Application of Brassica Receptor Gene BnRLP26 in Sclerotinia Disease Control

Abstract

The application provides application of a Brassica napus class receptor gene BnRLP26 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 BnRLP26 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 BnRLP26 gene in obtaining high-sclerotinia-disease-resistant oilseed rape material by creating super-expression oilseed rape, and application of the BnRLP26-CRISPR gene knockout oilseed rape in obtaining sclerotinia disease resistance weakened sclerotinia disease oilseed rape material. The BnRLP26 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 napus receptor-like gene BnRLP26 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 functions 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 the 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 a decrease in the expression of the target gene. Gene knock-out is achieved by inserting a long non-plant sequence or removing the target gene in the plant genome, resulting in complete or nearly complete inhibition of the expression of the target gene. By constructing gene over-expression plants and / or RNAi plants and gene knock-out mutants, comparing and analyzing the differences in phenotypes and traits between the wild type / normal plants and the mutants, 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 the result of activating disease resistance signaling and activating a series of defense responses by plant receptors recognizing pathogen ligands. From the perspective of genetics, after the recognition of receptors-ligands, 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 pathogen molecular patterns in plants. It is expected that the use of RLP genes to create crop germplasm with enhanced disease resistance will have good results, 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 broad 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 exogenous disease-resistant regulatory genes into plants through Agrobacterium-mediated method, so that the plants obtain disease resistance that they originally do 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 receptor gene BnRLP26 in Sclerotinia disease prevention and control. The Brassica napus receptor gene BnRLP26 has a regulatory function on Sclerotinia disease resistance and can be applied to the creation of Sclerotinia-resistant crop germplasm.

[0011] The application is the application of transgenic rape obtained by creating transgenic rape to obtain disease-resistant rape material with changed disease resistance. The application is the application of transgenic rape with overexpressed BnRLP26 to obtain rape material with increased Sclerotinia disease resistance, or the application of BnRLP26-CRISPR transgenic rape to obtain rape material with reduced Sclerotinia disease resistance.

[0012] The application takes Brassica napus double 11 variety cDNA as a template, and obtains Brassica napus gene BnRLP26 through PCR cloning, the nucleotide sequence of which is shown as SEQ ID:1, the open reading frame (ORF) of the gene is 2301 bp long, and the encoded protein is composed of 766 amino acids, the sequence of which is shown as SEQ ID:2. The BnRLP26 protein contains a transmembrane domain and a LRR (Leucine-rich repeat) domain. The nucleotide sequence cloned in the application is consistent with the LOC106383875 nucleotide sequence of Brassica napus variety ZS11 in the NCBI database; and the protein sequence cloned in the application is identical to XP_022553201.2.

[0013] Before the application, there is no any public report on the function of the gene. The application first clarifies the regulation effect and mechanism of the gene on the resistance of Brassica napus to sclerotinia disease through constructing super-expression and CRISPR knockout transgenic Brassica napus of the gene and analyzing the disease resistance of the transgenic Brassica napus. The analysis results of the inoculated leaf lesion area and pathogen biomass show that, compared with the wild-type Brassica napus plants, the super-expression transgenic Brassica napus plants are significantly more resistant to S. sclerotiorum, and the knockout transgenic plants are significantly more susceptible to S. sclerotiorum, which indicates that BnRLP26 positively regulates the resistance of Brassica napus to S. sclerotiorum.

[0014] Based on the clarified function of the BnRLP26 gene in the application, the application aims to provide the application of the Brassica napus BnRLP26 gene in obtaining the Brassica napus material with changed disease resistance through creating transgenic Brassica napus, including (1) the application in obtaining the Brassica napus material with increased resistance to sclerotinia disease through creating the transgenic Brassica napus with super-expressed BnRLP26 (Example 1); and (2) the application in obtaining the Brassica napus material with reduced resistance to sclerotinia disease through creating the transgenic Brassica napus with BnRLP26-CRISPR gene knockout (Example 2).

