Application of Brassica napus gene BnSKIP31 in sclerotinia prevention and control

Abstract

The application provides application of a Brassica napus gene BnSKIP31 in sclerotinia disease prevention and control, and is obtained by creating a gene edited Brassica napus to obtain a Brassica napus material with enhanced disease resistance. A CRISPR gene knockout Brassica napus of BnSKIP31 is constructed, the negative regulation function of the gene on sclerotinia disease resistance is first revealed, and the application of the BnSKIP31 gene in creating a CRISPR gene knockout Brassica napus to obtain a sclerotinia disease resistant Brassica napus material with increased sclerotinia disease resistance is provided. The BnSKIP31 gene provided by the application is a new gene resource suitable for creating and breeding new materials and new varieties of sclerotinia disease resistant Brassica napus, and the disease resistance of the created Brassica napus germplasm is durable.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of the rapeseed gene BnSKIP31 in the control of sclerotinia stem rot. Background Technology

[0002] 1. F-box proteins and plant disease resistance

[0003] Plant disease resistance results from the activation of resistance signal transduction and a series of defense responses through the recognition of pathogen ligands by plant receptors. From a genetic perspective, after receptor-ligand recognition, genes involved in resistance regulation include early resistance signal transduction genes and late defense-related genes. Generally, regulating resistance through early resistance signal transduction genes is more effective.

[0004] E3 ubiquitin ligases play a crucial role in the ubiquitin-proteasome protein degradation system, directly mediating the degradation of target proteins, including early disease resistance signaling proteins, thereby affecting the stability of these proteins. Knocking out the E3 ubiquitin ligase gene can alter its regulation of certain disease resistance-related proteins, thus affecting plant disease resistance. F-box proteins are a core component of the SCF (Skp1–Cullin–F-box) type E3 ubiquitin ligase complex, responsible for targeting substrate proteins. After binding to other subunits such as Cullin and Rbx1, F-box proteins can promote the transfer of ubiquitin molecules to target proteins, thereby labeling and degrading them. Our research has found that the rapeseed F-box protein BnSKIP31 directly binds to and targets the degradation of the early disease resistance signaling 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 that improves disease resistance, but there are currently no public reports on this strategy.

[0005] 2. Control techniques for plant sclerotinia stem rot

[0006] Sclerotinia sclerotiorum is a plant rot caused by infection with the fungus *Sclerotinia sclerotiorum*. *Sclerotinia sclerotiorum* is a necrotrophic pathogen with a very wide host range and is a major disease of oilseed and vegetable crops, causing significant economic losses annually. Due to the lack of highly resistant varieties, chemical control remains an important method. However, given the problems of some pesticides such as ecological pollution, toxicity to humans and animals, and the ease with which pathogens develop resistance, identifying important regulatory genes for sclerotinia sclerotiorum resistance and creating and utilizing resistant varieties are crucial for the green control of sclerotinia sclerotiorum.

[0007] 3. Plant disease resistance breeding technology

[0008] Plant disease resistance breeding techniques are mainly divided into two categories: traditional disease resistance breeding and disease resistance breeding through genetic engineering and molecular modification. Traditional disease resistance breeding is significantly limited by natural genetic isolation, restricting the availability of disease-resistant resources to those with closely related genetic relationships. It also requires multiple hybridizations and backcrosses, resulting in a long breeding cycle and significant human and material resources. In contrast, genetic engineering and molecular modification breeding methods introduce exogenous disease resistance regulatory genes or modified genes into plants using techniques such as Agrobacterium-mediated transformation, or knock out or increase the expression of target genes through gene editing, thereby acquiring or enhancing disease resistance that was not previously present. Therefore, genetic engineering and molecular modification breeding methods overcome the limitations of natural genetic isolation, broaden the range of available disease resistance resources, and are relatively simple and convenient to operate, with a short breeding cycle and minimal human and material resources required. Furthermore, by modifying broad-spectrum disease resistance regulatory genes or introducing multiple genes with different disease resistance spectra, varieties with broad-spectrum disease resistance can be created. Therefore, they are particularly suitable for breeding varieties with broad-spectrum and durable disease resistance. Summary of the Invention

[0009] The purpose of this invention is to provide an application of the rapeseed (Brassica napus) F-box gene BnSKIP31 in the control of sclerotinia disease. The rapeseed F-box gene BnSKIP31 has a negative regulatory function on sclerotinia disease resistance and can be applied to the creation of germplasm for crops resistant to Sclerotinia sclerotiorum.

