Application of Brassica Receptor Gene BnRLP41 in Sclerotinia Disease Prevention and Control

Abstract

The application provides application of a Brassica napus class receptor gene BnRLP41 in sclerotinia disease prevention and control, and is obtained by creating transgenic oilseed rape to change the disease resistance of the oilseed rape material. The application constructs a BnRLP41 super-expression and CRISPR gene knockout transgenic oilseed rape, first discloses the positive regulation function of the gene on sclerotinia disease resistance, provides application of the BnRLP41 gene in obtaining high-sclerotinia-disease-resistant oilseed rape material by creating super-expression oilseed rape, and application of the BnRLP41-CRISPR gene knockout oilseed rape in obtaining sclerotinia disease resistance weakened sclerotinia disease oilseed rape material. The BnRLP41 gene provided by the application is a new gene resource suitable for creating and breeding new sclerotinia disease-resistant oilseed rape materials and new varieties.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to application of a Brassica receptor gene BnRLP41 in sclerotinia disease prevention and control. BACKGROUND

[0002] 1. Plant gene function analysis technology

[0003] Plant gene function is usually judged and clarified by comparing and analyzing phenotypes or function exertion in the case of super-normal expression and normal expression of the gene. The super-normal expression of the gene includes two categories of higher-than-normal expression and lower-than-normal expression. The higher-than-normal expression is mainly over-expression, which is mainly achieved by connecting a strong promoter to drive expression of the target gene. The lower-than-normal expression is mainly achieved by RNA interfering (RNAi) and gene knock-out. RNAi is achieved by constructing and transforming a hairpin structure containing a fragment of the same sequence inserted in opposite directions or other non-expressed sequences, resulting in reduction of expression of the target gene. Gene knock-out is achieved by inserting a long non-plant sequence into the target gene in the plant genome or excising the target gene, resulting in complete or nearly complete inhibition of expression of the target gene. By constructing gene over-expression plants and / or RNAi plants and gene knock-out mutants, comparing and analyzing differences in phenotypes and traits between the plants and wild-type / normal plants, the regulatory function of the target gene on the traits can be clarified.

[0004] 2. Plant disease resistance genetic regulation technology

[0005] Plant disease resistance is a result of activation of disease resistance signaling and activation of a series of defense responses by plant receptors recognizing ligands of pathogens. From the perspective of genetics, after recognition of the receptor-ligand, disease resistance signaling is activated, and immune disease resistance is activated. When plants face pathogen invasion, the first layer of innate immune system is started by specific recognition of conserved molecular patterns of pathogenic bacteria by pattern recognition receptors on the surface of plant cell membranes. Recognition of pathogens and signal transduction and activation of intracellular disease resistance pathways are crucial for disease resistance. Receptor-like protein (RLP) is one of the two major receptors known to recognize pathogenic molecular patterns in plants. It is expected that RLP genes can be used to create crop germplasm with enhanced disease resistance, but there are few public reports on this aspect.

[0006] 3. Plant sclerotinia disease prevention and control technology

[0007] Sclerotinia sclerotiorum is a necrotrophic pathogen with a very wide host range, and is a major disease of oilseed crops and vegetable crops, causing huge economic losses every year. Due to the lack of highly resistant varieties, chemical control is still an important means. Due to the ecological pollution, toxicity to humans and animals, and easy to make pathogens resistant to some pesticides, identifying important Sclerotinia resistance regulatory genes, creating and using disease-resistant varieties are of great importance to the green prevention and control of Sclerotinia.

[0008] 4. Plant disease-resistant breeding technology

[0009] Plant disease-resistant breeding technology mainly includes traditional disease-resistant breeding and disease-resistant breeding through genetic engineering. Traditional disease-resistant breeding is significantly limited in the range of available disease-resistant resources due to natural genetic isolation, and can only use disease-resistant resources with close genetic relationship. In addition, it needs multiple crosses and backcrosses, and thus has a long breeding cycle and requires a large amount of manpower and material resources. Genetic engineering breeding method is to introduce an exogenous disease-resistant regulatory gene into a plant through Agrobacterium-mediated method, so that the plant obtains disease resistance that it originally does not have. Therefore, genetic engineering breeding method breaks the limitation of natural genetic isolation, widens the range of available disease-resistant resources, and has the characteristics of relatively simple and convenient operation, short breeding cycle, and no need for a large amount of manpower and material resources. In addition, a broad-spectrum disease-resistant regulatory gene or multiple genes with different disease resistance spectrum can be introduced to create a variety with broad-spectrum disease resistance. Therefore, it is particularly suitable for breeding broad-spectrum and durable disease-resistant varieties. SUMMARY

[0010] The purpose of the present application is to provide an application of Brassica napus class receptor gene BnRLP41 in Sclerotinia disease prevention and control. The Brassica napus class receptor gene BnRLP41 has a regulatory function on disease resistance, and can be applied to the creation of Sclerotinia sclerotiorum crop germplasm.

