Rapeseed gene BnaGH3 and its application in the prevention and control of rapeseed sclerotinia disease
By identifying and utilizing the BnaGH3 gene of rapeseed and using CRISPR/Cas9 technology for gene editing, the problem of insufficient resistance to sclerotinia in rapeseed has been solved, and the effect of significantly improving the resistance to sclerotinia in rapeseed has been achieved, providing a basis for disease-resistant breeding in rapeseed. Provides new genetic resources.
Patent Information
- Application Number
- CN202311833124.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-01-21
- Estimated Expiration
- 2043-12-28
AI Technical Summary
Germplasm resources that are completely immune to sclerosis have not been found in rapeseed. The existing technology has limited signaling pathways and gene mining for rapeseed to respond to sclerosis infection, which hinders the disease-resistant breeding process of rapeseed.
By identifying the key antibacterial sclerotinia regulatory gene BnaGH3 in rapeseed and using CRISPR/Cas9 technology for gene editing, homozygous mutant transgenic rapeseed was obtained and its role in antibacterial sclerotinia disease in rapeseed was studied.
It was found that the BnaGH3 gene plays a positive regulatory role in the resistance of rapeseed to Sclerotinia sclerotiorum. By constructing transgenic rapeseed with this gene, the resistance of rapeseed to Sclerotinia sclerotiorum can be significantly improved, providing a new way to create disease-resistant varieties and germplasm.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a Brassica napus BnaGH3 gene and an application thereof in the prevention and control of rapeseed sclerotinia disease. Background Art
[0002] Rapeseed is one of the most important oil crops in the world, a traditional oil crop in my country, and an important source of edible oil in my country. Rapeseed will be attacked by a variety of pests and diseases during its growth and development, among which sclerotinia is the most common disease. Sclerotinia is a global disease caused by infection with Sclerotinia sclerotiorum, which seriously restricts the high and stable yield of rapeseed. In recent years, with the changes in ecological conditions and the large-scale promotion of double-low rapeseed, the incidence of sclerotinia in rapeseed has shown an increasing trend. my country maintains an average annual incidence of sclerotinia of 10% to 30%. In years with high incidence, the incidence of field plants reaches more than 80%. Sclerotinia causes a 10% to 20% reduction in rapeseed production in my country, and up to 70% in severe disease years. After infection with sclerotinia, the oil content and quality of rapeseed decrease significantly.
[0003] At present, no germplasm resources that are completely immune to Sclerotinia sclerotiorum have been found in rapeseed or even the entire cruciferous plant family, which has brought great challenges to the selection of Sclerotinia sclerotiorum-resistant varieties and hindered the process of rapeseed disease-resistant breeding. In addition, the current exploration of the signaling pathways and genes of rapeseed in response to Sclerotinia sclerotiorum infection is very limited, and the potential molecular mechanism of plant response to the pathogen is still unclear. Therefore, it is particularly important to further explain the mechanism of rapeseed's response to Sclerotinia sclerotiorum infection and fully understand the response pattern of plants when pathogens harm them, so as to provide a theoretical basis for revealing the interaction pattern of rapeseed with Sclerotinia sclerotiorum and provide guidance for the improvement of Sclerotinia sclerotiorum resistance.
[0004] After the CRISPR / Cas9 technology was successfully developed and applied to gene editing in animal cells, it was quickly applied to plants. Currently, CRISPR / Cas9 technology can be used for genetic improvement of polyploid crops such as wheat, switchgrass, soybeans and strawberries. Therefore, using the efficient CRISPR / Cas9 technology to obtain rapeseed mutants will not only help the study of rapeseed gene function, but also provide a new direction for the genetic improvement of rapeseed in the future. Summary of the invention
[0005] Identifying key anti-sclerotinia sclerotinia regulatory genes and creating and rationally utilizing disease-resistant varieties through biotechnology are of great significance for the prevention and control of rapeseed sclerotinia sclerotinia and green development. Based on this, the present invention provides a new rapeseed anti-sclerotinia sclerotinia gene, the rapeseed BnaGH3 gene, and studies its role in rapeseed resistance to sclerotinia sclerotinia.
[0006] The present invention uses the cDNA of rapeseed Westar variety as a template and obtains the full length of rapeseed gene BnaGH3 by PCR cloning. The open reading frame (ORF) of the gene is 1854bp long, the nucleic acid sequence is shown in SEQ ID NO.1, the encoded protein consists of 617 amino acids, and the encoded protein sequence is shown in SEQ ID NO.2.