[0010] The application is in the creation of rapeseed with the BnSKIP31 gene knocked out to obtain rapeseed materials with increased resistance to sclerotinia stem rot.

[0011] This invention uses cDNA from the Shuang 11 variety of rapeseed (Brassica napus) as a template to clone the rapeseed gene BnSKIP31 via PCR. Its nucleotide sequence is shown in SEQ ID No. 1. The open reading frame (ORF) of this gene is 945 bp long, encoding a protein of 314 amino acids, the sequence of which is shown in SEQ ID No. 2. The BnSKIP31 protein contains an F-box domain and a Herpes domain. The nucleotide sequence cloned in this invention is completely identical to the nucleotide sequence ZS11C07G030650 of the rapeseed variety ZS11 in the Brassica_napus cv.ZS11 PacBio V1.0 database.

[0012] Prior to this invention, the function of this gene had not been publicly reported. This invention, for the first time, elucidates the regulatory role of this gene in rapeseed resistance to Sclerotinia sclerotinia by constructing a CRISPR knockout of this gene and analyzing its disease resistance. Analysis of lesion area and pathogen biomass on inoculated leaves showed that, compared to wild-type rapeseed plants, the knockout plants were significantly more resistant to Sclerotinia sclerotinia, indicating that BnSKIP31 negatively regulates rapeseed resistance to Sclerotinia sclerotinia.

[0013] Based on the BnSKIP31 gene function explained in this invention, the purpose of this invention is to provide the application of the aforementioned rapeseed BnSKIP31 gene in obtaining rapeseed materials with increased resistance to sclerotinia stem rot by creating CRISPR gene knockout rapeseed (Example 1).

[0014] The application of the rapeseed BnSKIP31 gene in obtaining rapeseed materials with increased resistance to sclerotinia stem rot by creating CRISPR gene knockout rapeseed is achieved through the following steps:

[0015] (1) Construction and acquisition of CRISPR knockout structure of BnSKIP31 gene: Two 20bp knockout targets were pre-defined based on the BnSKIP31 genome sequence. The target sequences were cloned into the plant expression vector pBSbdcas9i. The recombinant structure pBSbdcas9i-BnSKIP31 was obtained by colony PCR identification and sequencing.

[0016] (2) Obtaining Agrobacterium that has transformed the BnSKIP31 gene CRISPR knockout structure: The BnSKIP31 gene CRISPR knockout structure (pBSbdcas9i-BnSKIP31) was transformed into Agrobacterium strains with strong infectivity to rapeseed by electroporation and other methods.

[0017] (3) Creation and acquisition of rapeseed with BnSKIP31 gene CRISPR knockout structure: The BnSKIP31 gene CRISPR knockout structure was introduced into rapeseed by Agrobacterium-mediated transformation to obtain rapeseed with BnSKIP31 gene CRISPR knockout structure.

[0018] (4) Obtaining homozygous rapeseed lines with BnSKIP31 gene CRISPR knockout structure: Using antibiotic resistance screening, the phenotypic segregation of the offspring of transgenic plants was detected, and homozygous rapeseed lines with BnSKIP31 gene CRISPR knockout structure that no longer segregate in the offspring and can be stably inherited were obtained.

[0019] (5) Screening, identification and acquisition of homozygous rapeseed lines with CRISPR knockout structure of BnSKIP31 gene that have increased resistance to sclerotinia stem rot: Using homozygous rapeseed lines with CRISPR knockout structure of BnSKIP31 gene (pBSbdcas9i-BnSKIP31) as materials, the resistance to sclerotinia stem rot was detected and analyzed, and rapeseed with CRISPR knockout structure of BnSKIP31 gene that has increased resistance to sclerotinia stem rot was obtained.

