Application of rape gene BnSKIP31 in prevention and control of sclerotiniose

Abstract

The invention provides an application of a Brassica napus gene BnSKIP31 in prevention and control of sclerotiniose, which is an application in obtaining a disease-resistant enhanced rape material by creating a gene to edit rape. According to the invention, CRISPR gene knockout rape of BnSKIP31 is constructed, the negative regulation function of the gene on sclerotiniose resistance is disclosed for the first time, and application of the BnSKIP31 gene to acquisition of sclerotiniose rape materials with increased sclerotiniose resistance by creating CRISPR gene knockout rape is provided. The BnSKIP31 gene provided by the invention is a new gene resource suitable for creating and breeding new sclerotiniose-resistant rape materials and new varieties, and the created rape germplasm has lasting disease resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to application of rapeseed gene BnSKIP31 in the prevention and control of sclerotinia disease. Background Art

[0002] 1. F-box proteins and plant disease resistance Plant disease resistance is the result of plant receptors recognizing pathogen ligands, activating disease resistance signal transduction, and activating a series of defense responses. From a genetic point of view, after receptor-ligand recognition, the genes involved in resistance regulation include early disease resistance signal transduction genes and late defense-related genes. Generally speaking, the effect of regulating disease resistance through early disease resistance signal transduction genes is better.

[0003] E3 ubiquitin ligase plays a key role in the ubiquitin-proteasome protein degradation system and can directly mediate the degradation of target proteins including early disease resistance signal transduction proteins, thereby affecting the stability of early disease resistance signal transduction-related proteins. By knocking out the E3 ubiquitin ligase gene, its regulation of certain disease resistance-related proteins can be changed, thereby affecting the disease resistance of plants. F-box protein is one of the core components of the SCF (Skp1–Cullin–F-box) class E3 ubiquitin ligase complex, responsible for target substrate proteins. After binding with other subunits such as Cullin and Rbx1, F-box protein can promote the transfer of ubiquitin molecules to target proteins, thereby marking target proteins for degradation. The inventors have found that the rapeseed F-box protein BnSKIP31 directly binds to and targets the degradation of the early disease resistance signal transduction protein, the calcium ion channel protein BnCNGC3, thereby inhibiting disease resistance. Therefore, using gene editing technology to create gene knockout materials based on the F-box gene BnSKIP31 may be an effective strategy for creating crop germplasm with improved disease resistance, but there are currently no public reports of this strategy.

[0004] 2. Plant Sclerotinia disease prevention and control technology Plant sclerotinia is caused by infection by Sclerotinia sclerotiorum. Sclerotinia sclerotiorum is a necrotrophic pathogenic fungus with a very wide host range. It is a major disease of crops such as oilseeds and vegetables. It causes huge economic losses every year. Due to the lack of highly resistant varieties, chemical control is still an important means. Since some pesticides have problems such as ecological pollution, human and animal toxicity, and easy to make pathogens resistant, it is crucial to identify important anti-sclerotinia regulatory genes and create and use disease-resistant varieties for green prevention and control of sclerotinia.

[0005] 3. Plant disease resistance breeding technology Plant disease resistance breeding technology is mainly divided into two categories: traditional disease resistance breeding and disease resistance breeding designed by genetic engineering and molecular modification. Traditional disease resistance breeding has a natural genetic isolation phenomenon, which significantly limits the scope of available disease resistance resources. Only disease resistance resources with close genetic relationships can be used, and multiple hybridization and backcrossing are required. Therefore, the breeding cycle is long and requires a lot of manpower and material resources. The genetic engineering and molecular modification design breeding method is to introduce exogenous disease resistance regulatory genes or modified genes into plants through Agrobacterium-mediated methods and other technologies, or to knock out or increase the expression of plant target genes through gene editing and other technologies, so that they can obtain disease resistance that they did not originally have or improve disease resistance. Therefore, the genetic engineering and molecular modification design breeding method breaks the limitations of natural genetic isolation, broadens the scope of available disease resistance resources, and has the characteristics of relatively simple and convenient operation, short cultivation cycle, and no need for a lot of manpower and material resources. In addition, varieties with broad-spectrum disease resistance can be created by modifying broad-spectrum disease resistance regulatory genes or introducing multiple genes with different disease resistance spectra. Therefore, it has the advantages of being particularly suitable for cultivating broad-spectrum and long-lasting disease-resistant varieties. Summary of the invention

[0006] The invention aims to provide an application of a rapeseed (Brassica napus) F-box gene BnSKIP31 in the prevention and control of sclerotinia sclerotiorum. The rapeseed F-box gene BnSKIP31 has a negative regulatory function on sclerotinia sclerotiorum resistance and can be applied to the creation of Sclerotinia sclerotiorum resistance crop germplasm.