[0007] The present invention obtains homozygous mutant transgenic rapeseed by gene editing the rapeseed gene BnaGH3, and finds that the transgenic rapeseed has changed its resistance to sclerotinia. The gene editing to obtain homozygous mutant transgenic rapeseed is achieved by the following steps:
[0008] (1) sgRNA target selection and successful construction of CRISPR / Cas9 vector (CRISPR / Cas9-BnaGH3). Design sgRNA targeting the BnaGH3 gene and clone the sequence into the CRISPR / CAS9 vector; the sgRNA sequence is shown in SEQ ID NO.3, and the protein sequence encoded by the BnaGH3 gene is shown in SEQ ID NO.2;
[0009] (2) Obtaining Agrobacterium. The gene mutation expression vector CRISPR / Cas9-BnaGH3 was transformed into the Agrobacterium strain GV3101, which has strong infectivity to rapeseed, by electroporation. The strains were screened on LB solid medium containing kanamycin, and identified by PCR and sequencing to obtain Agrobacterium carrying the BnaGH3 gene for the next step of genetic transformation of rapeseed.
[0010] (3) Obtaining the BnaGH3 mutant of Brassica napus. The above Agrobacterium vector was transferred into rapeseed by infecting the hypocotyls of rapeseed with Agrobacterium to obtain transgenic rapeseed plants. The specific steps are as follows:
[0011] a. Seed sterilization and sowing: Select full Westar rapeseed seeds, sterilize them, place them in a sterile box, and culture them at 25℃ for 5-6 days.
[0012] b. Preparation and infection of explants. Cultivate the target strain in LB liquid medium containing Kan resistance. Shake overnight at 28℃ and 220rpm until the OD600 of the bacterial solution is 0.6. Cut the hypocotyl into 0.8-1.0cm lengths. Pour the resuspended bacterial solution into the culture dish containing the hypocotyls and infect for 10 minutes. Transfer the hypocotyls to a culture dish containing filter paper, place for 20-30 minutes, and then transfer to M1 medium and culture at 25℃ for 2 days.
[0013] c. Select induced callus: Transfer the explants in M1 medium to M2 medium and culture at 25°C for two weeks.
[0014] d. Induce differentiation, bud regeneration and rooting. Transfer the callus tissue in the M2 medium to the M3 medium. If green buds appear, cut the tissue with a sterile blade and transfer it to the M4 medium for rooting.
[0015] e. Screening and identification of homozygous mutants of BnaGH3 of Brassica napus. Design primers for identifying mutants, amplify DNA of positive plants, recover fragments and perform sequencing detection. Compare the sequencing result sequence with the original sequence, analyze the mutation type and mutation site, wherein the mutant identification primer sequence is shown in SEQ ID NO.4.
[0016] The obtained BnaGH3 gene rapeseed homozygous mutant strains can be used as materials, and their resistance to rapeseed sclerotinia sclerotinia can be detected and analyzed by inoculating with Sclerotinia sclerotiorum, so as to clarify the regulatory role of the BnaGH3 gene on rapeseed sclerotinia sclerotinia resistance.
[0017] Prior to the present invention, there was no public report on the function of this gene. The present invention, for the first time, knocked out BnaGH3 by constructing a gene mutation vector of this gene, obtained a single gene mutant rapeseed, analyzed its disease resistance, and clarified the regulatory effect and mechanism of this gene on rapeseed resistance to Sclerotinia sclerotiorum. Compared with the non-transgenic wild-type control plants, the single gene mutant had a reduced ability to resist Sclerotinia sclerotiorum and was more susceptible to Sclerotinia sclerotiorum, indicating that BnaGH3 strongly positively regulates rapeseed's resistance to Sclerotinia sclerotiorum.
[0018] The BnaGH3 gene provided by the present invention is a high-quality anti-bacterial sclerotinia control gene resource, and the disease-resistant material obtained by using the gene has the advantages of strong disease resistance, etc. The BnaGH3 gene is an immune receptor gene and a gating gene for disease resistance signal transduction. Therefore, it has a good resistance control effect and is a high-quality disease resistance control gene resource.
[0019] Beneficial Effects
[0020] Rapeseed sclerotinia resistance is a quantitative trait resistance, which is controlled by multiple genes. Generally speaking, the degree of regulation of this resistance by a single gene is low. Therefore, rapeseed sclerotinia resistance materials are very scarce worldwide, and there is no stable high-resistant material. The present invention discovered the BnaGH3 gene through screening, which is of great significance for the prevention and control of rapeseed sclerotinia and the cultivation of disease-resistant varieties. Using BnaGH3 to create disease-resistant varieties and germplasm is an economical, effective and safe way to greenly prevent and control diseases. The BnaGH3 gene is a brand-new gene resource suitable for creating and breeding new materials and new varieties of rapeseed resistant to sclerotinia.