[0015] The application of the Brassica napus BnRLP26 gene in obtaining the Brassica napus material with increased resistance to sclerotinia disease through creating the transgenic Brassica napus with super-expressed BnRLP26 is specifically realized through the following steps:

[0016] (1) Construction and acquisition of BnRLP26 gene super-expression structure: cloning the open reading frame (ORF) of the BnRLP26 gene into a plant expression vector to make it express under the drive of a strong promoter;

[0017] (2) Acquisition of Agrobacterium with BnRLP26 gene super-expression structure: transforming the constructed BnRLP26 gene super-expression structure into an Agrobacterium strain with strong invasiveness to Brassica napus through methods such as electroporation;

[0018] (3) Transgenic rape plants overexpressing BnRLP26 are created and obtained by introducing the BnRLP26 gene overexpression structure into rape plants through Agrobacterium-mediated method;

[0019] (4) Homozygous lines of transgenic rape plants overexpressing BnRLP26 are obtained by detecting the trait segregation of the transgenic plant offspring using antibiotic resistance and BnRLP26 gene expression as detection indexes, and obtaining homozygous lines of transgenic rape plants overexpressing BnRLP26 that no longer segregate traits and can stably inherit;

[0020] (5) Homozygous lines of transgenic rape plants overexpressing BnRLP26 with increased resistance to sclerotinia disease are screened, identified and obtained by detecting the resistance to sclerotinia disease of the homozygous lines of transgenic rape plants overexpressing BnRLP26, and obtaining transgenic rape plants overexpressing BnRLP26 with increased disease resistance.

[0021] Application of rape BnRLP26 gene in obtaining rape materials with reduced resistance to sclerotinia disease through creating transgenic rape plants with BnRLP26-CRISPR gene knockout. The following steps are implemented:

[0022] (1) Construction and obtaining of BnRLP26 gene CRISPR knockout structure: three 20bp knockout targets are preset according to the BnRLP26 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-BnRLP26;

[0023] (2) Obtaining of Agrobacterium transformed with BnRLP26 gene CRISPR knockout structure: the BnRLP26 gene CRISPR knockout structure (pBSbdcas9i-BnRLP26) is transformed into Agrobacterium strains with strong invasion ability to rape by electroporation and other methods;

[0024] (3) Creation and obtaining of transgenic rape plants with BnRLP26 gene CRISPR knockout structure: the BnRLP26 gene CRISPR knockout structure is introduced into rape plants through Agrobacterium-mediated method, and transgenic rape plants with BnRLP26 gene CRISPR knockout structure are obtained;

[0025] (4) Obtaining of homozygous lines of transgenic rape plants with BnRLP26 gene CRISPR knockout structure: antibiotic resistance is used for screening, and the trait segregation of the transgenic plant offspring is detected to obtain homozygous lines of transgenic rape plants with BnRLP26 gene CRISPR knockout structure that no longer segregate traits and can stably inherit;

[0026] (5) Screening and obtaining of homozygous line of BnRLP26 gene CRISPR knockout structure rapeseed with weakened resistance to sclerotinia disease: using homozygous line of BnRLP26 gene CRISPR knockout structure (pBSbdcas9i-BnRLP26) rapeseed as material, detecting and analyzing the resistance to sclerotinia disease, and obtaining BnRLP26 gene CRISPR knockout rapeseed with weakened resistance to sclerotinia disease.

[0027] Advantages of the present application: (1) the BnRLP26 gene provided by the present application is a high-quality resistance to sclerotinia disease regulatory 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 BnRLP26 gene encodes a receptor-like protein, which rapidly triggers an immune response after recognizing the pathogenic molecular pattern, and has good resistance triggering and regulation effect, so it is a high-quality disease-resistant gene resource. Using RLP to create disease-resistant germplasm is an economical, effective and safe way for green disease control. Therefore, the BnRLP26 gene is a new gene resource suitable for creating and breeding new materials and 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 genetic engineering breeding method using disease-resistant regulatory genes. The traditional breeding method has the disadvantages of limited range of available disease-resistant resources due to natural genetic isolation, long breeding period, and the need for a large amount of labor and material. The genetic engineering breeding method has the advantages of wide range of available disease-resistant resources, relatively simple and convenient operation, short breeding period, no need for a large amount of labor and material, and is particularly suitable for breeding broad-spectrum, durable and highly disease-resistant varieties. The present application uses the disease-resistant regulatory gene BnRLP26 to create and breed rapeseed material with high resistance to sclerotinia disease by using genetic engineering method, which has the characteristics of short period and rapid breeding. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1This invention relates to the detection of gene expression in BnRLP26 overexpressing plants and the identification of gene editing in CRISPR gene knockout plants. It provides evidence of various transgenic rapeseed plants obtained in this invention, showing the gene expression results in BnRLP26 overexpressing plants and the gene editing pattern in gene knockout plants. The expression level of BnRLP26 in overexpressing (OE) plants was detected using real-time quantitative PCR (qRT-PCR), with rapeseed BnACTIN7 as an internal reference gene and ZS11 expression set to 1. The qRT-PCR analysis was performed in triplicate (biological replicates, each containing three technical replicates). The expression results were statistically analyzed using GraphPad Prism with Student's t-test. Data are expressed as mean ± standard deviation. Significance is indicated by * (**, P < 0.01). The results showed that the expression level of BnRLP26 was significantly increased in both overexpressing lines #4 and #13, reaching 62.4-fold and 183.3-fold higher than the control, respectively (A). Two successfully knockout mutant lines, #16 and #19, each had a 25bp deletion at target site 1 and a 1bp insertion at target site 2 of the BnRLP26 gene, respectively, resulting in the inability to properly encode the functional BnRLP26 gene (B). This indicates that these plants are true BnRLP26 gene overexpression plants and CRISPR gene knockout plants.