[0020] Advantages of the present invention: (1) The BnSKIP31 gene provided by the present invention is a high-quality regulatory gene resource for resistance to sclerotinia stem rot. The BnSKIP31 gene encodes an F-box protein, which directly targets the degradation of early disease resistance signaling proteins such as BnCNGC3, and is a negative regulatory protein for plant disease resistance. By knocking out this gene through gene editing technology to obtain disease-resistant germplasm, it will not lead to rapid co-evolution of pathogens' reverse defense response, thus the resistance is long-lasting. Using this gene to create disease-resistant germplasm is an economical, effective and safe way to control diseases in a green manner. Therefore, the BnSKIP31 gene is a novel gene resource suitable for creating and breeding new materials and varieties of rapeseed resistant to sclerotinia stem rot. (2) Short cycle for obtaining disease-resistant materials. The main methods for obtaining disease-resistant plant materials and varieties are conventional traditional breeding methods and genetic engineering breeding methods using disease resistance regulatory genes. Traditional breeding methods have disadvantages such as the range of available disease-resistant resources being limited by natural genetic isolation, long breeding cycles, and the need for a large amount of artificial resources. Genetic engineering breeding methods offer advantages such as a wide range of available disease-resistant resources, relatively simple and convenient operation, short breeding cycle, no need for large amounts of manpower and resources, and particular suitability for breeding broad-spectrum, durable, and highly disease-resistant varieties. This invention utilizes the disease-resistant regulatory gene BnSKIP31 and employs genetic engineering methods to create and breed rapeseed materials with high resistance to Sclerotinia stem rot, featuring a short cycle and rapid breeding. Attached Figure Description

[0021] Figure 1 This invention provides evidence of gene-edited rapeseed plants obtained through this invention, identifying gene editing in BnSKIP31 CRISPR gene knockout plants and demonstrating the editing pattern of this gene in BnSKIP31 gene knockout plants. Two knockout mutant lines, #4 and #20, were successfully obtained, exhibiting different editing patterns in four BnSKIP31 homologous genes in the ZS11 variety background. Mutant line #4 shows BnSKIP31 on chromosome A06. A06 Gene target sites 1 and 2 are inserted together by 2 bp into BnSKIP31 on chromosome C07. C07 A 1bp deletion at two target sites on chromosome A09, specifically BnSKIP31. A09 An 8bp sequence is deleted at target site 2 of the gene, in BnSKIP31 on chromosome C09. C09Two gene target sites were deleted by 1 bp; mutant line #20 was found to have BnSKIP31 on chromosome A06. A06 A 1bp insertion is performed at target site 1 of the gene, in chromosomes C07, A09, and C09, using BnSKIP31. C07 BnSKIP31 A09 BnSKIP31 C09 Insertion of 1 bp at each of the two target sites of the gene resulted in the inability to properly encode the functional BnSKIP31 protein. Figure 1 This indicates that these plants are true BnSKIP31 gene knockout plants.

[0022] Figure 2 Evidence was provided regarding the anti-sclerotinia stem rot function of the BnSKIP31 gene, showing that BnSKIP31 negatively regulates the resistance of rapeseed to Sclerotinia stem rot. Inoculation analysis of rapeseed BnSKIP31 gene knockout plants with Sclerotinia stem rot UF-1 strain was performed. The figure shows the quantitative statistical analysis results of phenotype, lesion area, and mycelial biomass of the gene knockout mutant plants after inoculation. The inoculation experiment was repeated three times. Student's t-test was used to statistically analyze the lesion area using GraphPad Prism. Data are expressed as mean ± standard deviation. Significance differences are indicated by different numbers of asterisks (*, P < 0.05; ****, P < 0.0001). The results showed that the gene knockout mutant plants exhibited a significantly more resistant phenotype than the control (ZS11) plants. Quantitative analysis of lesion area showed that the lesion area of ​​the two knockout mutant lines was 158.3 mm². 2 and 173.8mm 2 It was significantly lower than the control group's 242.1 mm. 2 The bacterial counts were only 65.4% and 71.8% of those in the control group, respectively. Bacterial counts also showed that the bacterial count in lesions of the gene knockout mutant plants was significantly lower than that in the control plants. These results indicate that BnSKIP31 negatively regulates the resistance of rapeseed to Sclerotinia sclerotiorum. Detailed Implementation

[0023] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0024] Example 1

[0025] This invention, for the first time, elucidates the negative regulatory function of the BnSKIP31 gene on resistance to Sclerotinia sclerotinia in rapeseed by constructing a CRISPR gene knockout of the BnSKIP31 gene and combining it with inoculation analysis. Knockout of the BnSKIP31 gene leads to a significant increase in rapeseed resistance to Sclerotinia sclerotinia. Therefore, new rapeseed materials with enhanced resistance to Sclerotinia sclerotinia can be created and obtained by constructing a CRISPR gene knockout of BnSKIP31 gene, which can be used for the creation and breeding of Sclerotinia sclerotinia resistant rapeseed varieties, and for analyzing the function and mechanism of action of the BnSKIP31 gene. The main steps of analyzing the function and mechanism of the BnSKIP31 gene and creating and obtaining new rapeseed materials with enhanced resistance to Sclerotinia sclerotinia include:

