4. Studies have found that inhibiting the expression of the AOP-like gene significantly improves the plant's resistance to Verticillium wilt. In a specific embodiment of this invention, using the cotton AOP protease gene AOP-like as the target gene, the expression level of the AOP-like gene in plants is reduced by virus-induced gene silencing (VIGS), significantly improving the plant's resistance to Verticillium wilt. Inhibition of AOP-like gene expression can be used for improving cotton resistance to Verticillium wilt or creating new resistant germplasm in production applications.

[0041] The beneficial effects of this invention are:

[0042] (1) The AOP protease gene AOP-like cloned in this invention has not been studied in cotton before. The AOP-like gene in cotton was identified for the first time and its sequence structure, expression pattern and function were analyzed systematically, clarifying the important role of AOP-like in the resistance of cotton plants to Verticillium wilt.

[0043] (2) Tissue expression pattern analysis showed that AOP-like genes were predominantly expressed only in root tissues. Transcriptional profiling analysis showed that the expression level of this gene was significantly upregulated at 6 h and 12 h after induction of Verticillium wilt in both the sea island cotton and upland cotton genetic standard lines Hai7124 and TM-1 compared with before induction (0 h). This result indicates that the gene is closely related to Verticillium wilt resistance.

[0044] (3) Virus-induced gene silencing (VIGS) was used to suppress the expression of this gene, and phenotypic studies were conducted using the standard genetic lines of sea island cotton and upland cotton (G. barbadense L. acc. Hai7124, G. hirsutum L. acc. TM-1) as recipients. Expression analysis of the VIGS materials revealed that, compared with the control, the expression of AOP-like was significantly reduced in the lines with suppressed gene expression. Moreover, plants with suppressed AOP-like expression exhibited a phenotype of increased resistance to Verticillium wilt, indicating that the expression level of this gene affects cotton resistance to Verticillium wilt. This provides new evidence for understanding the function of AOP family members in plant resistance to Verticillium wilt.

[0045] (4) Inhibition of AOP-like expression significantly improved the resistance of cotton to Verticillium wilt. Molecular biological experiments confirmed that AOP-like expression was upregulated by the transcription factor MYB15. Attached Figure Description

[0046] Figure 1 AOP-like feature description;

[0047] The study included: A) Visualization of the three-dimensional structure of cotton AOP-like proteins; B) Phylogenetic analysis of AOP-like genes in the cotton genetic standard lines Hai7124, TM-1, and Arabidopsis thaliana; C) Tissue expression pattern analysis of AOP-like proteins in Hai7124 and TM-1; D) Expression pattern analysis of cotton AOP-like proteins induced by *Verticillium dahliae*. Error bars represent standard deviation (SD). p-values ​​were determined by one-way ANOVA; p < 0.05 indicated significant difference, while p > 0.05 indicated no significant difference, expressed in ns; E) Subcellular localization of AOP-like proteins in tobacco epidermal cells, with overlapping green and red fluorescence positions indicating that green fluorescence is localized in chloroplasts. Red fluorescence is chloroplast autofluorescence.

[0048] Figure 2 AOP-like silencing can enhance the resistance of upland cotton (TM-1) to Verticillium wilt;

[0049] TRV is a vector for Tobacco Rattle Virus (TRV), comprising pTRV1 and pTRV2. pTRV1 is an auxiliary vector. The CLA (cloroplastos alterados 1) gene encodes 1-deoxyxylulose 5-phosphate synthase, involved in chloroplast development; pTRV is a publicly available vector. pTRV:CLA served as a positive control. After CLA-silenced plants developed an albino phenotype, the silencing efficiency of AOP-like genes was assessed. Specifically, A, RT-qPCR was used to detect the expression levels of AOP-like genes in AOP-like silenced plants and control plants. B, at 11, 15, 20, and 25 days after inoculation with Verticillium wilt, AOP-like silenced plants showed significantly enhanced disease resistance compared to control plants. Error bars represent standard deviation (SD). The p-value was determined by the t-test. A p-value was considered statistically significant (p < 0.05, significant; p < 0.01, highly significant). C. Disease phenotypes of AOP-like silent plants and control plants 15 and 20 days after inoculation with Verticillium wilt. D. Stem obliquely cut 11 days after inoculation with Verticillium wilt; AOP-like silent plants showed less browning than control plants.