[0011] The application is the application of the transgenic Brassica napus in the Brassica napus material with changed disease resistance obtained by creating the transgenic Brassica napus. The application is the application of the transgenic Brassica napus with overexpressed BnRLP41 in the Brassica napus material with increased Sclerotinia disease resistance, or the application of the transgenic Brassica napus with BnRLP41-CRISPR gene knockout in the Brassica napus material with reduced Sclerotinia disease resistance.

[0012] The application takes Brassica napus double 11 variety cDNA as a template, and obtains Brassica napus gene BnRLP41 through PCR cloning, the nucleotide sequence of which is shown as SEQ ID:1, the open reading frame (ORF) of the gene is 2655 bp long, the encoded protein is composed of 884 amino acids, and the sequence is shown as SEQ ID:2. The BnRLP41 protein contains an LRR domain. The cloned nucleotide sequence of the application is consistent with the LOC106447094 nucleotide sequence of Brassica napus variety ZS11 in the NCBI database, and the protein sequence is identical to XP_013744403.2.

[0013] Before the application, there is no any public report on the function of the gene. The application first clarifies the regulation of the gene on the resistance of Brassica napus to Sclerotinia sclerotiorum by constructing super-expression and CRISPR knockout transgenic Brassica napus of the gene and analyzing the resistance of the transgenic Brassica napus. The results show that the super-expression transgenic Brassica napus is significantly more resistant to Sclerotinia sclerotiorum than the wild-type Brassica napus, and the knockout transgenic Brassica napus is significantly more susceptible to Sclerotinia sclerotiorum, indicating that BnRLP41 positively regulates the resistance of Brassica napus to Sclerotinia sclerotiorum.

[0014] Based on the clarified function of the BnRLP41 gene in the application, the application aims to provide the application of the Brassica napus BnRLP41 gene in obtaining Brassica napus materials with changed disease resistance by creating transgenic Brassica napus, including (1) the application in obtaining Brassica napus materials with increased resistance to Sclerotinia sclerotiorum by creating transgenic Brassica napus with super-expressed BnRLP41 (Example 1); and (2) the application in obtaining Brassica napus materials with reduced resistance to Sclerotinia sclerotiorum by creating transgenic Brassica napus with BnRLP41-CRISPR gene knockout (Example 2).

[0015] The application of the Brassica napus BnRLP41 gene in obtaining Brassica napus materials with increased resistance to Sclerotinia sclerotiorum by creating transgenic Brassica napus with super-expressed BnRLP41 is specifically realized by the following steps:

[0016] (1) Construction and acquisition of BnRLP41 gene super-expression structure: cloning the open reading frame (ORF) of the BnRLP41 gene into a plant expression vector to make it express under the drive of a strong promoter;

[0017] (2) Acquisition of Agrobacterium transformed with the BnRLP41 gene super-expression structure: transforming the constructed BnRLP41 gene super-expression structure into an Agrobacterium strain with strong invasiveness to Brassica napus by electroporation or other methods;

[0018] (3) Creation and acquisition of transgenic Brassica napus with super-expressed BnRLP41: introducing the BnRLP41 gene super-expression structure into Brassica napus through Agrobacterium-mediated method to obtain Brassica napus with the BnRLP41 gene super-expression structure.

[0019] (4) Obtaining a homozygous line of transgenic rapeseed overexpressing BnRLP41: the segregation of the traits of the offspring of the transgenic plants is detected by using antibiotic resistance and BnRLP41 gene expression as detection indexes, and a homozygous line of transgenic rapeseed overexpressing BnRLP41 which is no longer segregated and can be stably inherited is obtained;

[0020] (5) Screening, identification and obtaining of a homozygous line of transgenic rapeseed overexpressing BnRLP41 with increased resistance to sclerotinia blight: the resistance to sclerotinia blight of the homozygous line of transgenic rapeseed overexpressing BnRLP41 is detected and analyzed, and transgenic rapeseed overexpressing BnRLP41 with increased resistance to sclerotinia blight is obtained.