[0026] (1) Construction and acquisition of the CRISPR knockout structure of the BnSKIP31 gene

[0027] Two suitable target sites were designed based on the BnSKIP31 genome sequence. Each gRNA unit contains 20bp gRNA and a gRNA backbone. The two gRNA units were combined to form 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 italicized part is a sequence containing the Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (sequence shown in SEQ ID NO.3) and BnSKIP31-CR-R1 (5'-cagtggtctc aaa acg tgg ggt cta atg ccttca atg cac-3', the italicized part is a sequence containing the Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (sequence shown in SEQ ID NO.4). The vector pBSbdcas9i was digested with Bsa I (Eco31 I), and recombinant ligation was performed using T4 ligase. Subsequent transformation and screening on kanamycin agar plates were conducted, and positive single clones were picked. PCR identification was performed using pBSbdcas9i identification primers M13F (5'-gta aaa cga cgg cca gt-3') (sequence shown in SEQ ID NO. 5) and zmpl: (5'-cca gaa att gaa cgc cga ag-3') (sequence shown in SEQ ID NO. 6). Further sequencing identification yielded the CRISPR knockout structure pBSbdcas9i-BnSKIP31.

[0028] (2) Obtaining Agrobacterium tumefaciens transformed with the BnSKIP31 gene CRISPR knockout structure

[0029] The CRISPR knockout structure of the BnSKIP31 gene (pBSbdcas9i-BnSKIP31) was transformed into Agrobacterium strains with strong infectivity against rapeseed, such as EHA105, by methods such as electroporation. Transformants were screened on YEP medium containing kanamycin and then identified by PCR to obtain Agrobacterium carrying the CRISPR knockout structure of the BnSKIP31 gene, pBSbdcas9i-BnSKIP31.

[0030] (3) Creation and acquisition of rapeseed with BnSKIP31 gene CRISPR knockout structure

[0031] The CRISPR knockout structure pBSbdcas9i-BnSKIP31 of the BnSKIP31 gene was introduced into rapeseed using Agrobacterium-mediated transformation to obtain the T0 generation of rapeseed transgenic pBSbdcas9i-BnSKIP31. The specific operational steps are as follows:

[0032] (i) Seed washing and germination

[0033] 75% ethanol for 30-60 seconds, sterile water once, 1 min / time; 0.15% mercuric chloride for 10 min, sterile water rinse twice, 1 min / time; sterile water rinse for 30 min, inoculate onto sterile filter paper and air dry; inoculate seeds into culture flasks and incubate in the dark at 23℃ for 5-6 days.

[0034] (ii) Pre-culture

[0035] Cut the hypocotyls of germinating rapeseed seedlings into 0.4-0.6 cm segments, inoculate them into pre-culture medium, and culture at 23°C under light for 2-3 days.

[0036] (iii) Agrobacterium infection and co-culture

[0037] Agrobacterium was picked into the infection solution to prepare OD. 600 The explants were inoculated into a 0.2 μL Agrobacterium suspension and incubated for 10 min. After inoculation, the explants were inoculated onto sterile filter paper and air-dried, then inoculated onto co-culture medium and incubated in the dark at 23°C for 48-72 h.

[0038] (iv) Destermination (extended screening)

[0039] The co-cultured explants were inoculated onto sterile culture medium and cultured at 23°C under light for 6 days.

[0040] (v) Screening / Differentiation

[0041] The sterilized explants were inoculated onto selection / differentiation medium, with 30 explants per plate, and cultured at 23°C under light. The plates were changed every 15 days.

[0042] (vi) Rooting culture

[0043] The differentiated buds were inoculated into rooting medium and cultured under light at 23°C until roots were formed.

[0044] (vii) Detection

[0045] Genomic DNA was extracted from rapeseed using the CTAB method, and resistance genes were detected by PCR.

[0046] (viii) Soil cultivation

[0047] After the kanamycin-resistant seedlings have grown to full root system, the T0 generation sterile seedlings are cultured and hardened off at 27℃ for 2 days with the cover off. Then they are transplanted into the soil and placed in an incubator for routine management. Finally, T0 seeds are harvested.