[0050] Figure 3 AOP-like silencing can enhance the resistance of Sea Island cotton (Hai7124) to Verticillium wilt;

[0051] In this study, A) RT-qPCR was used to detect the expression level of the AOP-like gene in AOP-like silent plants and control plants. B) At 11, 15, 20, 25, and 30 days after inoculation with Verticillium wilt, AOP-like silent plants showed significantly enhanced disease resistance compared to control plants. Error bars represent standard deviation (SD). The p-value was determined by the t-test. A p-value was considered statistically significant (p < 0.05, significant; p < 0.01, highly significant). C) Disease phenotypes of AOP-like silent plants and control plants 20 and 30 days after inoculation with Verticillium wilt. D) Stem oblique sections were cut 15 days after inoculation with Verticillium wilt; AOP-like silent plants showed less browning than control plants.

[0052] Figure 4 MYB15 transcription factor positively regulates AOP-like expression;

[0053] In section A, the binding motif of MYB15 in the AOP-like promoter is located at -1112 to -1132 bp upstream of the ATG in GhAOP-like and at -1102 to -1122 bp upstream of the ATG in GbAOP-like. Section B analyzes the expression pattern of MYB15 in Hai7124 and TM-1 after induction by *V. dahliae*. Error bars represent standard deviation (SD). p-values ​​were determined by one-way ANOVA; p < 0.05 indicated significant difference, and p > 0.05 indicated no significant difference, expressed in ns. Sections C and D show the yeast one-hybrid assay (C), where the minimum concentration of basidiomycin inhibiting the growth of pEmpty-AbAi and pAOP-like_motif-AbAi bait bacteria was 300 ng / mL. Furthermore, pGADT7-MYB15 transformed into pAOP-like_motif-AbAi and inoculated onto SD / -Leu / -Ura AbA (300 ng / mL) cultured at 30℃ for 3-5 days showed normal growth. In a dual-luciferase reporter assay (D), the fluorescence signal of tobacco leaves co-injected with 35S::MYB15 / AOP-like_pro-luc was significantly enhanced compared to 35S::Empty / AOP-like_pro-luc. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0055] Example 1: Planting and Growth Conditions of Plant Materials

[0056] The study used the Verticillium wilt-resistant island cotton genetic standard line (G. barbadense L. acc. Hai7124), the susceptible upland cotton genetic standard line (G. hirsutum L. acc. TM-1), and Nicotiana benthamiana as research subjects, all of which were grown in the growth chamber of Nanjing Agricultural University. Seedlings cultured for one week in a greenhouse (16 h: 8 h, light: dark, 23-25℃) were used for VIGS experiments, and cotton seedlings cultured for three weeks were used for inoculation with Verticillium wilt pathogen.

[0057] The verticillium wilt pathogen strain (V. dahliae, V991) was cultured on potato dextrose agar (PDA) at 25°C for one week, and then placed in Czapek's liquid medium (25°C, 180 rpm) for 3-5 days. Cotton seedlings were inoculated using the root-damage method, with a spore suspension concentration of 1×10⁻⁶. 7 mL -1 .