[0021] Application of rapeseed BnRLP41 gene in obtaining rapeseed material with reduced resistance to sclerotinia blight through creating transgenic rapeseed with BnRLP41-CRISPR gene knockout. The following steps are taken to achieve this:

[0022] (1) Construction and obtaining of BnRLP41 gene CRISPR knockout structure: three 20-bp knockout targets are preset according to the BnRLP41 genomic sequence, and the target sequence is cloned into the plant expression vector pBSbdcas9i. Colony PCR identification and sequencing are performed to obtain the recombination structure pBSbdcas9i-BnRLP41;

[0023] (2) Obtaining Agrobacterium transformed with BnRLP41 gene CRISPR knockout structure: the BnRLP41 gene CRISPR knockout structure (pBSbdcas9i-BnRLP41) is transformed into an Agrobacterium strain with strong invasion ability to rapeseed by electroporation or other methods;

[0024] (3) Creation and obtaining of transgenic rapeseed with BnRLP41 gene CRISPR knockout structure: the BnRLP41 gene CRISPR knockout structure is introduced into rapeseed by Agrobacterium-mediated method, and transgenic rapeseed with BnRLP41 gene CRISPR knockout structure is obtained;

[0025] (4) Obtaining a homozygous line of transgenic rapeseed with BnRLP41 gene CRISPR knockout structure: antibiotic resistance is used for screening, the segregation of the traits of the offspring of the transgenic plants is detected, and a homozygous line of transgenic rapeseed with BnRLP41 gene CRISPR knockout structure which is no longer segregated and can be stably inherited is obtained;

[0026] (5) Screening and obtaining of homozygous line of BnRLP41 gene CRISPR knockout structure rapeseed with weakened resistance to sclerotinia disease: taking homozygous line of BnRLP41 gene CRISPR knockout structure (pBSbdcas9i-BnRLP41) rapeseed as material, detecting and analyzing resistance to sclerotinia disease, and obtaining BnRLP41 gene CRISPR knockout rapeseed with weakened resistance to sclerotinia disease.

[0027] Advantages of the present application: (1) the BnRLP41 gene provided by the present application is a high-quality sclerotinia disease resistance gene resource, and the disease-resistant material obtained by using the gene has the advantages of strong disease resistance. The resistance of rapeseed to sclerotinia disease is quantitative resistance, which is controlled by multiple genes. Generally speaking, the degree of regulation of a single gene on the resistance is low. Therefore, the rapeseed material with resistance to sclerotinia disease is very scarce worldwide, and there is no high-resistance material. The BnRLP41 gene encodes a receptor-like protein, which is a receptor gene for recognizing pathogenic molecular patterns, and has good resistance triggering effect, so it is a high-quality disease resistance gene resource. Using RLP to create disease-resistant varieties and germplasm is an economical, effective and safe way for green disease control. Therefore, the BnRLP41 gene is a new gene resource suitable for creating and breeding new materials and new varieties of rapeseed resistant to sclerotinia disease. (2) The period for obtaining disease-resistant material is short. The methods for obtaining disease-resistant plant material and variety mainly include conventional traditional breeding method and gene engineering breeding method using disease resistance regulatory gene. The traditional breeding method has the disadvantages of limited range of available disease resistance resources due to natural genetic isolation, long breeding period, and the need for a large amount of manual labor. The gene engineering breeding method has the advantages of wide range of available disease resistance resources, relatively simple and convenient operation, short breeding period, no need for a large amount of manual labor, and is particularly suitable for breeding broad-spectrum, durable and highly disease-resistant varieties. The present application uses the disease resistance regulatory gene BnRLP41 and adopts the gene engineering method to create and breed rapeseed material with high resistance to sclerotinia disease, which has the characteristics of short period and rapid breeding. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1The identification evidence of various types of transgenic rape plants obtained by the present application is provided, which shows the detection results of BnRLP41 gene expression in overexpression plants and the gene editing form of gene knockout plants. The expression level of BnRLP41 gene in overexpression (OE) plants in which BnRLP41 gene is silenced is detected by real-time fluorescent quantitative PCR (A), with rape BnACTIN7 as an internal reference gene, and the expression amount of ZS11 is set as 1. The qRT-PCR analysis is performed for three biological repeats, each of which includes three technical repeats, and the expression results are statistically analyzed by Student's t-test using GraphPad Prism, and the data are represented by mean ± standard deviation. The significant difference is indicated by * (**, P < 0.01; ***, P < 0.001). The results show that the expression amount of BnRLP41 in two overexpression lines #7 and #18 is significantly increased, which is 10.5 times and 189.5 times of the control respectively (A). The knockout mutant line lacks a 10 bp sequence at the target site 1 of BnRLP41 gene, which causes the inability to normally encode functional BnRLP41 gene (B). It is shown that these plants are true BnRLP41 gene overexpression plants and CRISPR knockout plants.