[0048] (4) Obtaining homozygous lines of rapeseed with BnSKIP31 gene CRISPR knockout structure

[0049] Antibiotic resistance screening was used to detect phenotypic segregation in the progeny of transgenic plants. Rapeseed seeds were screened on Basta-resistant plates to observe whether they could grow into healthy seedlings normally. Homozygous rapeseed lines transgenic with the BnSKIP31 gene CRISPR knockout structure pBSbdcas9i-BnSKIP31, whose progeny no longer segregated and could be stably inherited, were obtained. All of these homozygous rapeseed lines grew into healthy seedlings normally on Basta-resistant plates.

[0050] This invention obtained a homozygous line of rapeseed with the BnSKIP31-CRISPR gene knockout, which can grow into healthy seedlings normally on Basta resistance plates. Using BnSKIP31... A06 -CR-F2(5'-ctc tca tct ctc tgt ccc tt-3') (sequence shown in SEQ ID NO.7) and BnSKIP31 A06 The -CR-R2(5'-atc aac aac tat gag atg ca-3') primer pair (sequence shown in SEQ ID NO. 8) detects 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 The -CR-R3(5'-cag ctg tga tat gca cat gt-3') primer pair (sequence shown in SEQ ID NO.10) detects BnSKIP31 on chromosome C07. C07Gene 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 The -CR-R4(5'-gct tct gac aag ttc tat cat a-3') primer pair (sequence shown in SEQ ID NO.12) detects 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 The -CR-R5(5'-gct tct gac cag ttc aat cat a-3') primer pair (sequence shown in SEQ ID NO.14) detects BnSKIP31 on chromosome C09. C09 Gene editing was performed to determine if the transformed seedlings were gene-edited. Sequence sequencing was then conducted on the target site of the gene-edited seedlings, ultimately confirming them as mutant plants with successful BnSKIP31 gene knockout. Sequencing results showed that two BnSKIP31 gene knockout mutant lines, #4 and #20, were successfully obtained, exhibiting different editing patterns in the four BnSKIP31 homologous genes within the ZS11 cultivar background: mutant line #4 showed different editing patterns in the BnSKIP31 gene on chromosome A06. A06 Gene target sites 1 and 2 are inserted together by 2 bp into BnSKIP31 on chromosome C07. C07 A 1bp deletion at two target sites on chromosome A09, specifically BnSKIP31. A09 An 8bp sequence is deleted at target site 2 of the gene, in BnSKIP31 on chromosome C09. C09 Two gene target sites were deleted by 1 bp; mutant line #20 was found to have BnSKIP31 on chromosome A06. A06 A 1bp insertion is performed at target site 1 of the gene, in chromosomes C07, A09, and C09, using BnSKIP31. C07 BnSKIP31 A09 BnSKIP31 C09 Insertion of 1 bp at each of the two target sites of the gene resulted in the inability to properly encode the functional BnSKIP31 protein. Figure 1 This indicates that these plants are true BnSKIP31 CRISPR gene knockout plants.

[0051] (5) Disease resistance detection and analysis of CRISPR knockout structures of transgenic BnSKIP31 gene in homozygous rapeseed lines resistant to sclerotinia stem rot

[0052] Using homozygous rapeseed lines transfected with the CRISPR knockout structure of the BnSKIP31 gene (pBSbdcas9i-BnSKIP31) as materials, we investigated and analyzed their resistance to Sclerotinia sclerotiorum by inoculating them with the bacteria. This clarified the regulatory role of the BnSKIP31 gene in Sclerotinia sclerotiorum resistance and laid the foundation for creating and obtaining Sclerotinia resistant rapeseed using this gene.

[0053] Activation culture of Sclerotinia sclerotiorum: Select plump and uncontaminated Sclerotinia sclerotiorum sclerotia, cut the sclerotia in half with a sterile blade that has been flammed with an alcohol lamp, place them cut side down on a PDA solid plate, and incubate at 23°C in the dark for 3 days. Use a 4mm diameter punch to take a piece of hyphae 3-5mm inward from the edge of the colony, and inoculate it onto a new PDA solid plate with the hyphae side down. Incubate at 23°C in the dark for about 36 hours before inoculation.

[0054] Inoculation of Sclerotinia sclerotiorum: Select rapeseed plants with uniform growth for inoculation. Use a 4mm diameter punch to collect mycelial blocks 3-5mm inward from the edge of the colony. Inoculate the mycelial blocks with the mycelial side down into the middle of a fully developed leaf, symmetrically inoculating two mycelial blocks on each half of the leaf. Cover with a film to retain moisture and incubate in a greenhouse at 23℃. After an appropriate time (about 30 hours), take photos and record the data. Analyze the lesion area using ImageJ software.