[0029] Figure 2 BnRLP26 positively regulates rapeseed resistance to Sclerotinia sclerotiorum. Evidence is provided regarding the anti-Sclerotinia sclerotiorum function of the BnRLP26 gene, showing that BnRLP26 positively regulates rapeseed resistance to Sclerotinia sclerotiorum. Inoculation analysis of BnRLP26 transgenic rapeseed plants with Sclerotinia sclerotiorum UF-1 was performed. The figure shows the phenotype, lesion area, and mycelial biomass of overexpressing plants (A) and knockout mutant plants (B) 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 is indicated by different numbers of asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The results showed that the overexpressing plants exhibited a significantly more resistant phenotype than the control (ZS11) plants (A), while the knockout mutant plants were significantly more susceptible to the disease than the control (B). Quantitative analysis of lesion area showed that the lesion area of ​​the two overexpressing lines was 109.6 mm². 2 and 94.1mm 2 It was significantly lower than the control group's 169.2 mm. 265% and 56% respectively; the bacterial amount determination also showed that the bacterial amount in the lesion of the overexpression plants was significantly lower than that of the control plants. The lesion area of the two knock-out mutant lines was 179.3 mm 2 and 178.3 mm 2 , which was significantly higher than that of the control plants (126.9 mm 2 , 1.41 times of the control); the bacterial amount determination also showed that the bacterial amount in the lesion of the knock-out mutant plants was significantly higher than that of the control plants. These results showed that BnRLP26 positively regulated the resistance of oilseed rape 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 cloned an oilseed rape gene BnRLP26, and for the first time clarified the function of the gene in conferring resistance to Sclerotinia sclerotiorum in oilseed rape by constructing overexpression transgenic oilseed rape. The overexpression of BnRLP26 gene resulted in significantly increased resistance of oilseed rape to Sclerotinia sclerotiorum, and therefore, a new material of oilseed rape with increased resistance to Sclerotinia sclerotiorum can be created by constructing homozygous line of overexpression of BnRLP26 gene, which can be used for creating and breeding oilseed rape varieties resistant to Sclerotinia sclerotiorum, analyzing the function and mechanism of BnRLP26 gene, etc. The main steps for cloning, function and mechanism analysis of BnRLP26 gene, creating and obtaining a new material of oilseed rape with increased resistance to Sclerotinia sclerotiorum include:

[0033] 1) Cloning and preservation of BnRLP26 gene of oilseed rape

[0034] The BnRLP26 gene of Brassica napus provided in the present application is cloned through the following steps. First, primers BnRLP26-F (5'-atg tcg cta tcg cgt cta cat tcg-3') (the sequence is shown as SEQ ID: 3) and BnRLP26-R (5'-tta tcc gca cca acc aaa ata cc-3') (the sequence is shown as SEQ ID: 4) are designed according to the BnRLP26 sequence in the Brassica napus genome database. Total RNA is extracted from the leaves of Brassica napus variety Shuang 11 by using the TRIZOL reagent, and BnRLP26 cDNA is obtained by using the high-fidelity enzyme pfu-mediated RT-PCR method. The PCR product is purified by cutting the gel after 1% agarose gel electrophoresis, and is connected to the pEASY-Blunt Cloning Vector carrier. Escherichia coli DH5α is transformed by heat shock, and is cultured in LB medium overnight. Whether the extracted plasmid contains the BnRLP26 gene is tested by using the BnRLP26-F / BnRLP26-R primer pair for PCR, and finally the company is sent for sequencing verification, so that the full-length sequence of the BnRLP26 gene cDNA is successfully cloned and obtained. The BnRLP26 nucleotide sequence is shown as SEQ ID: 1, the open reading frame (ORF) of the gene is 2301 bp long, and the encoded protein is composed of 766 amino acids, the sequence of which is shown as SEQ ID: 2. The gene product contains a transmembrane domain and a LRR domain. It is found through BLAST analysis that the cloned nucleotide sequence of the present application is consistent with the LOC106383875 nucleotide sequence of Brassica napus variety ZS11 in the NCBI database, and the protein sequence is identical to XP_022553201.2. There is no report on the function of the gene before the present application.