[0058] Example 2: Transcriptional analysis of cotton AOP-like genes

[0059] Table 1: Primers used for amplification

[0060]

[0061] The published cotton transcription profile reveals that AOP-like genes are expressed only in the roots (e.g. Figure 1 (as shown in C), and AOP-like transcription profiles of root tissues at 6 h, 12 h, 24 h, 48 h and 72 h after inoculation with Verticillium wilt in island cotton materials Hai7124 and upland cotton TM-1 (e.g., AOP-like). Figure 1 (As shown in D in the figure). The results showed that, compared with 0 h, the expression of AOP-like proteins was significantly upregulated at both 6 h and 12 h (as shown in the figure). Figure 1(As shown in D in the table). For subcellular localization, the commercially available pBinGFP vector was selected, with Kpn I and BamH1 restriction enzyme sites. A 35S::AOP-like-GFP vector was constructed using homologous recombination. Amplification primers are shown in Table 1. Red fluorescence indicates chloroplast autofluorescence. Fluorescence signals in tobacco epidermal cells were detected 2-3 days post-injection using a Leica SP8 confocal microscope (Leica, Germany).

[0062] Example 3: Construction of cotton pTRV: AOP-like vector

[0063] TRV is a publicly available vector for the cotton-tobacco brittle virus. Virus-induced gene silencing (VIGS) experiments were conducted in cotton using pTRV1 and pTRV2 vectors. pTRV1 is an auxiliary vector into which a specific AOP-like target fragment (313 bp, as shown in SEQ ID NO:5) was inserted. The restriction enzyme sites were EcoR1 and Xhol. The TRV:AOP-like vector was constructed using homologous recombination, and its primers were designed using Primer 6 software (primer sequences are shown in Table 2). PCR amplification was performed using cDNA from the island cotton material Hai7124 as a template. After amplification, 2 μL of 10 × Loading buffer was added, followed by agarose gel electrophoresis. The target band was then excised and recovered from the gel; specific steps were described in the gel recovery kit instructions. The pTRV2 vector plasmid was digested with enzymes, and then incubated with the target fragment at 37°C for 30 min in a PCR instrument for recombination. Immediately after 30 minutes, remove the sample and place it on ice. Transform the recombinant product into competent *E. coli* cells, plate it, and incubate it upside down at 37°C for 12 hours. Pick single colonies from the plates and place them in 700 μL of liquid LB medium containing the corresponding antibiotic. Incubate at 37°C and 200 rpm for 5-6 hours. Using the bacterial culture as a template, perform PCR amplification with universal primers on the vector. After gel electrophoresis, send positive bacterial cultures to a sequencing company for sequencing. Bacterial cultures with correct sequencing sequences are considered to have successfully constructed the vector. Extract the plasmid, store it at -20°C, and use it for subsequent *Agrobacterium* transformation experiments to finally obtain *Agrobacterium* containing TRV: AOP-like structures.

[0064] Table 2: Primers used for amplification

[0065]

[0066] Example 4: Identification of Verticillium wilt resistance in AOP-like silent plants

[0067] To elucidate the role of AOP-like genes in cotton's resistance to Verticillium wilt, a virus-induced gene silencing (VIGS) assay was used to analyze the function of cotton in resisting Verticillium wilt. Agrobacterium tumefaciens suspensions of pTRV1 and pTRV2, stored at -80°C, were streaked onto plates containing Kan and Rif antibiotics and incubated upside down at 28°C for 2 days. Single colonies from the plates were picked and transferred to 700 μL of liquid LB medium containing the corresponding antibiotics and incubated at 28°C with a shaker at 200 rpm for approximately 24 h. The bacterial suspension was then cultured in 50 mL of liquid LB medium containing Kan and Rif antibiotics and incubated overnight at 28°C with a shaker at 200 rpm for 12 h until OD (dose elapsed). 600 The bacterial cells were collected by centrifugation at 4000 rpm for 10 min to approximately 1.5 μL. The supernatant (LB) was discarded, and 10 mL of the prepared resuspension solution was added to each tube to resuspend the bacteria at the bottom of the tube. After resuspending Agrobacterium, the cells were centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. The Agrobacterium was resuspended again with the resuspension solution, and the OD was adjusted. 600 The concentration was set at 1.2-1.4, and the prepared bacterial solution was then incubated at 28℃ in the dark for 3 hours. Agrobacterium strains containing pTRV1 and pTRV2 vectors were combined in a 1:1 ratio and co-injected into the underside of the cotyledons of cotton seedlings. The injection targets were the cotyledons of 7-day-old cotton seedlings. TRV: CLA1 (Cloroplastos alterados1) served as a positive control. When cotton plants exhibited an albino phenotype after injection of TRV: CLA1 (approximately 2 weeks), the silencing efficiency of AOP-like expression was tested. TRV: 00 served as a negative control. The expression of AOP-like expression in the AOP-like silencing group was significantly lower than that in the control group (e.g., ...). Figure 2 (As shown in A in 3 and A in 3). Disease analysis of cotton seedlings 11-30 days after V991 inoculation showed that the disease leaf rate of AOP-like silent plants was significantly lower than that of control plants, indicating that AOP-like silencing enhances the resistance of cotton to Verticillium wilt (e.g., Figure 2 B and C in Figure 3 (As shown in B and C).

