Application of GhBBX20 gene in improving cotton resistance to verticillium wilt

Abstract

The application belongs to the technical field of biology, and particularly relates to application of GhBBX20 gene in improving cotton resistance to verticillium wilt. The full-length sequence of CDS of the GhBBX20 gene is constructed on a overexpression vector WMV068 to obtain a recombinant overexpression vector WMV068-GhBBX20, which is then transformed into agrobacterium, and cotton is transformed by using a stem tip transformation method to obtain a transgenic cotton material overexpressing the GhBBX20. Protein detection results show that the expression level of the GhBBX20 gene in the transgenic cotton material is significantly increased; metabolic group analysis results show that overexpression of the GhBBX20 can significantly induce synthesis of flavonoids; meanwhile, the transgenic cotton material overexpressing the GhBBX20 shows strong resistance to verticillium wilt. The application provides a new gene resource and breeding material for cotton breeding against verticillium wilt, has important application value for solving the problem of cotton verticillium wilt, and is suitable for promotion and application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the GhBBX20 gene in improving cotton resistance to Verticillium wilt. Background Technology

[0002] By Verticillium dahliae ( Verticilliumdahliae Verticillium wilt, caused by cotton, is known as the "cancer" of cotton and seriously threatens cotton production. Discovering new disease-resistant genes in cotton and using them to create new disease-resistant materials is a pressing problem that needs to be solved in cotton bio-breeding.

[0003] Verticillium dahliae infects the roots, then spreads through the vascular bundles of the stem to the above-ground tissues, ultimately causing leaf wilting and even plant death, severely threatening cotton yield and quality. In the process of plant defense against pathogens, lignin acts as an important physical barrier, effectively preventing the invasion and spread of pathogens. Regulating lignin accumulation helps improve plant disease resistance. Lignin and flavonoids are both key products of the phenylpropanoid metabolic pathway, with flavonoids playing important roles in plant disease defense, including antibacterial, antioxidant, and signal transduction functions. However, how to improve Verticillium wilt resistance by regulating flavonoid synthesis has not yet been reported.

[0004] Therefore, screening regulatory factors that regulate the synthesis pathway of flavonoids and elucidating the mechanism by which flavonoids mediate resistance to Verticillium wilt and promote growth and development will help solve the "cancer" problem in cotton production. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of the GhBBX20 gene in improving cotton resistance to Verticillium wilt.

[0006] To achieve the above objectives, one aspect of the technical solution of the present invention is to provide the application of the GhBBX20 gene in improving the resistance of cotton to Verticillium wilt.

[0007] Furthermore, the GhBBX20 gene was obtained by amplification using cotton cDNA as a template and primers shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] Furthermore, the nucleotide sequence of the GhBBX20 gene is shown in SEQ ID NO.3.

[0009] Furthermore, the amino acid sequence encoded by the GhBBX20 gene is shown in SEQ ID NO.4.

[0010] Furthermore, the GhBBX20 gene was constructed into the WMV068 vector and transformed into cotton to enhance the expression level of the GhBBX20 gene in cotton materials, induce the synthesis of flavonoids, and thus improve the resistance of cotton to Verticillium wilt.

[0011] Furthermore, the WMV068 vector sequence is shown in SEQ ID NO.5.

[0012] Furthermore, the cotton in question is upland cotton.

[0013] On the other hand, the technical solution of the present invention is to provide a recombinant expression vector containing the GhBBX20 gene and genetically engineered bacteria.

[0014] On the other hand, the technical solution of the present invention is to provide a recombinant expression vector containing the GhBBX20 gene and the application of genetically engineered bacteria in improving cotton resistance to Verticillium wilt.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is the first to apply the GhBBX20 gene to the regulation of resistance to Verticillium wilt in cotton. The full-length CDS sequence of the GhBBX20 gene was constructed into the plant overexpression vector WMV068 to obtain the recombinant overexpression vector WMV068-GhBBX20. Cotton was then transformed using Agrobacterium-mediated shoot tip transformation to obtain transgenic cotton material overexpressing GhBBX20. Western blotting protein detection confirmed the successful overexpression of the GhBBX20 protein in the transgenic cotton.

[0016] 2. This invention verifies the application of overexpression of the GhBBX20 gene in improving cotton resistance to Verticillium wilt. The results of Verticillium wilt resistance identification showed that, compared with the wild-type control, the disease index of transgenic cotton plants overexpressing GhBBX20 was significantly reduced and the symptoms of disease were significantly alleviated, indicating that overexpression of GhBBX20 can significantly improve cotton resistance to Verticillium wilt.