[0029] Figure 2 The evidence of the antibacterial sclerotinia function of BnRLP41 gene is provided, which shows that BnRLP41 positively regulates the resistance of rape to sclerotinia. The rape BnRLP41 transgenic plants are subjected to inoculation analysis of sclerotinia UF-1. The results of phenotype, lesion area and hyphal biomass quantitative statistical analysis of BnRLP41 overexpression plants (A) and knockout mutant plants (B) after inoculation are shown in the figure. The inoculation experiment is repeated three times. Student's t-test is used for statistical analysis of lesion area by GraphPad Prism, and the data are represented by mean ± standard deviation. The significant difference is indicated by different numbers of * (*, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001). The results show that the overexpression plants exhibit a more resistant phenotype than the control (ZS11) plants (A), while the knockout mutant plants are significantly more susceptible than the control (B). The quantitative analysis results of lesion area show that the lesion area of two overexpression lines is 85.1 mm 2 and 74.9 mm 2 , which is significantly smaller than 159.1 mm 2 of the control, only 53% and 47% of the control; the hyphal biomass determination also shows that the hyphal biomass contained in the lesion area of the overexpression plants is significantly lower than that of the control plants. The lesion area of the knockout mutant line is 170.6 mm 2 , which is significantly higher than 129.4 mm 2The resistance of the knockout mutant plants to S. sclerotiorum was 1.32 times that of the control, and the amount of bacteria in the lesion of the knockout mutant plants was significantly higher than that of the control, as determined by bacterial amount determination. These results indicate that BnRLP41 positively regulates the resistance of Brassica napus to S. sclerotiorum. DETAILED DESCRIPTION

[0030] The present application is further illustrated in conjunction with the accompanying drawings and examples.

[0031] Example 1

[0032] The present application clones a Brassica napus gene BnRLP41, and first clarifies the positive regulation function of the gene to the resistance to sclerotinia by constructing an overexpression transgenic Brassica napus. The overexpression of the BnRLP41 gene leads to a significant increase in the resistance of Brassica napus to sclerotinia, and thus, a new Brassica napus material with enhanced resistance to sclerotinia can be created by constructing a homozygous line of the overexpression Brassica napus of the BnRLP41 gene, and the new material can be used for the creation and selection of Brassica napus varieties resistant to sclerotinia, the analysis of gene function and mechanism of action, and the like. The main steps of cloning, function and mechanism analysis of the BnRLP41 gene, and the creation and acquisition of a new Brassica napus material with enhanced resistance to sclerotinia include:

[0033] 1) Cloning and preservation of Brassica napus BnRLP41 gene

[0034] The BnRLP41 gene of Brassica napus provided in the present application is cloned through the following steps. First, primers BnRLP41-F (5'-atg tct gaa tcc cgt gtg cgt ttg-3') (the sequence is shown as SEQ ID: 3) and BnRLP41-R (5'-cta acg gtt tct gct ctt gt-3') (the sequence is shown as SEQ ID: 4) are designed according to the BnRLP41 sequence in the Brassica napus genome database. Total RNA is extracted from the leaves of Brassica napus variety Zhongshuang 11 by using TRIZOL reagent, and BnRLP41 cDNA is obtained by using high-fidelity pfu enzyme-mediated RT-PCR method. The PCR product is purified by cutting the gel after 1% agarose gel electrophoresis, and then connected with pEASY-Blunt Cloning Vector. Escherichia coli DH5α is transformed by heat shock, and then cultured in LB medium overnight. Whether the extracted plasmid contains the BnRLP41 gene is tested by PCR method using BnRLP41-F / BnRLP41-R as primers, and finally sent to a company for sequencing verification, so as to successfully clone and obtain the full-length sequence of BnRLP41 gene cDNA. The BnRLP41 nucleotide sequence is shown as SEQ ID: 1, the open reading frame (ORF) of the gene is 2655 bp long, and the encoded protein is composed of 884 amino acids, the sequence of which is shown as SEQ ID: 2. The gene product contains an LRR domain. It is found by BLAST analysis that the cloned nucleotide sequence of the present application is consistent with the LOC106447094 nucleotide sequence of Brassica napus variety ZS11 in the NCBI database, and the protein sequence is the same as XP_013744403.2. There is no report on the function of the gene before the present application.