[0035] The Escherichia coli carrying the pEASY-BnRLP26 vector is transformed and stored in a -80℃ refrigerator. At any time, the strain can be activated, the plasmid can be extracted, the BnRLP26 gene can be amplified by PCR, and the BnRLP26 gene can be subcloned into a target vector for transgenic research.

[0036] 2) Construction and acquisition of BnRLP26 gene overexpression structure

[0037] The BnRLP26 sequence cloned according to the present application is used to design primers BnRLP26-F2 (5'-gacgatgataagggcggtacctcgctatcgctactcat tcg-3', the italicized part is a Kpn I enzyme cutting site, the sequence consistent with the vector pCAMBIA1300) (the sequence is shown as SEQ ID: 5) and BnRLP26-R2 (5'-gtcctaggctacgtaggatccttatccgcaccaaccaaaatacc-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-BnRLP26 plasmid obtained in 1) of Example 1 is used as a template, and BnRLP26-F2 / BnRLP26-R2 is used as a primer pair to obtain the target gene through PCR amplification.

[0038] The pCAMBIA1300 plasmid is double-enzyme cut with Kpn I and Bam HI, and the obtained target gene is connected to the enzyme-cut pCAMBIA1300 vector through homologous recombination. The reagents used in the experiment are PCR one-step directional cloning kit. The connection product is subjected to E. coli transformation, kanamycin plate screening, PCR identification and sequencing identification, etc. to obtain the BnRLP26 gene super-expression structure pCAMBIA1300-BnRLP26. 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 BnRLP26 gene super-expression structure pCAMBIA1300-BnRLP26

[0040] The BnRLP26 gene super-expression structure pCAMBIA1300-BnRLP26 is transformed into Agrobacterium strains with strong invasiveness to rape, such as EHA105, by methods such as electroporation, and the transformants are screened on YEP medium containing kanamycin. Agrobacterium carrying the BnRLP26 gene super-expression structure pCAMBIA1300-BnRLP26 is obtained through double enzyme cutting of Kpn I and Bam HI and PCR identification, which is used for the genetic transformation of rape in the next step.

[0041] 4) Creation and obtaining of rape transformed with the BnRLP26 gene super-expression structure pCAMBIA1300-BnRLP26

[0042] The BnRLP26 gene super-expression structure pCAMBIA1300-BnRLP26 was introduced into rape by Agrobacterium-mediated method to obtain the T0 generation of the rape with pCAMBIA1300-BnRLP26.

[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 was inoculated on a culture bottle and cultured in dark at 23°C for 5-6d.

[0045] (ii) Pre-culture

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

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

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

[0049] (iv) Decontamination (delayed screening)

[0050] The co-cultured explant was inoculated on the decontamination medium and cultured in light at 23°C for 6d.

[0051] (v) Screening / differentiation

[0052] The decontaminated explant was inoculated on the screening / differentiation medium, 30 explants per dish, and cultured in light at 23°C, and the plate was changed every 15d.

[0053] (vi) Rooting culture

[0054] The differentiated bud was inoculated on the rooting culture medium and cultured in light at 23°C until rooting.

[0055] (vii) Detection

[0056] The rape genomic DNA was extracted by CTAB method, and the resistance gene was detected by PCR.

[0057] (viii) Soil planting

[0058] 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.

[0059] 5) Screening, identification, and acquisition of homozygous rapeseed lines overexpressing the BnRLP26 gene with the structure pCAMBIA1300-BnRLP26.

[0060] Kanamycin resistance and BnRLP26 gene expression were used as indicators to detect phenotypic segregation in the offspring of transgenic plants. Kanamycin resistance screening was conducted on rapeseed seeds on kanamycin-containing plates to observe whether they could grow into healthy seedlings normally on kanamycin-resistant plates. Gene expression was detected using methods such as real-time quantitative PCR. Homozygous rapeseed lines with the BnRLP26 gene overexpression structure pCAMBIA1300-BnRLP26 that showed no further phenotypic segregation in offspring and could be stably inherited were obtained.

[0061] This invention obtained two homozygous rapeseed lines, OE-line 4 and OE-line 13, both of which were able to grow normally into healthy seedlings on kanamycin resistance plates, and the expression level of the BnRLP26 gene was significantly higher than that of the control plants, being 62.4 times and 183.3 times that of the control, respectively. Figure 1 A). This indicates that these plants are true BnRLP26 gene overexpression plants.