[0068] Example 5: Yeast One-Hybrid Experiment (YIH)

[0069] To further explore the upstream molecular mechanisms regulating AOP-like expression, transcription factor binding site prediction analysis was conducted based on AOP-like promoter sequences. It was found that upstream of the AOP-like start codon, specifically in GhAOP-like sequences from upland cotton TM-1 (-1132 bp ~ -1112 bp) and GbAOP-like sequences from sea island cotton Hai7124 (-1122 bp ~ -1102 bp), both contain the MYB15 transcription factor binding motif AGGCATCCTCAACACCAACAA (e.g., ...). Figure 4 (As shown in A in the figure). The binding ability of MYB15 to the motif was tested using the Y1H technique.

[0070] The experiment used the T4 ligation method to construct the vector pAOP-like_motif-AbAi. The vector pAbAi was linearized with SmaI, and the motif was set with three tandem repeat sequences (primer sequences are shown in SEQ ID NO:6 and SEQ ID NO:7). Simultaneously, GhMYB15 and GbMYB15 showed 100% amino acid sequence homology, and the vector pGADT7-MYB15 (primer sequences are shown in Table 3), i.e., AD-MYB15, was constructed using homologous recombination, with EcoRI and BamHI restriction sites. pAOP-like_motif-AbAi was transformed into yeast Y1H Gold to obtain bait yeast, and the bait yeast was subjected to gold basidiomycin (AbA) assay. r The minimum inhibitory concentration (MIC) test was performed. p53-AbAi was used as a positive control, with a MIC of 300 ng / mL. pAOP-like_motif-AbAi bacteria were inoculated into (SD) / -Ura (AbA) culture medium. r The samples were placed at 30 ℃ for 2–3 days. The results showed that the lowest inhibitory concentration of apobasididin for the bait bacteria pAOP-like_motif-AbAi and pEmpty-AbAi was 300 ng / mL. After transforming pAOP-like_motif-AbAi bait bacteria with AD-MYB15, the samples were plated onto (SD) / -Leu / -Ura AbA (300 ng / mL) and incubated at 30 ℃ for 3–5 days to detect the binding of MYB15 to the target element. pGADT7-53, i.e., AD-53 transformed into p53-AbAi bait bacteria, served as a positive control, while AD-MYB15 transformed into pEmpty-AbAi bait bacteria served as a negative control. The results showed that AD-MYB15 transformed into pAOP-like_motif-AbAi bait bacteria could grow normally on (SD) / -Leu / -Ura AbA (300 ng / mL), while AD-MYB15 transformed into pEmpty-AbAi bait bacteria could not grow on (SD) / -Leu / -Ura AbA (300 ng / mL). Figure 4 As shown in C), this indicates that MYB15 can bind to the motif AGGCATCCTCAACACCAACAA.