[0035] The Escherichia coli carrying the pEASY-BnRLP41 vector is transformed and stored in a -80℃ refrigerator. At any time, the strain can be activated, the plasmid is extracted, PCR amplification is performed, and the BnRLP41 gene is subcloned into the target vector for transgenic research.

[0036] 2) Construction and acquisition of BnRLP41 gene overexpression structure

[0037] The BnRLP41 sequence cloned according to the present application is used to design primers BnRLP41-F2 (5'-gacgatgataagggcggtacctctgaatccgtgtgcgtttg-3', the italicized part is a Kpn I enzyme cutting site, the sequence consistent with the linearized vector pCAMBIA1300) (the sequence is shown as SEQ ID: 5) and BnRLP41-R2 (5'-gtcctaggctacgtaggatccctaacggtttctgctcttttcg-3', the italicized part is a Bam HI enzyme cutting site, the sequence consistent with the linearized vector pCAMBIA1300) (the sequence is shown as SEQ ID: 6). The pEASY-BnRLP41 plasmid obtained in 1) of Example 1 is used as a template, and BnRLP41-F2 / BnRLP41-R2 is used as a primer pair to obtain the target gene by PCR amplification.

[0038] The pCAMBIA1300 plasmid is double-enzyme cut by Kpn I and Bam HI, and the obtained target gene is ligated with the enzyme-cut pCAMBIA1300 vector for homologous recombination. The reagents used in the experiment are PCR one-step directional cloning kit. The ligation product is subjected to E. coli transformation, kanamycin plate screening, PCR identification and sequencing identification, etc. to obtain the BnRLP41 gene super-expression structure pCAMBIA1300-BnRLP41. The super-expression vector pCAMBIA1300 drives the expression of the target gene with the CaMV 35S promoter, and carries a FLAG tag, which is convenient for molecular identification and gene function research.

[0039] 3) Obtaining Agrobacterium carrying the BnRLP41 gene super-expression structure pCAMBIA1300-BnRLP41

[0040] The BnRLP41 gene super-expression structure pCAMBIA1300-BnRLP41 is transformed into Agrobacterium strains with strong invasiveness to Brassica napus, such as EHA105, by electroporation or other methods, and the transformants are screened on YEP medium containing kanamycin. Agrobacterium carrying the BnRLP41 gene super-expression structure pCAMBIA1300-BnRLP41 is obtained by double enzyme cutting of Kpn I and Bam HI and PCR identification, which is used for the genetic transformation of Brassica napus in the next step.

[0041] 4) Creation and obtaining of Brassica napus transformed with the BnRLP41 gene super-expression structure pCAMBIA1300-BnRLP41

[0042] The BnRLP41 gene super-expression structure pCAMBIA1300-BnRLP41 is introduced into rape through Agrobacterium-mediated method to obtain T0 generation of the rape with pCAMBIA1300-BnRLP41.

[0043] (i) Seed cleaning and germination

[0044] 75% ethanol 30-60s, sterile water 1 time, 1 min / time; 0.15% mercury 10 min, sterile water cleaning 2 times, 1 min / time; sterile water cleaning 30 min, inoculated on sterile filter paper and dried; the seed is inoculated on a culture bottle, and dark culture is carried out at 23°C for 5-6d.

[0045] (ii) Pre-culture

[0046] The hypocotyl of the germinated rape seedling is cut into 0.4-0.6cm segments, inoculated on pre-culture medium, and cultured at 23°C under light for 2-3d.

[0047] (iii) Agrobacterium infection and co-culture

[0048] The Agrobacterium is picked up in the infection liquid to prepare an Agrobacterium resuspension liquid with OD 600 =0.2, and the explant is inoculated in the Agrobacterium suspension liquid for 10 min. The infected explant is inoculated on sterile filter paper and dried, and inoculated on co-culture medium, and dark culture is carried out at 23°C for 48-72h.