[0021] The methods for obtaining disease-resistant plant materials and varieties mainly include conventional traditional breeding methods and genetic engineering breeding methods using disease-resistant regulatory genes. Traditional breeding methods have the disadvantages that the range of available disease-resistant resources is limited by natural genetic isolation, the breeding cycle is long, and a large amount of manpower and material resources are required. The genetic engineering breeding method has the advantages of a wide range of available disease-resistant resources, relatively simple and convenient operation, a short breeding cycle, no need for a large amount of manpower and material resources, and is particularly suitable for breeding broad-spectrum, durable, and highly disease-resistant varieties. The present invention utilizes the jasmonic acid isoleucine synthase gene BnaGH3 and adopts a genetic engineering method to create and cultivate rapeseed materials, which have the characteristics of a short cycle and rapid breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Figure 2 is a diagram of the nucleotide sequence of the sgRNA site and its targeting site.
[0023] Figure 2 The results of inoculating Sclerotinia sclerotiorum with the wild type and the BnaGH3 mutant in the examples of the present invention are shown. Figure 2 A is the phenotypic result of inoculation with Sclerotinia sclerotiorum, and B is the statistical quantitative analysis of the lesion area. The results show that the BnaGH3 mutant plants are significantly more susceptible to the disease than the wild-type plants (WT). This indicates that the resistance of the BnaGH3 mutant plants to Sclerotinia sclerotiorum is significantly lower than that of the wild-type plants. The deletion mutation of the BnaGH3 gene leads to a significant reduction in the resistance of rapeseed to Sclerotinia sclerotiorum. DETAILED DESCRIPTION
[0024] Unless otherwise specified, the relevant reagents in the present invention are all conventional reagents, and the relevant operation steps are all conventional operations, which can be performed according to the operating instructions of the reagent supplier or the guidance of classic reference books such as "Molecular Cloning Experiment Guide".
[0025] Embodiment 1:
[0026] The present invention cloned a Brassica napus jasmonate isoleucine synthase (BnaGH3), and for the first time obtained transgenic rapeseed, clarified the anti-sclerotinia disease regulatory function of the gene, and revealed that the gene has a positive regulatory function on rapeseed sclerotinia disease resistance. The mutation of the BnaGH3 gene leads to a significant decrease in rapeseed's resistance to sclerotinia disease. Therefore, by constructing homozygous mutant plants of the gene and using genetic engineering technology, new materials for rapeseed resistant to sclerotinia disease can be created and obtained. The main steps include:
[0027] 1.1sgRNA target selection and successful construction of CRISPR / Cas9 vector
[0028] After obtaining all the targets of the full sequence of the BnaGH3 gene through the CRISPR / P online target design website, suitable targets were screened. According to the genome sequence, two pairs of conservative primers were designed for the expression vector. After the sequence was synthesized by the company, PCBC was used as a template to connect to the intermediate vector PCBC through PCR. Then, the PCR product was recovered and purified after 1% agarose gel electrophoresis. The recovered product was purified by BsaI restriction endonuclease (Thermo Scientific TM ) and T4 ligase (ThermoScientific TM ) was spliced into the CRISPR / CAS9 vector, and the enzyme-ligation product was transformed into E. coli DH5α competent cells by heat shock method. After activation in liquid LB medium at 37°C for 40 minutes, it was spread on the corresponding resistance plate and cultured overnight. After PCR verification and sequencing comparison, the gene mutation expression vector CRISPR / CAS9-BnaGH3 was obtained.
[0029] 1.2. Agrobacterium transformation of CRISPR / CAS9 vectors
[0030] The gene mutation expression vector CRISPR / Cas9 was transformed into the Agrobacterium strain GV3101, which has strong infectivity to rapeseed, by electroporation, and screened on LB solid culture medium containing kanamycin. Agrobacterium carrying the BnaGH3 gene was obtained by PCR and sequencing identification for the next step of genetic transformation of rapeseed.
[0031] 1.3. Obtaining the BnaGH3 mutant of Brassica napus
[0032] (1) Seed disinfection and sowing
[0033] Select full Westar rapeseed seeds and place them in a sterile box. Add 70% alcohol and soak for 2 minutes. After pouring out the alcohol, add sterile water to wash. After pouring out the sterile water, add 50% sodium hypochlorite disinfectant and wash for 15 minutes. Use sterile tweezers to sow the disinfected seeds in a sterile box containing MS culture medium and culture at 25℃ for 5-6 days.