[0071] Table 3: Primers required for vector construction

[0072]

[0073] Example 6 Dual-Luciferase Reporter Assay (Dual-LUC)

[0074] The binding affinity of MYB15 to the AOP-like promoter (164 bp, sequence shown in SEQ ID NO:8) was tested using Dual-LUC technology. A 35S::MYB15 vector was constructed using homologous recombination with BamHI and SacI restriction sites. The AOP-like_pro-mini35S-luc vector (AOP-like_pro-luc) was constructed using T4 ligation with KpnI and HindIII restriction sites. The mini35S promoter, CAMV35S, is the smallest promoter driving LUC reporter gene expression. The mini35S sequence was inserted into pGreenII0800-LUC to generate the pGreenII0800-LUC (mini35S) vector with BamHI and HindI restriction sites. Nicotiana benthamiana was transformed with 35S::MYB15 / AOP-like_pro-luc, and Nicotiana benthamiana was co-transformed with 35S::Empty / AOP-like_pro-luc as a negative control. Each experiment was conducted in triplicate. Fluorescence imaging was performed using a Tanon 5200 CCD imaging system. Dual-LUC results showed that tobacco leaves injected with 35S::Empty / AOP-like_pro-luc emitted a very weak fluorescence signal, while tobacco leaves injected with 35S::MYB15 / AOP-like_pro-luc emitted a stronger fluorescence (e.g., ...). Figure 4 As shown in Figure D), this indicates that MYB15 can bind to AOP-like promoters.

[0075] Table 4: Primers required for vector construction

[0076]

[0077] Verticillium wilt poses a significant threat to the global crop industry. In this study, we identified an AOP-like protease; silencing this gene enhances cotton's resistance to Verticillium wilt, thus negatively regulating cotton's resistance to the disease. Figure 2-3 Furthermore, transcriptional regulation is a key level controlling gene expression. Transcriptome-based combined with transcription factor binding prediction analysis showed that the transcription factor MYB15, after being induced by vitamin D, exhibits a co-expression trend with AOP-like genes. Molecular interaction experiments further demonstrated that MYB15 positively regulates AOP-like expression. This invention provides a novel approach to cotton resistance to Verticillium wilt, offering a new perspective on the mechanism of AOP-like enzymes, a key enzyme in cotton glucosinolate metabolism, in resisting Verticillium wilt.

[0078] sequence list

[0079] SEQ ID NO:1 (AOP-like cDNA ORF sequence in Hai7124 sea island cotton)

[0080] ATGGGTGTCAATGCTGAGATTGAGTTTCCAGTCATTCAGTTCCGTTCATCAGATTTGGAGCGAGGGACCGATGGATGGCACTGTTTGTGCAAGAGGGTTCGAGAGGCTTGCGAGACTTTTGGCTGTTTCGAGGTGGTGTACGAAAAGATATCAACAAAAGTTCGAGAAGAGACATTTGGGTTGATGAAAGAACTGGTTGAGCTCCCATTGGAGAGGAAACAGAAGAACGCTAGTCCCATGCCTTACCATGGATGGGTTGGACCATGCAATCAGGTTTCTTTGTTGTATGAAGGCTTCGGACTTGGAGATGCCTCCAACTATGACTCTGTTAAAAGTTTTGCTCAACTTATGTGGCCTGATGGTCACCCACGCTTCTGCAACACTGTACATACCATGGCGACTCAAATAGAGGAGTTGAACAAGTTAATATGGTTAATGTTAATTGATAGCTATGGATTAGGAGAAAAATGGGAGTCAGTGATGATAAACTACAAAACGCTAGTGCGGTTTATGAAATACATGGCCCCTCCACCTGGGGAGTACGAGAGAGGACTCTTTGCTCATACTGATAAACCTGTTAGCACAATCATTTGTGATGATCATGTTTCAGGGCTTGAAATTAAGGTCAATGATGGTCAATGGATCAAGTTGTCTTTATCTCCTTCTTCCTTTTGTTTTGTTGTTGGAGATCCTCTCAAGGCATGGAGTAATGGAAGATTAAAAGCAGTTAATCACAGAGTAATGATGAGTGGAGATAAAGATCGATTTTCTCTAGCAGCCTTTGCCATTCCAGTTGAGGGCACCATAATCAAGGCACCCGAAGAGCTTATAGATGAGCAGCATCCTCAGCTTTACAAGGATTTTGATTTCATGGACTTCTTCCTGTTTGCCTTCTCTGACCCAGCAAAGCACATTGACTCCGGCGAGCAGCTCCAAGCCTATGCTTCTCTCTCACCACCGATTTCTCATTGA