[0049] (iv) Decontamination (delayed screening)

[0050] The co-cultured explant is inoculated on decontamination medium, and light culture is carried out at 23°C for 6d.

[0051] (v) Screening / differentiation

[0052] The decontaminated explant is inoculated on screening / differentiation medium, 30 explants per dish, and light culture is carried out at 23°C, and the plate is replaced every 15d.

[0053] (vi) Rooting culture

[0054] The differentiated bud is inoculated on rooting culture medium, and light culture is carried out at 23°C until rooting.

[0055] (vii) Detection

[0056] The CTAB method is used to extract rape genomic DNA, and PCR detection is carried out on the resistance gene.

[0057] (viii) Soil planting

[0058] After the kanamycin-resistant seedlings grow to the full root system, the T0 generation of sterile seedlings is cultured at 27℃ for 2 days, and then transplanted into the soil and placed in a culture box for routine management, and finally the T0 seeds are obtained.

[0059] 5) Screening and obtaining of the homozygous line of the BnRLP41 gene overexpression structure pCAMBIA1300-BnRLP41 in rape

[0060] The trait segregation of the transgenic plant offspring is detected by taking kanamycin resistance and BnRLP41 gene expression as the detection indexes. The kanamycin resistance screening is performed on the plate containing kanamycin and aimed at the rape seeds, and whether the seeds can grow into healthy seedlings on the kanamycin-resistant plate is observed. The gene expression is detected by using the real-time fluorescent quantitative PCR and other methods. The homozygous line of the BnRLP41 gene overexpression structure pCAMBIA1300-BnRLP41 in rape which has no segregation of the offspring traits and can stably inherit is obtained.

[0061] The two BnRLP41 overexpression homozygous lines of rape, OE-line 7 and OE-line 18, are obtained in the application, and all of them can grow into healthy seedlings on the kanamycin-resistant plate, and the average expression of the BnRLP41 gene is significantly higher than that of the control plants, being 10.5 times and 189.5 times (A) of the control plants respectively. Figure 1 A). It is shown that these plants are the true BnRLP41 gene overexpression plants.

[0062] 6) Detection and analysis of the disease resistance of the BnRLP41 gene overexpression homozygous line of rape

[0063] The BnRLP41 gene overexpression homozygous line of rape obtained in 5) is taken as the material, the resistance of the material to the sclerotinia rot of rape is detected and analyzed by inoculating Sclerotinia sclerotiorum, so that the regulation of the BnRLP41 gene on the sclerotinia rot resistance of rape is determined, and a foundation for creating and obtaining the sclerotinia rot-resistant rape by using the gene is laid.

[0064] The activated culture of Sclerotinia sclerotiorum: full and uncontaminated sclerotia of Sclerotinia sclerotiorum are selected, the sclerotia are cut into two halves by using a sterile knife blade burned by an alcohol lamp, the cut surfaces are placed on PDA solid plates, and the plates are cultured at 23℃ in the dark for 3 days; a puncher with a diameter of 4 mm is used to punch the mycelium block of 3-5 mm from the edge of the colony, and the mycelium side is inoculated on a new PDA solid plate, and the plate is cultured at 23℃ in the dark for about 36 hours.

[0065] Inoculation of S. sclerotiorum: The oilseed rape plants with the same growth vigor were selected for inoculation. The mycelium block with 3-5 mm inside from the colony edge was punched by a puncher with 4 mm diameter, and inoculated to the middle of the fully developed leaf with the mycelium facing down. Each leaf was inoculated with one mycelium block on the left and right half of the leaf symmetrically, and covered with film for moisturizing, and placed in a 23℃ greenhouse for culture. After appropriate time (about 24 h), the photograph was recorded, and the lesion area was analyzed by ImageJ software.

[0066] The inoculation experiment was repeated three times. The lesion area was statistically analyzed by Student's t-test. The results showed that the BnRLP41 overexpression plants were significantly more resistant to disease than the non-transgenic control plants (ZS11) Figure 2 A). The quantitative analysis results of the lesion area showed that the lesion area of the ZS11 control plants was 159.1 mm 2 , and the lesion areas of the two overexpression plants were 85.1 mm 2 and 74.9 mm 2 , which were only 53% and 47% of the control Figure 2 B).