[0034] (2) Explant preparation and infection
[0035] The target strain was cultured in LB liquid medium containing Kan resistance. Shake overnight at 28°C and 220 rpm until the bacterial solution OD600 reached 0.6. Cut the hypocotyl into small pieces of 0.8-1.0 cm in length, pour the resuspended bacterial solution into the culture dish containing the hypocotyl, and culture at 25°C for 2 days.
[0036] (3) Selecting and inducing callus
[0037] The explants in the M1 medium were transferred to the M2 medium (MS2.2 g / L, Sucrose 30 g / L, Agarose 6 g / L) and cultured at 25°C for two weeks. The above operations were all performed in a clean bench, and the experimental instruments and reagents used were sterilized.
[0038] (4) Inducing differentiation, bud regeneration, and rooting
[0039] Transfer the callus in M2 medium to M3 (MS 2.2 g / L, glucose 30 g / L, xylose 0.25 g / L, MES 0.6 g / L, Agarose 6 g / L) medium. If green buds appear, cut the tissue with a sterile blade and transfer it to M4 (MS 4.4 g / L, Sucrose 30 g / L, Agarose 6 g / L) medium. When the callus grows into a complete plant, move it into soil.
[0040] (5) Screening and identification of homozygous mutants of BnaGH3 in Brassica napus
[0041] The plant genomic DNA was extracted by CTAB method, and the positive plants were detected by PCR amplification. Primers for mutant identification were designed, and the positive plant DNA was amplified and the fragments were recovered for sequencing detection. The sequencing result sequence was compared with the original sequence to analyze the mutation type and mutation site. The mutant identification primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.5.
[0042] (6) Disease resistance analysis of BnaGH3 homozygous mutant strains of Brassica napus
[0043] The homozygous mutant strain of rapeseed with the BnaGH3 gene obtained above was used as the material. By inoculating Sclerotinia sclerotiorum, its resistance to rapeseed sclerotinia was detected and analyzed, so as to clarify the regulatory effect of the BnaGH3 gene on rapeseed sclerotinia resistance and whether it is possible to create and obtain new materials of rapeseed resistant to sclerotinia by constructing transgenic rapeseed with this gene and using genetic engineering technology.
[0044] a. Activation culture of Sclerotinia sclerotiorum
[0045] Use an alcohol lamp to burn the laboratory-stored Sclerotinia bacterial plate and disinfect it. Then place the inoculation loop with the bacterial surface facing down in the middle of the PDA solid culture medium and culture it at 25℃ in the dark for about 2 days.
[0046] (2) Inoculation of Sclerotinia sclerotiorum
[0047] Select rapeseed plants with consistent growth for inoculation. Use a 5mm diameter puncher to punch out mycelium blocks, inoculate them into fully developed leaves, keep them moist, and place them in a 25℃ greenhouse for culture. After an appropriate time (about 36h), take photos and record them. The lesion area is analyzed using ImageJ software, and the inoculation experiment is repeated three times. The lesion area data were analyzed, and the data are expressed as mean ± SE. The results showed that BnaGH3 mutant plants were significantly more susceptible to the disease than wild-type plants (WT). This shows that the resistance of BnaGH3 mutant plants to sclerotinia is significantly lower than that of wild-type plants. The deletion mutation of the BnaGH3 gene leads to a significant reduction in rapeseed's resistance to sclerotinia, so BnaGH3 plays a positive regulatory role in rapeseed's resistance to sclerotinia.
[0048] In summary, the present invention uses CRISPR / Cas9 technology to target BnaGH3, obtains BnaGH3 homozygous mutant plants by genetic transformation, and identifies and analyzes the resistance phenotype of the mutant to Sclerotinia sclerotiorum. BnaGH3 has a strong positive regulatory effect on the resistance of rapeseed to sclerotinia sclerotiorum. New rapeseed materials can be created and obtained by constructing transgenic rapeseed of BnaGH3 and using genetic engineering technology.
[0049] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.
Claims
1. BnG3 The application of the gene in the prevention and control of Brassica napus sclerotinia is characterized in that: BnG3 The deletion mutation of the gene leads to a decrease in the resistance of rapeseed to sclerotinia disease, which is caused by infection with Sclerotinia sclerotiorum. The nucleic acid sequence of the BnaGH3 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.
2.
2. A method for screening rapeseed that is resistant to sclerotinia sclerotiorum, characterized in that: Detection of rapeseed BnaGH3 gene, If rapeseed appears BnG3 If the gene is deleted or mutated, the variety has poor resistance to bacterial sclerotinia. The nucleic acid sequence of the BnaGH3 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2.