[0055] The inoculation experiment was repeated three times. Student's t-test was used for statistical analysis of lesion area. Results showed that BnSKIP31 CRISPR gene knockout plants were significantly more resistant to the disease than the non-transgenic plant (ZS11) control. Figure 2 A). Quantitative analysis of lesion area showed that the lesion area of ​​the ZS11 control plant was 242.1 mm². 2 The lesion areas of the two knockout mutant lines were 158.3 mm². 2 and 173.8mm 2 Significantly lower than the control plants ( Figure 2 B). This indicates that BnSKIP31 CRISPR gene knockout plants exhibit significantly higher resistance to Sclerotinia stem rot than non-transgenic control plants. Inhibition of BnSKIP31 gene expression leads to a significant increase in rapeseed resistance to Sclerotinia stem rot. This invention successfully created a new rapeseed material with enhanced resistance to Sclerotinia stem rot by constructing BnSKIP31 CRISPR gene knockout rapeseed.

[0056] In summary, this invention combines Figures 1-2The results revealed for the first time the negative regulatory effect of the rapeseed F-box gene BnSKIP31 on rapeseed resistance to Sclerotinia stem rot, provided the application pathways, application technologies and examples of BnSKIP31 in the creation of germplasm for Sclerotinia stem rot resistant crops, and successfully obtained rapeseed with high resistance to Sclerotinia stem rot.

Claims

1. Use of a Brassica napus (canola) gene Brassica napus BnSKIP31 in the control of Sclerotinia sclerotiorum (Lib.) de Bary Sclerotinia sclerotiorum caused by Sclerotinia sclerotiorum (Lib.) de Bary, characterized in that, The gene BnSKIP31 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the protein sequence encoded by the gene is shown as SEQ ID NO.

2. ​ 2. Use according to claim 1, characterized in that, In the creation of knock-out BnSKIP31 genes in Brassica napus to obtain Brassica napus material with increased resistance to Sclerotinia disease.

3. Use according to claim 2, characterized in that, The use of Brassica napus material with increased resistance to Sclerotinia sclerotiorum is achieved by the following steps: (1) BnSKIP31 Construction and acquisition of gene CRISPR knockout structure Will BnSKIP31 A specific sequence fragment of the gene was cloned into the CRISPR vector pBSbdcas9i to obtain the CRISPR knockout structure pBSbdcas9i- BnSKIP31 ; (2) Transformation BnSKIP31 Agrobacterium acquisition of CRISPR knock-out constructs Will BnSKIP31 Gene CRISPR knock-out construct pBSbdcas9i- BnSKIP31 Agrobacterium strains with strong infection ability on Brassica napus were transformed by the electric shock method; (3) trans BnSKIP31 Creation and acquisition of a genetically CRISPR-knocked-out structured oilseed rape The gene CRISPR knockout structure is introduced into rapeseed by Agrobacterium-mediated method to obtain transgenic rapeseed BnSKIP31 The gene CRISPR knockout structure is introduced into rapeseed by Agrobacterium-mediated method to obtain transgenic rapeseed BnSKIP31 The gene CRISPR knockout structure is introduced into rapeseed by Agrobacterium-mediated method to obtain transgenic rapeseed (4) trans BnSKIP31 Obtaining a homozygous line of rapeseed with a CRISPR knock-out of the gene The transgenic plant offspring is detected by means of antibiotic resistance screening, and a transgenic plant offspring with stable inheritance and no segregation of traits is obtained BnSKIP31 A homozygous line of rapeseed with a CRISPR knockout structure (5) Anti-phythophthora BnSKIP31 Screening and obtaining of homozygous lines of oilseed rape with CRISPR knock-out of the gene With the trans BnSKIP31 CRISPR knockout structure pBSbdcas9i- BnSKIP31 The homozygous line of Brassica napus with the gene CRISPR knockout is used as the material to detect and analyze the resistance to sclerotinia stem rot, and the homozygous line of Brassica napus with the gene CRISPR knockout with increased resistance to sclerotinia stem rot is obtained. BnSKIP31 The homozygous line of Brassica napus with the gene CRISPR knockout is used as the material to detect and analyze the resistance to sclerotinia stem rot, and the homozygous line of Brassica napus with the gene CRISPR knockout with increased resistance to sclerot

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