[0081] SEQ ID NO:2 (AOP-like cDNA ORF sequence in upland cotton TM-1)

[0082] ATGGGTGTCAATGCTGAGATTGAGTTTCCAGTCATTCAGTTCCGTTCATCAGATTTGGAGCGAGGGACCGATGGATGGCGCTGTTTGTGCAAGAGGGTTCGAGAGGCTTGCGAGACTTTTGGCTGTTTCGAGGTGGTGTACGAAAAGATATCAACAAAAGTTCGAGAAGAGACATTTGGGTTGATGAAAGAACTGGTTGAGGTCCCATTGGAGAGGAAACAGAAGAACGCTAGTCCCATGCCTTACCATGGATGGGTTGGACCATGCAATCAGGTTTCTTTGTTGTATGAAGGCTTCGGACTTGGAGATGCCTCCAACTATGACTCTGTTAAAAGTTTTGCTCAACTTATGTGGCCTGATGGTCACCCACGCTTCTGCAACACTGTACATACCATGGCGACTCAAATAGAGGAGTTGAACAAGTTAATATGGTTAATGTTAATTGATAGCTATGGATTAGGAGAAAAATGGGAGTCAGTGATGATAAACTACAAAACGCTAGTGCGGTTTATGAAATACATGGCCCCTCCACCTGGGGAGTACGAGAGAGGACTCTTTGCTCATACTGATAAACCTGTTAGCACAATCATTTGTGATGATCATGTTTCAGGGCTTGAAATTGAGGTCAATGATGGTCAATGGATCAAGTTGTCTTTATCTCCTTCTTCCTTTTGTTTTGTTGTTGGAGATCCTCTCAAGGCATGGAGTAATGGAAGATTAAAAGCAGTTAATCACAGAGTAATGATGAGTGGAAATAAAGATCGATTTTCTCTAGCAGCCTTTGCCATTCCAGTTGAGGGCACCATAATCAAGGCACCCGAAGAGCTTATAGATGAGAAGCATCCTCAGCTTTACAAGGATTTTGATTTCATGGACTTCTTCCTGTTTGCCTTCTCTGACCCAGCAAAGCACATTGACTCCGGCGAGCAGCTCCAAGCCTATGCTTCTCTCTCACCACCGATTTCTCATTGA

[0083] SEQ ID NO:3 (Amino acid sequence of AOP-like in Gossypium barbadense Hai7124)

[0084] MGVNAEIEFPVIQFRSSDLERGTDGWHCLCKRVREACETFGCFEVVYEKISTKVREETFGLMKELVELPLERKQKNASPMPYHGWVGPCNQVSLLYEGFGLGDASNYDSVKSFAQLMWPDGHPRFCNTVHTMATQIEELNKLIWLMLIDSYGLGEKWESVMINYKTLVRFMKYMAPPPGEYERGLFAHTDKPVSTIICDDHVSGLEIKVNDGQWIKLSLSPSSFCFVVGDPLKAWSNGRLKAVNHRVMMSGDKDRFSLAAFAIPVEGTIIKAPEELIDEQHPQLYKDFDFMDFFLFAFSDPAKHIDSGEQLQAYASLSPPISH

[0085] SEQ ID NO:4 (Amino acid sequence of AOP-like in Gossypium hirsutum TM-1)