[0007] The application is to obtain rapeseed materials with enhanced resistance to sclerotinia sclerotinia by creating BnSKIP31 gene knockout rapeseed.

[0008] The present invention uses the cDNA of the double 11 variety in rapeseed (Brassica napus) as a template, and obtains the rapeseed gene BnSKIP31 by PCR cloning, the nucleotide sequence of which is shown in SEQ ID No.1, the open reading frame (ORF) of the gene is 945bp long, and the encoded protein consists of 314 amino acids, and its sequence is shown in SEQ ID No.2. The BnSKIP31 protein contains an F-box domain and a Herpes domain. The nucleotide sequence cloned by the present invention is completely consistent with the ZS11C07G030650 nucleotide sequence of the rapeseed variety ZS11 in the Brassica_napus cv.ZS11 PacBio V1.0 database.

[0009] Prior to the present invention, there was no public report on the function of this gene. The present invention, for the first time, constructed a CRISPR knockout rapeseed with this gene and analyzed its disease resistance, clarifying the regulatory role of this gene in rapeseed resistance to Sclerotinia sclerotiorum. The analysis results of the lesion area of ​​inoculated leaves and the biomass of pathogens showed that compared with wild-type rapeseed plants, the knockout plants were significantly more resistant to Sclerotinia sclerotiorum, indicating that BnSKIP31 negatively regulates rapeseed resistance to Sclerotinia sclerotiorum.

[0010] Based on the BnSKIP31 gene function explained in the present invention, the application purpose of the present invention is to provide the application of the rapeseed BnSKIP31 gene in obtaining rapeseed materials with increased resistance to sclerotinia disease by creating CRISPR gene knockout rapeseed (Example 1).

[0011] The application of the rapeseed BnSKIP31 gene in obtaining rapeseed materials with enhanced resistance to sclerotinia sclerotiorum by creating CRISPR gene knockout rapeseed is achieved by the following steps: (1) Construction and acquisition of the CRISPR knockout structure of the BnSKIP31 gene: Two 20 bp knockout targets were preset based on the BnSKIP31 genome sequence, and the target sequences were cloned into the plant expression vector pBSbdcas9i. The recombinant structure pBSbdcas9i-BnSKIP31 was obtained through colony PCR identification and sequencing; (2) Obtaining Agrobacterium transformed with the CRISPR knockout structure of the BnSKIP31 gene: The CRISPR knockout structure of the BnSKIP31 gene (pBSbdcas9i-BnSKIP31) was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation or other methods; (3) Creation and acquisition of rapeseed with CRISPR knockout structure of BnSKIP31 gene: The CRISPR knockout structure of BnSKIP31 gene was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed with CRISPR knockout structure of BnSKIP31 gene; (4) Obtaining homozygous lines of rapeseed with the CRISPR knockout structure of the BnSKIP31 gene: Screening with antibiotic resistance, detecting the trait segregation of the offspring of transgenic plants, and obtaining homozygous lines of rapeseed with the CRISPR knockout structure of the BnSKIP31 gene, in which the offspring traits no longer segregate and can be stably inherited; (5) Screening, identification and acquisition of homozygous rapeseed lines with CRISPR knockout structure of BnSKIP31 gene and enhanced resistance to sclerotinia sclerotinia: The homozygous rapeseed lines with CRISPR knockout structure of BnSKIP31 gene (pBSbdcas9i-BnSKIP31) were used as materials to detect and analyze resistance to sclerotinia sclerotinia, and obtain CRISPR knockout rapeseed with BnSKIP31 gene and enhanced resistance to sclerotinia sclerotinia.