[0017] 3. This invention elucidates the mechanism by which the GhBBX20 gene enhances cotton resistance to Verticillium wilt. Metabolomics analysis showed that, compared with the wild-type control, transgenic cotton plants overexpressing GhBBX20 significantly accumulated flavonoids, indicating that the GhBBX20 gene enhances cotton resistance to Verticillium wilt by inducing flavonoid synthesis. This invention provides new theoretical basis for elucidating the molecular mechanism of cotton resistance to Verticillium wilt, and also provides new gene resources and breeding materials for cotton breeding against Verticillium wilt. It has significant application value in solving this "cancer" problem of cotton Verticillium wilt and is suitable for widespread application. Attached Figure Description

[0018] Figure 1This is a map of the plant expression vector WMV068.

[0019] Figure 2 A represents the expression level analysis of the GhBBX20 gene in transgenic cotton overexpressing GhBBX20; Figure 2 B represents the result of the Verticillium wilt resistance assessment.

[0020] Figure 3 Metabolomics analysis for transgenic cotton overexpressing GhBBX20. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] The following embodiments define the present invention and describe how the present invention isolates GhBBX20 from cotton, constructs a vector, creates transgenic cotton that overexpresses GhBBX20 through transgenic technology, and performs metabolomics analysis and Verticillium wilt resistance analysis.

[0023] Example 1: Construction of GhBBX20 overexpression vector Using Primer Premier 5 software, homologous recombination primers were designed based on the CDS sequence of the GhBBX20 gene, and the CDS sequence of this gene was constructed into the WMV068 vector. Figure 1 The primer sequences are as follows: WMV068-GhBBX20-SalI-F: GAGAGAACACGGGGGACgtcgacATGGAACAAAAAAGAAAGATG (SEQ ID NO.1) WMV068-GhBBX20-BamHI-R: GAACATCGTATGGGTACATggatccACGTGCAGCATATGATGAAGG (SEQ ID NO.2) Using cotton cDNA as a template, PCR amplification was performed using the primers described above. The PCR amplification system is shown in Table 1.

[0024] Table 1 PCR amplification system PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 52℃ annealing for 15 s, 68℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 5 min. After PCR amplification, 20 μL of product was analyzed by 1% agarose gel electrophoresis. After confirming the correct fragment size, the target fragment was purified and recovered using a gel extraction kit, following the kit instructions. The WMV068 vector was digested with Sal I and Bam HI restriction endonucleases to obtain a linearized vector. The purified and recovered GhBBX20 gene PCR product was ligated into the WMV068 linearized vector for homologous recombination. The recombination reaction system is shown in Table 2.

[0025] Table 2 Recombination Reaction System After gently mixing the above system, incubate at 37°C for 30 min, then on ice for 5 min to obtain the recombinant product. Transform 10 μL of the recombinant product into *E. coli* DH5α competent cells, plate on LB agar plates containing 50 mg / L kanamycin, and incubate overnight at 37°C. Pick single colonies, extract plasmid, and after PCR and sequencing verification, obtain the recombinant plasmid, named WMV068-GhBBX20.

[0026] In this embodiment, the nucleotide (CDS) sequence of the GhBBX20 gene is shown in SEQ ID NO: 3, and the amino acid sequence it encodes is shown in SEQ ID NO: 4; the WMV068 vector sequence is shown in SEQ ID NO: 5.