[0086] MGVNAEIEFPVIQFRSSDLERGTDGWRCLCKRVREACETFGCFEVVYEKISTKVREETFGLMKELVEVPLERKQKNASPMPYHGWVGPCNQVSLLYEGFGLGDASNYDSVKSFAQLMWPDGHPRFCNTVHTMATQIEELNKLIWLMLIDSYGLGEKWESVMINYKTLVRFMKYMAPPPGEYERGLFAHTDKPVSTIICDDHVSGLEIEVNDGQWIKLSLSPSSFCFVVGDPLKAWSNGRLKAVNHRVMMSGNKDRFSLAAFAIPVEGTIIKAPEELIDEKHPQLYKDFDFMDFFLFAFSDPAKHIDSGEQLQAYASLSPPISH。

[0087] SEQ ID NO:5 (Target fragment sequence):

[0088] GGGACCGATGGATGGCACTGTTTGTGCAAGAGGGTTCGAGAGGCTTGCGAGACTTTTGGCTGTTTCGAGGTGGTGTACGAAAAGATATCAACAAAAGTTCGAGAAGAGACATTTGGGTTGATGAAAGAACTGGTTGAGCTCCCATTGGAGAGGAAACAGAAGAACGCTAGTCCCATGCCTTACCATGGATGGGTTGGACCATGCAATCAGGTTTCTTTGTTGTATGAAGGCTTCGGACTTGGAGATGCCTCCAACTATGACTCTGTTAAAAGTTTTGCTCAACTTATGTGGCCTGATGGTCACCCACGCTTCT

[0089] SEQ ID NO:6 (Upstream primer sequence for constructing pAOP-like_motif-AbAi vector)

[0090] agcttATAGGCGAATAAAACGAAGAAAAACAAAAGTCAATAGGGGCCAAACAAAGCAGGCATCCTCAACACCAACAAGAGTCAATCCAACATCACAGTACACGCCACTAAATAAGACATAAAATCAGggtac

[0091] SEQ ID NO:7 (Downstream primer sequence for constructing pAOP-like_motif-AbAi vector)

[0092] cCTGATTTTATGTCTTATTTAGTGGCGTGTACTGTGATGTTGGATTGACTCTTGTTGGTGTTGAGGATGCCTGCTTTGTTTGGCCCCTATTGACTTTTGTTTTTCTTCGTTTTATTCGCCTATaSEQ ID NO:8 (AOP-like_pro (164 bp))

[0093] GAAAGATAAATAGGCGAATAAAACGAAGAAAAACAAAAGTCAATAGGGGCCAAACAAAGCAGGCATCCTCAACACCAACAAGAGTCAATCCAACATCACAGTACACGCCACTAAATAAGACATAAAATCAGACATGAACGACAGAAAACAATTACAGATATTTA。

Claims

1. The AOP-like gene with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2 is used in at least one of the following (a1)-(a3): (a1) Improve cotton's resistance to Verticillium wilt; (a2) Prepare products to improve cotton resistance to Verticillium wilt; (a3) Develop new cotton germplasm with improved resistance to Verticillium wilt.