[0012] Advantages of the present invention: (1) The BnSKIP31 gene provided by the present invention is a high-quality anti-bacterial sclerotinia regulatory gene resource. The BnSKIP31 gene encodes an F-box protein, which directly targets the degradation of early disease resistance signal transduction proteins such as BnCNGC3, and is a negative regulatory protein for plant disease resistance. Knocking out the gene through gene editing technology to obtain disease-resistant germplasm will not lead to rapid co-evolution of pathogen counter-defense reactions, so the resistance is long-lasting. Using this gene to create disease-resistant germplasm is an economical, effective and safe way to green disease prevention and control. Therefore, the BnSKIP31 gene is a brand-new gene resource suitable for creating and breeding new materials and new varieties of rapeseed resistant to bacterial sclerotinia. (2) The cycle for obtaining disease-resistant materials is short. 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, short breeding cycle, no need for a lot of manpower and material resources, and is particularly suitable for breeding broad-spectrum, durable, and highly disease-resistant varieties. The present invention utilizes the disease-resistant regulatory gene BnSKIP31 and adopts genetic engineering methods to create and cultivate rapeseed materials with high resistance to bacterial sclerotinia, which has the characteristics of short cycle and rapid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The present invention provides evidence of gene-edited rapeseed plants obtained by the present invention, which is the gene editing identification of BnSKIP31 CRISPR gene knockout plants, showing the editing form of the gene in BnSKIP31 gene knockout plants. Two knockout mutant lines #4 and #20 were successfully obtained. The four BnSKIP31 homologous genes in the ZS11 variety background produced different editing forms. Mutant line #4 had BnSKIP31 on chromosome A06. A06 The target sites 1 and 2 of the gene were inserted into BnSKIP31 on chromosome C07. C07 The target site 2 of the gene is missing 1 bp, and the BnSKIP31 in chromosome A09 A09 The target site 2 of the gene lacks an 8 bp sequence, which is located at BnSKIP31 on chromosome C09. C09 The target site 2 of the gene is missing 1bp; mutant line #20 is on chromosome A06 BnSKIP31 A06 Insert 1 bp at the target site 1 of the gene, BnSKIP31 on chromosomes C07, A09, and C09 C07 、BnSKIP31 A09 、BnSKIP31 C09 1 bp was inserted into each of the two target sites of the gene, resulting in the inability to encode functional BnSKIP31 protein ( Figure 1 ). This indicates that these plants are true BnSKIP31 gene knockout plants.

[0014] Figure 2 The evidence of the anti-bacterial sclerotinia function of the BnSKIP31 gene was provided, indicating that BnSKIP31 negatively regulates rapeseed's resistance to Sclerotinia. The rapeseed BnSKIP31 gene knockout plants were inoculated with the Sclerotinia UF-1 strain. The figure shows the quantitative statistical analysis results of the phenotype, lesion area and mycelial biomass of the gene knockout mutant plants after inoculation. The inoculation experiment was repeated three times. The Student's t-test test statistical analysis of the lesion area was performed using GraphPad Prism, and the data are expressed as mean ± standard deviation. The significance of the difference is indicated by different numbers of * (*, P<0.05; ****, P<0.0001). The results showed that the gene knockout mutant plants showed a significantly more disease-resistant phenotype than the control (ZS11) plants. The results of quantitative analysis of the lesion area showed that the lesion areas of the two knockout mutant lines were 158.3 mm 2 and 173.8mm 2 , significantly lower than the control group of 242.1 mm 2 , only 65.4% and 71.8% of the control; the bacterial count results also showed that the bacterial count in the lesions of the gene knockout mutant plants was significantly lower than that of the control plants. These results indicate that BnSKIP31 negatively regulates rapeseed's resistance to Sclerotinia sclerotiorum. DETAILED DESCRIPTION

[0015] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0016] Example 1 The present invention, for the first time, constructs CRISPR gene knockout rapeseed of the BnSKIP31 gene, and combines it with inoculation analysis to clarify the negative regulatory function of the gene on rapeseed resistance to sclerotinia. Knockout of the BnSKIP31 gene leads to a significant increase in rapeseed resistance to sclerotinia. Therefore, by constructing CRISPR gene knockout rapeseed of BnSKIP31, new rapeseed materials with enhanced resistance to sclerotinia can be created and obtained, which can be used for the creation and breeding of sclerotinia-resistant rapeseed varieties, analysis of the function and mechanism of action of the BnSKIP31 gene, and other uses. The main steps for analyzing the function and mechanism of the BnSKIP31 gene, and creating and obtaining new rapeseed materials with increased resistance to sclerotinia include:

[0017] (1) Construction and acquisition of CRISPR knockout construct of BnSKIP31 gene Two suitable targets were designed based on the BnSKIP31 genome sequence. Each gRNA unit contained a 20 bp gRNA and a gRNA backbone. The two gRNA units were combined as a complete knockout functional sequence. Using it as a template, amplification was performed using primers BnSKIP31-CR-F1 (5'-cagtggtctc atg cac ctc gaa tca gaa gtc tcc tgt ttc ag-3', the italic part is a sequence containing a Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (the sequence is shown in SEQ ID NO.3) and BnSKIP31-CR-R1 (5'-cagtggtctc aaa acg tgg ggt cta atg ccttca atg cac-3', the italic part is a sequence containing a Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (the sequence is shown in SEQ ID NO.4). The vector pBSbdcas9i was digested with Bsa I (Eco31 I), and recombined with T4 ligase, followed by transformation and kanamycin medium plate screening. Positive single clones were picked and PCR identified using pBSbdcas9i identification primers M13F (5'-gta aaa cga cgg cca gt-3') (sequence as shown in SEQ ID NO.5) and zmpl: (5'-cca gaa att gaa cgc cga ag-3') (sequence as shown in SEQ ID NO.6). Further sequencing and identification was performed to obtain the CRISPR knockout structure pBSbdcas9i-BnSKIP31.

[0018] (2) Obtaining Agrobacterium transformed with the CRISPR knockout construct of the BnSKIP31 gene The BnSKIP31 gene CRISPR knockout structure (pBSbdcas9i-BnSKIP31) was transformed into an Agrobacterium strain with strong infection ability to rapeseed, such as EHA105, by electroporation and other methods. The transformants were screened on YEP medium containing kanamycin and then identified by PCR to obtain Agrobacterium carrying the BnSKIP31 gene CRISPR knockout structure pBSbdcas9i-BnSKIP31.

[0019] (3) Creation and acquisition of BnSKIP31 gene CRISPR knockout rapeseed The BnSKIP31 gene CRISPR knockout structure pBSbdcas9i-BnSKIP31 was introduced into rapeseed by Agrobacterium-mediated method to obtain the rapeseed T0 generation transformed with pBSbdcas9i-BnSKIP31. The specific operation steps are as follows:

[0020] (i) Seed cleaning and germination 75% ethanol for 30-60s, sterile water once, 1min / time; 0.15% mercuric chloride for 10min, wash twice with sterile water, 1min / time; wash with sterile water for 30min, inoculate on sterile filter paper and dry; inoculate the seeds in culture bottles and culture in the dark at 23℃ for 5-6d.

[0021] (ii) Pre-culture The germinated hypocotyls of rapeseed seedlings were cut into 0.4-0.6 cm segments, inoculated into pre-culture medium, and cultured at 23°C for 2-3 days.

[0022] (iii) Agrobacterium infection and co-cultivation Pick Agrobacterium in the infection solution and prepare OD 600 = 0.2 Agrobacterium resuspension, inoculate the explants in the Agrobacterium suspension for 10 minutes, inoculate the infected explants on sterile filter paper to dry, inoculate on co-culture medium, and culture at 23°C in the dark for 48-72 hours.

[0023] (iv) Degerming (extended screening) The co-cultivated explants were inoculated on sterile culture medium and cultured at 23°C under light for 6 days.

[0024] (v) Screening / differentiation The sterilized explants were inoculated on screening / differentiation medium, 30 explants per dish, cultured at 23°C under light, and the plate was changed every 15 days.

[0025] (vi) Rooting culture The differentiated buds were inoculated into rooting medium and cultured at 23°C under light until roots formed.

[0026] (vii) Detection The CTAB method was used to extract rapeseed genomic DNA, and PCR was used to detect resistance genes.

[0027] (viii) Soil planting After the kanamycin-resistant seedlings have grown a complete root system, the T0 generation sterile seedlings are incubated with the lid open at 27°C for 2 days, then transplanted into the soil, placed in an incubator for routine management, and finally harvested to obtain T0 seeds.

[0028] (4) Obtaining homozygous rapeseed lines with CRISPR knockout of BnSKIP31 gene The segregation of traits of the offspring of transgenic plants was detected by antibiotic resistance screening. The test was carried out on rapeseed seeds on plates containing Basta resistance to observe whether they can grow normally into healthy seedlings on Basta resistance plates. The homozygous rapeseed lines with the BnSKIP31 gene CRISPR knockout structure pBSbdcas9i-BnSKIP31, whose offspring traits no longer separated and could be stably inherited, were obtained. All these rapeseed homozygous lines can grow normally into healthy seedlings on Basta resistance plates.