[0027] The nucleotide sequence (CDS) of the GhBBX20 gene is as follows: ATGGAACAAAAAAGAAAGATGAAGATTTGGTGTGAAGTGTGTGACAAAGAAGAAGCCACAGTGTTTTGCCCAGCTGATGAAGCTGCTCTTTGTGGTGTGTGTGATCATAATGTTCATCATGCAAATAAGCTTGCAACTAAACACTGTCGTTTTGCTCTTCTTCAACCCGATGAATCCCCTCTTTGTGATATCTGTCAGGAGAAAAGAGCGCTTCTGTTTTGTCAAGAAGATAGAGCAATACTCTGTAGGGAATGTGATATTCCAATTCACAAAGCCAATGAACATACCACGAAACATAACAGGTTTCTTCTCACAGGCGTTAAGCTTTCTTCTTCTTCTTCTTGTTCTTCTGCTAGTGGTTTCCCTCCTGCTTTAAACACAGTTTCATCTTCCTCTAATGGCGGTTATGGTGGTCAAACCATTTGTTCTGAAGCTGAAACTACTTCACAGTCCAATAACGAAATTGAGAAGCCATTGACGACTTACAGGATCGAACATAATTACACCATCGGCGACGACGACGTTTTGGTTTCAATGAGTATATCGGAGTACTTGATGGAAACTTTACCTGGTTGGCGTGTCGATGATTTTCTTGAACCTTCATCATATGCTGCACGTTAA (SEQ ID NO.3) The amino acid sequence encoded by GhBBX20 is as follows: MEQKRKMKIWCEVCDKEEATVFCPADEAALCGVCDHNVHHANKLATKHCRFALLQPDESPLCDICQEKRALLFCQEDRAILCRECDIPIHKANEHTTKHNRFLLTGVKLSSSSSCSSASGFPPALNTVSSSSNGGYGGQTICSEAETTSQSNNEIEKPLTTYRIEHNYTIGDDDVLVSMSISEYLMETLPGWRVDDFLEPSSYAAR (SEQ ID NO.4) The vector sequence of WMV068 is as follows: Example 2: Obtaining GhBBX20-overexpressing transgenic cotton through Agrobacterium-mediated genetic transformation of cotton shoot tips. Cotton seeds (upland cotton, Xinluzao 36) were delinted with sulfuric acid, then disinfected by soaking in 70% (v / v) ethanol solution for 1 min and 0.1% mercuric chloride solution for 15 min, followed by washing five times with sterile water. The disinfected seeds were then placed in sterile Petri dishes lined with double-layered filter paper, with an appropriate amount of sterile water added, and germinated in a 30℃ plant culture incubator. After 24 hours, the seed coat and two cotyledons were removed to expose the apical meristem (explant).

[0028] The WMV068-GhBBX20 recombinant plasmid constructed in Example 1 was transformed into Agrobacterium GV3101 competent cells to obtain a genetically engineered strain. The strain was plated on LB agar plates containing 50 mg / L rifampin and 50 mg / L kanamycin and cultured at 28°C for 48 h. Positive Agrobacterium clones containing the GhBBX20 recombinant plasmid were picked and inoculated into YEP liquid medium containing 50 mg / L rifampin and 50 mg / L kanamycin, and cultured overnight at 28°C with shaking at 180 rpm. 1 mL of the above Agrobacterium culture was inoculated into fresh YEP liquid medium containing 50 mg / L rifampin and 50 mg / L kanamycin and cultured at 28°C with shaking at 180 rpm until OD... 600 Centrifuge at 0.6-0.8, 4000 r / min for 5 min, collect the bacterial cells and resuspend them in M1 resuspension (M1 resuspension consists of infection medium without AS and Silwet-L77) for later use.

[0029] Before infection, 0.1% (v / v) of 20 mg / mL butyryl eugenol (AS) and 0.2% (v / v) of surfactant Silwet-L77 were added to the Agrobacterium resuspension to prepare the infection medium. The explants were then immersed in the infection medium, and the integration of the exogenous gene into the meristematic stem cells was promoted by ultrasound and vacuum treatment (ultrasound time 40 s, ultrasound frequency 40 kHz, infection time 50 min). After infection, the plants were cultured in co-culture medium at 35℃ for 3 days, followed by transfer to recovery medium for 15 days. Positive shoots were then induced in selection medium, and shoot elongation was induced in shoot elongation medium. Finally, positive seedlings were induced to root and transplanted into flowerpots to obtain T0 generation transgenic cotton plants.

[0030] The infection medium consisted of: 10 ml / L CA stock solution, 30 g / L glucose, 4.2 g / L MES (Sigma-Aldrich product, catalog number V900336), 0.1 ml / L Vitamin B5 (PhytoTech product, catalog number G219), 1 mg / L 6-BA, 0.1 mg / L NAA, 0.2 mM acetylsuccinone (AS), and pH 5.4.

[0031] The recovery medium (R0) consisted of: a mixture of 4.4 g / L MS salt and vitamin B5 (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, and pH 5.6.

[0032] The selection medium consisted of: a mixture of 4.4 g / L MS salt and vitamin B5 (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L spectinomycin, and pH 5.6.

[0033] The bud elongation medium consisted of: a mixture of 4.4 g / L MS salt and vitamin B5 (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L spectinomycin, and pH 5.6.

[0034] Example 3: Molecular identification of transgenic cotton materials overexpressing GhBBX20 Leaves from the T0 generation transgenic cotton plants (OE-GhBBX20-5, OE-GhBBX20-7, OE-GhBBX20-10) transplanted in Example 2 and wild-type cotton plants (WT, Xinluzao 36) were used for protein extraction and Western blotting.