2. Application of biological materials associated with AOP-like genes with nucleotide sequences as shown in SEQ ID NO:1 or SEQ ID NO:2 in at least one of the following (a1)-(a3): (a1) Improve cotton's resistance to Verticillium wilt; (a2) Prepare products to improve cotton resistance to Verticillium wilt; (a3) Develop new cotton germplasm with improved resistance to Verticillium wilt; The biomaterials associated with AOP-like genes are the biomaterials described in (b1) or (b2) below: (b1) The protein AOP-like encoded by the AOP-like gene; (b2) Biological materials for silencing, interfering with, or inhibiting the AOP-like genes; The biological material used for silencing, interfering with, or inhibiting the AOP-like gene is at least one of the following (c1) to (c10): (c1) Nucleic acid molecules used to silence, interfere with, or inhibit the expression of said AOP-like genes; (c2) Primers used to amplify the nucleic acid molecule described in (c1); (c3) An expression cassette containing the nucleic acid molecule described in (c1); (c4) A recombinant vector containing the nucleic acid molecule described in (c1), or a recombinant vector containing the expression cassette described in (c3); (c5) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c3), or a recombinant microorganism containing the recombinant vector described in (c4); (c6) A transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c3), or a transgenic plant cell line containing the recombinant vector described in (c4); (c7) A transgenic plant tissue containing the nucleic acid molecule described in (c1), or a transgenic plant tissue containing the expression cassette described in (c3), or a transgenic plant tissue containing the recombinant vector described in (c4); (c8) A transgenic plant organ containing the nucleic acid molecule described in (c1), or a transgenic plant organ containing the expression cassette described in (c3), or a transgenic plant organ containing the recombinant vector described in (c4); (c9) A transgenic plant containing the nucleic acid molecule described in (c1), or a transgenic plant containing the expression cassette described in (c3), or a transgenic plant containing the recombinant vector described in (c4); (c10) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c9); (c11) Propagation material of the transgenic plant as described in (c9).

3. Use according to claim 2, characterized in that, The AOP-like protein encoded by the AOP-like gene described in (b1) is at least one of the proteins described in (d1)-(d3) below: (d1) a protein having an amino acid sequence as shown in SEQ ID NO: 3; (d2) a protein having an amino acid sequence as shown in SEQ ID NO: 4; (d3) a fusion protein having the same function obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein as described in (d1) or (d2).

4. Use according to claim 1 or 2, characterized in that, With the AOP-like gene as the target gene, the expression of the AOP-like gene is silenced, inhibited or interfered, or the activity or content of the AOP-like protein encoded by the AOP-like gene is reduced in the target cotton through a genetic engineering method, so as to improve the Verticillium wilt resistance of cotton or cultivate new cotton germplasm with improved Verticillium wilt resistance.

5. A method of increasing resistance to Verticillium wilt in cotton plants, comprising introducing into a cotton plant a nucleic acid molecule encoding a polypeptide having the amino acid sequence of SEQ ID NO:

2. With the AOP-like gene as the target gene, the expression of the AOP-like gene is silenced, inhibited or interfered, or the activity or content of the AOP-like protein encoded by the AOP-like gene is reduced in the target cotton through a genetic engineering method, so as to improve the Verticillium wilt resistance of cotton.

6. The method of claim 5, wherein, The AOP-like protein encoded by the AOP-like gene is at least one of the following (d1)-(d3): (d1) a protein having an amino acid sequence as shown in SEQ ID NO: 3; (d2) a protein having an amino acid sequence as shown in SEQ ID NO: 4; (d3) a fusion protein having the same function obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein as described in (d1) or (d2).

7. A method of breeding a new germplasm of cotton with improved Verticillium wilt resistance, characterized in that, With the AOP-like gene as the target gene, the expression of the AOP-like gene is silenced, inhibited or interfered, or the activity or content of the AOP-like protein encoded by the AOP-like gene is reduced in the target cotton through a genetic engineering method, so as to improve the Verticillium wilt resistance of cotton or cultivate new cotton germplasm with improved Verticillium wilt resistance.

8. An AOP-like gene related to the Verticillium wilt resistance of cotton, which has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

9. Protein AOP-like encoded by the AOP-like gene according to claim 8, characterized in that, The AOP-like protein encoded by the AOP-like gene is at least one of the following (d1)-(d3): (d1) a protein having an amino acid sequence as shown in SEQ ID NO: 3; (d2) a protein having an amino acid sequence as shown in SEQ ID NO: 4; (d3) a fusion protein having the same function obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein as described in (d1) or (d2).

10. Biomaterials containing the AOP-like gene of claim 8 or biomaterials for silencing, interfering or inhibiting the AOP-like gene of claim 8, characterized in that, The biological material is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

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