[0029] The present invention obtains a homozygous rapeseed line of BnSKIP31-CRISPR gene knockout, which can grow normally into healthy seedlings on a Basta-resistant plate. A06 -CR-F2 (5'-ctc tca tct ctc tgt ccc tt-3') (sequence shown in SEQ ID NO.7) and BnSKIP31 A06 -CR-R2 (5'-atc aac aac tat gag atg ca-3') (sequence as SEQ ID NO.8) primer pair for detecting BnSKIP31 on chromosome A06 A06 Gene editing format, using BnSKIP31 C07 -CR-F3 (5'-ctc tca tct ctc tgt ccc tt-3') (sequence shown in SEQ ID NO.9) and BnSKIP31 C07 -CR-R3 (5'-cag ctg tga tat gca cat gt-3') (sequence as SEQ ID NO.10) primer pair to detect BnSKIP31 on chromosome C07 C07 Gene editing format, using BnSKIP31 A09 -CR-F4 (5'-ccc tct cgt ttc tct atc cc-3') (sequence shown in SEQ ID NO.11) and BnSKIP31 A09 -CR-R4 (5'-gct tct gac aag ttc tat cat a-3') (sequence as SEQ ID NO.12) primer pair to detect BnSKIP31 on chromosome A09 A09 Gene editing format, using BnSKIP31 C09 -CR-F5 (5'-ccc tct cgt tac tct acc cc-3') (sequence shown in SEQ ID NO.13) and BnSKIP31 C09-CR-R5 (5'-gct tct gac cag ttc aat cat a-3') (sequence as SEQ ID NO.14) primer pair to detect BnSKIP31 on chromosome C09 C09 Gene editing form, detect whether the transformed seedlings are gene-edited seedlings. Then the gene-edited seedlings were sequenced near the target site, and finally determined to be mutant plants with successful knockout of the BnSKIP31 gene. The sequencing results showed that two BnSKIP31 gene knockout mutant lines #4 and #20 were successfully obtained, and the four BnSKIP31 homologous genes in the ZS11 variety background produced different editing forms: mutant line #4 had BnSKIP31 on chromosome A06. A06 The target sites 1 and 2 of the gene were inserted into BnSKIP31 on chromosome C07. C07 The target site 2 of the gene is missing 1 bp, and the BnSKIP31 in chromosome A09 A09 The target site 2 of the gene lacks an 8 bp sequence, which is located at BnSKIP31 on chromosome C09. C09 The target site 2 of the gene is missing 1bp; mutant line #20 is on chromosome A06 BnSKIP31 A06 Insert 1 bp at the target site 1 of the gene, BnSKIP31 on chromosomes C07, A09, and C09 C07 、BnSKIP31 A09 、BnSKIP31 C09 1 bp was inserted into each of the two target sites of the gene, resulting in the inability to encode functional BnSKIP31 protein ( Figure 1 ). This indicates that these plants are true BnSKIP31 CRISPR gene knockout plants.

[0030] (5) Disease resistance analysis of homozygous rapeseed lines with CRISPR knockout structure of BnSKIP31 gene resistant to Sclerotinia sclerotiorum The rapeseed homozygous line transformed with the CRISPR knockout structure of the BnSKIP31 gene (pBSbdcas9i-BnSKIP31) was used as the material, and its resistance to rapeseed sclerotinia sclerotiorum was detected and analyzed by inoculation with Sclerotinia sclerotiorum, thereby clarifying the regulatory role of the BnSKIP31 gene on rapeseed sclerotinia sclerotiorum resistance, laying the foundation for using this gene to create and obtain rapeseed resistant to sclerotinia sclerotiorum.

[0031] Activation culture of Sclerotinia sclerotiorum: Select full and uncontaminated sclerotia of Sclerotinia sclerotiorum, cut the sclerotia into two halves with a sterile blade burned by an alcohol lamp, place them with the cut surface facing down on a PDA solid plate, and culture them at 23℃ in the dark for 3 days. Use a 4mm diameter puncher to punch out mycelium blocks 3 to 5mm inward from the edge of the colony, and inoculate them with the mycelium side facing down on a new PDA solid plate. Culture them at 23℃ in the dark for about 36 hours before inoculation.