[0035] Total protein was extracted from cotton leaves using a plant total protein extraction kit, following the kit's instructions. An equal volume of protein sample was separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then incubated overnight at 4°C with Anti-HA primary antibody (1:1000 dilution). The next day, the membrane was washed three times with TBST, then incubated for 1 hour with HRP-labeled secondary antibody (1:5000 dilution) at room temperature. After washing three more times with TBST, the membrane was developed and the signal detected using ECL chemiluminescence immunoassay.

[0036] Protein test results as follows Figure 2 As shown in Figure A, specific GhBBX20 protein bands were detected in all three GhBBX20 overexpressing transgenic cotton lines: OE-GhBBX20-5, OE-GhBBX20-7, and OE-GhBBX20-10, while no such band was observed in the wild-type control (WT). These results indicate that GhBBX20 protein has been successfully overexpressed in transgenic cotton, yielding positive transgenic cotton plants overexpressing GhBBX20, which will be used for subsequent Verticillium wilt resistance evaluation and metabolomics analysis.

[0037] Example 4: Evaluation of Verticillium wilt resistance and metabolome analysis of transgenic cotton materials overexpressing GhBBX20 The transgenic cotton plants overexpressing GhBBX20 (OE-GhBBX20-5, OE-GhBBX20-7, OE-GhBBX20-10), which were identified as positive in Example 3, were used as the experimental group, while wild-type cotton plants (WT, Xinluzao 36) were used as the control group. The above cotton materials were planted in nutrient pots and cultured in an artificial climate chamber at 25℃ under a 16h / 8h (day / night) environment. After the cotton seedlings developed their first true leaves, they were inoculated with *Verticillium dahliae* Vd991 and cultured normally. The disease severity of the cotton seedlings, i.e., the disease index, was recorded on day 25 after inoculation. Disease incidence is classified into five levels: Level 0 (asymptomatic plants), Level 1 (cotyledon infection), Level 2 (one true leaf infection), Level 3 (two true leaves infection), and Level 4 (three or more leaves infection). The disease severity level is counted, and the disease severity index is calculated using the formula: Disease Severity Index = ∑(Number of diseased plants at each level × Disease Severity Level) / (Total Number of Plants × 4) × 100.

[0038] Results of Verticillium wilt resistance identification are as follows Figure 2 As shown in Figure B, compared with the wild-type control, the disease index of transgenic cotton plants overexpressing GhBBX20 was significantly reduced and the symptoms were significantly alleviated, indicating that overexpression of GhBBX20 can significantly improve the resistance of cotton to Verticillium wilt.

[0039] Metabolomics analysis results as follows Figure 3As shown, compared with the wild-type control, transgenic cotton plants overexpressing GhBBX20 accumulated flavonoids significantly, indicating that the GhBBX20 gene induces the synthesis of flavonoids, thereby improving the cotton's resistance to Verticillium wilt.

Claims

1. Application of GhBBX20 gene in improving cotton resistance to Verticillium wilt.

2. The application according to claim 1, characterized in that, The GhBBX20 gene was obtained by amplification using cotton cDNA as a template and primers shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The nucleotide sequence of the GhBBX20 gene is shown in SEQ ID NO.

3.

4. The application according to claim 1, characterized in that, The amino acid sequence encoded by the GhBBX20 gene is shown in SEQ ID NO.

4.

5. The application according to claim 1, characterized in that, The GhBBX20 gene enhances cotton's resistance to Verticillium wilt by inducing the synthesis of flavonoids.

6. The application according to claim 5, characterized in that, The GhBBX20 gene was constructed into an expression vector and transformed into cotton to enhance the expression level of the GhBBX20 gene in cotton materials and induce the synthesis of flavonoids.

7. The application according to claim 6, characterized in that, The expression vector is WMV068, and its sequence is shown in SEQ ID NO.

5.

8. The application according to any one of claims 1 to 7, characterized in that, The cotton in question is upland cotton.

9. The application according to claim 8, characterized in that, The pathogen causing the cotton Verticillium wilt is Verticillium dahliae.

10. The application of recombinant expression vectors containing the GhBBX20 gene and genetically engineered strains in improving cotton resistance to Verticillium wilt, characterized in that, The nucleotide sequence of the GhBBX20 gene is shown in SEQ ID NO.3, and the cotton is upland cotton.

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