[0067] These results showed that the resistance of the BnRLP41 overexpression plants to Sclerotinia was significantly higher than that of the non-transgenic control plants. The overexpression of the BnRLP41 gene led to a significant increase in the resistance of the oilseed rape to Sclerotinia, and therefore, BnRLP41 played a positive regulatory role in the resistance of the oilseed rape to Sclerotinia. The high Sclerotinia-resistant oilseed rape new material was successfully created by constructing the BnRLP41 overexpression oilseed rape according to the present application.

[0068] Example 2

[0069] According to the positive regulatory function of the oilseed rape gene BnRLP41 to Sclerotinia resistance as clarified in the present application, a technical system for creating and obtaining the new material of the oilseed rape with weakened resistance to Sclerotinia by constructing the CRISPR knockout transgenic oilseed rape of the gene and using the genetic engineering technology was established. The main steps include:

[0070] (1) Construction and acquisition of BnRLP41 gene CRISPR knockout structure

[0071] According to the genomic sequence of BnRLP41 gene, three suitable target sites were designed, each gRNA unit contained 20 bp gRNA and a gRNA backbone, three gRNA units were combined as a complete silencing sequence, and a primer BnRLP41-CR-F (5'-cag tgg tct cat gca cca ata cct cta gcc tgt tg-3', the italic part is a sequence containing a Bsa I or Eco31 I enzyme cutting site, consistent with the linearized vector pBSbdcas9i) (the sequence is shown as SEQ ID: 7) and BnRLP41-CR-R (5'-cag tgg tct caa aac gta aga tcg ttt cca ctt ag-3', the italic part is a sequence containing a Bsa I or Eco31 I enzyme cutting site, consistent with the linearized vector pBSbdcas9i) (the sequence is shown as SEQ ID: 8) were amplified. The vector pBSbdcas9i was cut with Bsa I or Eco31 I, recombined and connected with T4 ligase, followed by transformation, kanamycin medium plate screening, picking positive single colonies, using pBSbdcas9i identification primer Pbw2+ (5'-gca acg ctc tgt cat cgt tac aat-3') (the sequence is shown as SEQ ID: 9) and M49452 (470C):(5'-cta cgg ttc aag aaa atg taa gct gat-3') (the sequence is shown as SEQ ID: 10) for PCR identification, and further sequencing identification to obtain the CRISPR knockout structure pBSbdcas9i-BnRLP41.

[0072] (2) Obtaining Agrobacterium transformed with BnRLP41 gene CRISPR knockout structure

[0073] The BnRLP41 gene CRISPR knockout structure (pBSbdcas9i-BnRLP41) was transformed into Agrobacterium strains with strong invasion ability to Brassica napus, such as EHA105, by electroporation or other methods, and the transformants were screened on YEP medium containing kanamycin, and then identified by PCR to obtain Agrobacterium carrying the BnRLP41 gene CRISPR knockout structure pBSbdcas9i-BnRLP41.

[0074] (3) Creation and acquisition of BnRLP41 gene CRISPR knockout structure transformed Brassica napus

[0075] The BnRLP41 gene CRISPR knockout structure pBSbdcas9i-BnRLP41 was introduced into Brassica napus by Agrobacterium-mediated method, and T0 generation of Brassica napus transformed with pBSbdcas9i-BnRLP41 was obtained. The specific operation steps are as described in 4) of Example 1. Whether the transformed seedling is a BnRLP41 gene editing seedling was detected by BnRLP41-CR-F2 (5'-gaa ctc tga atc cca aca gta ct-3') (the sequence is shown as SEQ ID: 11) and BnRLP41-CR-R2 (5'-gca gtc tga taa ctg caa ata ctc-3') (the sequence is shown as SEQ ID: 12). The sequence of the target site of the gene editing seedling was sequenced, and finally the mutant plant with successfully knocked out BnRLP41 gene was determined.

[0076] (4) Obtaining homozygous lines of Brassica napus transformed with BnRLP41 gene CRISPR knockout structure

[0077] The trait segregation of the transgenic plant offspring was detected by antibiotic resistance screening method. Brassica napus seeds were subjected to Basta-resistant plates, and whether they could grow into healthy seedlings on the Basta-resistant plates was observed. Homozygous lines of Brassica napus transformed with BnRLP41 gene CRISPR knockout structure pBSbdcas9i-BnRLP41 were obtained, which had no trait segregation and could stably inherit. These homozygous lines of Brassica napus could all grow into healthy seedlings on the Basta-resistant plates.