[0032] Inoculation of Sclerotinia sclerotiorum: Select rape plants with consistent growth for inoculation. Use a 4mm diameter puncher to punch out hyphae blocks 3-5mm inward from the edge of the colony, and inoculate them with the hyphae facing down into the middle of fully developed leaves. Two hyphae blocks are symmetrically inoculated on the left and right halves of each leaf. Cover with film to keep moisture, and place in a 23℃ greenhouse for cultivation. After an appropriate period of time (about 30 hours), take photos and record them. The area of ​​lesions is analyzed using ImageJ software.

[0033] The inoculation experiment was repeated three times. The lesion area was statistically analyzed by Student's t-test. The results showed that the BnSKIP31 CRISPR gene knockout plants were significantly more resistant to disease than the non-transgenic plants (ZS11) control ( Figure 2 A). Quantitative analysis of the lesion area showed that the lesion area of ​​the ZS11 control plant was 242.1 mm 2 The lesion areas of the two knockout mutant strains were 158.3 mm 2 and 173.8mm 2 , which was significantly lower than that of the control plants ( Figure 2 B). It shows that the resistance of BnSKIP31 CRISPR gene knockout plants to sclerotinia is significantly higher than that of non-transgenic control plants. The inhibition of BnSKIP31 gene expression leads to a significant increase in the resistance of rapeseed to sclerotinia. The present invention successfully creates and obtains a new rapeseed material with increased resistance to sclerotinia by constructing BnSKIP31 CRISPR gene knockout rapeseed.

[0034] In summary, the present invention combines Figure 1-Figure 2 The results revealed for the first time the negative regulatory effect of rapeseed F-box gene BnSKIP31 on rapeseed resistance to Sclerotinia sclerotinia, provided the application approach, application technology and implementation example of BnSKIP31 in the creation of Sclerotinia sclerotinia-resistant crop germplasm, and successfully obtained rapeseed with high resistance to Sclerotinia sclerotinia.

Claims

1. Application of a Brassica napus gene BnSKIP31 in the prevention and control of sclerotinia sclerotiorum, characterized in that: The nucleotide sequence of the gene BnSKIP31 is shown in SEQ ID NO.1, and the protein sequences encoded by the gene BnSKIP31 are shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The application is to obtain rapeseed materials with altered disease resistance by creating gene-edited rapeseed (Brassica napus).

3. The use according to claim 2, characterized in that: Application in obtaining rapeseed materials with enhanced resistance to sclerotinia sclerotinia by creating BnSKIP31 CRISPR gene knockout rapeseed.

4. The use according to claim 3, characterized in that: The application of rapeseed materials with enhanced resistance to sclerotinia sclerotinia is achieved by the following steps: (1) Construction and acquisition of CRISPR knockout construct of BnSKIP31 gene A specific sequence fragment of the BnSKIP31 gene was cloned into the CRISPR vector pBSbdcas9i to obtain the CRISPR knockout structure pBSbdcas9i-BnSKIP31; (2) Obtaining Agrobacterium transformed with the CRISPR knockout construct of the BnSKIP31 gene The BnSKIP31 gene CRISPR knockout construct (pBSbdcas9i-BnSKIP31) was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation; (3) Creation and acquisition of BnSKIP31 gene CRISPR knockout rapeseed The CRISPR knockout structure of the BnSKIP31 gene was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed transformed with the CRISPR knockout structure of the BnSKIP31 gene; (4) Obtaining homozygous rapeseed lines with CRISPR knockout of BnSKIP31 gene The antibiotic resistance screening method was used to detect the segregation of traits in the offspring of transgenic plants, and the homozygous rapeseed lines with the CRISPR knockout structure of the BnSKIP31 gene were obtained, in which the offspring traits no longer separated and were stably inherited; (5) Screening, identification and acquisition of homozygous rapeseed lines with CRISPR knockout of the BnSKIP31 gene that is resistant to bacterial sclerotinia The rapeseed homozygous line transformed with the BnSKIP31 gene CRISPR knockout structure (pBSbdcas9i-BnSKIP31) was used as the material to detect and analyze the resistance to Sclerotinia sclerotinia, and to obtain the BnSKIP31 gene CRISPR knockout rapeseed homozygous line with increased resistance to Sclerotinia sclerotinia.

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