[0078] The BnRLP41-CRISPR gene knockout Brassica napus homozygous line was obtained, which could grow into a healthy seedling on the Basta-resistant plate. The gene knockout mutant line had a 10 bp sequence deletion at the target site 1 of the BnRLP41 gene, which resulted in the inability to normally encode functional BnRLP41 gene (B). It is shown that these plants are true BnRLP41 gene CRISPR knockout plants.

[0079] (5) Screening, identification and obtaining of Brassica napus homozygous lines transformed with BnRLP41 gene CRISPR knockout structure with weakened resistance to sclerotinia disease

[0080] The homozygous lines of Brassica napus transformed with BnRLP41 gene CRISPR knockout structure (pBSbdcas9i-BnRLP41) were used as materials to detect and analyze the resistance to sclerotinia disease. The inoculation method and disease resistance evaluation are as described in 6) of Example 1).

[0081] The results of the S. sclerotiorum inoculation analysis of the BnRLP41-CRISPR gene knockout plants constructed in the application show that the BnRLP41-CRISPR gene knockout plants are obviously more susceptible to the disease than the non-transgenic plant (ZS11) control Figure 2 B). The results of the lesion area quantitative analysis show that the lesion area of the ZS11 control plant is 129.4 mm 2 , and the lesion area of the knockout mutant strain is 170.6 mm 2 , which is significantly higher than that of the control plant Figure 2 B). It is shown that the resistance of the BnRLP41-CRISPR knockout plants to the S. sclerotiorum is significantly lower than that of the non-transgenic control plants. The inhibition of the BnRLP41 gene expression leads to a significant decrease in the resistance of the rape to the S. sclerotiorum. The BnRLP41-CRISPR gene knockout rape is successfully obtained by constructing the BnRLP41-CRISPR gene knockout rape in the application, and the new rape material with weakened resistance to the S. sclerotiorum is obtained.

[0082] In conclusion, the results of the application in combination with Figures 1-2 reveal for the first time that the rape receptor gene BnRLP41 positively regulates the resistance of the rape to the S. sclerotiorum, and the application approach, the application technology and the examples of the BnRLP41 in the creation of the S. sclerotiorum-resistant crop germplasm are provided, and the S. sclerotiorum-resistant rape is successfully obtained.

Claims

1. A type of rapeseed ( Brassica napus receptor-like genes BnRLP41 In the prevention and control of Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Its application in treating rapeseed sclerotinia stem rot caused by ), characterized in that, The gene BnRLP41 The nucleotide sequence is shown in SEQ ID: 1, and the protein sequence it encodes is shown in SEQ ID:

2.

2. The application according to claim 1, characterized in that, By creating hyperexpression BnRLP41 The application of genetically modified rapeseed to obtain rapeseed materials with increased resistance to sclerotinia stem rot.

3. The application according to claim 2, characterized in that, The following steps are used to obtain rapeseed materials with increased resistance to sclerotinia stem rot: (1) BnRLP41 Construction and acquisition of gene overexpression structures Will BnRLP41 The gene open reading frame was cloned into a plant expression vector, which was then expressed under the drive of a strong promoter. (2) Transformation BnRLP41 Obtaining Agrobacterium gene overexpression structures The built BnRLP41 Gene overexpression structures were transformed into Agrobacterium strains with strong infectivity against rapeseed using an electroporation method; (3) Overexpression BnRLP41 Creation and acquisition of genetically modified rapeseed Using Agrobacterium-mediated transformation BnRLP41 Gene overexpression structure introduced into rapeseed to obtain transgenic BnRLP41 Rapeseed with overexpression structure; (4) Overexpression BnRLP41 Obtaining homozygous lines of genetically modified rapeseed Antibiotic resistance and BnRLP41 Gene expression is used as a detection indicator to assess the segregation of traits in the offspring of transgenic plants, and to obtain overexpressed genes whose traits no longer segregate and can be stably inherited. BnRLP41 A genetically modified homozygous line of rapeseed; (5) Overexpression of sclerotinia stem rot resistance BnRLP41 Screening, identification and acquisition of transgenic rapeseed homozygous lines With hyperexpression BnRLP41 Using homozygous transgenic rapeseed lines as materials, resistance to sclerotinia stem rot was tested and analyzed to obtain transgenic lines with increased resistance. BnRLP41 Genetically modified rapeseed.

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