[0062] 6) Disease resistance detection and analysis of homozygous rapeseed lines overexpressing the BnRLP26 gene

[0063] Using the homozygous rapeseed line overexpressing the BnRLP26 gene obtained in step 5) as material, we detected and analyzed its resistance to Sclerotinia sclerotiorum by inoculating it with Sclerotinia sclerotiorum, thereby clarifying the regulatory role of the BnRLP26 gene in the resistance of rapeseed to Sclerotinia sclerotiorum, and laying the foundation for creating and obtaining rapeseed resistant to Sclerotinia sclerotiorum using this gene.

[0064] 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.

[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. One mycelium block was inoculated to each of 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 BnRLP26 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 169.2 mm 2 , and the lesion areas of the two overexpression plants were 109.6 mm 2 and 94.1 mm 2 , which were only 65% and 56% of the control Figure 2 B).

[0067] These results showed that the resistance of the BnRLP26 overexpression plants to sclerotinia was significantly higher than that of the non-transgenic control plants. The overexpression of the BnRLP26 gene led to a significant increase in the resistance of the oilseed rape to sclerotinia, and therefore, BnRLP26 positively regulated the resistance of the oilseed rape to sclerotinia. The high-sclerotinia-resistant oilseed rape new material was successfully created by constructing the BnRLP26-OE oilseed rape according to the present application.

[0068] Example 2

[0069] According to the positive regulation function of the oilseed rape gene BnRLP26 to the resistance to sclerotinia as clarified in the present application, a technical system for creating and obtaining the new oilseed rape material with weakened resistance to sclerotinia by constructing the CRISPR gene 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 BnRLP26 gene CRISPR knockout structure

[0071] According to the BnRLP26 genomic sequence, three suitable target sites were designed, each gRNA unit contained 20 bp gRNA and a gRNA backbone, and three gRNA units were combined as a complete knockout function sequence. As a template, primer BnRLP26-CR-F (5'-cag tgg tct cat gca aac ttc cag tga act cgt ta-3', italic part is a sequence containing Bsa I or Eco31 I enzyme cutting site, consistent with linearized vector pBSbdcas9i) (sequence as shown in SEQ ID: 7) and BnRLP26-CR-R (5'-cag tgg tct caa aac ttt cac cgg ttt gga agg tt-3', italic part is a sequence containing Bsa I or Eco31 I enzyme cutting site, consistent with linearized vector pBSbdcas9i) (sequence as shown in 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') (sequence as shown in SEQ ID: 9) and M49452 (470C):(5'-cta cgg ttc aag aaa atg taa gct gat-3') (sequence as shown in SEQ ID: 10) for PCR identification, and further sequencing identification to obtain the CRISPR knockout structure pBSbdcas9i-BnRLP26.

[0072] (2) Obtaining Agrobacterium transformed with BnRLP26 gene CRISPR knockout structure

[0073] The BnRLP26 gene CRISPR knockout structure (pBSbdcas9i-BnRLP26) was transformed into Agrobacterium strains with strong invasion ability to Brassica napus, such as EHA105, by electroporation and other methods, and the transformants were screened on YEP medium containing kanamycin, and then identified by PCR to obtain Agrobacterium carrying the BnRLP26 gene CRISPR knockout structure pBSbdcas9i-BnRLP26.

[0074] (3) Creation and acquisition of BnRLP26 gene CRISPR knockout structure transformed Brassica napus

[0075] The BnRLP26 gene CRISPR knockout structure pBSbdcas9i-BnRLP26 was introduced into Brassica napus by Agrobacterium-mediated method to obtain Brassica napus T0 generation transformed with pBSbdcas9i-BnRLP26. The specific operation steps are as described in 4) of Example 1. Whether the transformed seedlings are gene editing seedlings is detected by BnRLP26-CR-F2 (5'-cacttc ttc ttc act ccc ttc t-3') (the sequence is shown as SEQ ID: 11) and BnRLP26-CR-R2 (5'-gca aat ctg gag agg tat gtt c-3') (the sequence is shown as SEQ ID: 12). The gene editing seedlings are subsequently sequenced in the sequence near the target point, and finally the mutant plant with successfully knocked out BnRLP26 gene is determined.

[0076] (4) Obtaining homozygous lines of Brassica napus transformed with BnRLP26 gene CRISPR knockout structure

[0077] The trait segregation of the transgenic plant offspring is detected by antibiotic resistance screening method. Brassica napus seeds are subjected to the detection on the Basta-containing plate, and whether the seeds can grow into healthy seedlings on the Basta-resistant plate is observed. The homozygous lines of Brassica napus transformed with BnRLP26 gene CRISPR knockout structure pBSbdcas9i-BnRLP26 are obtained, which have no trait segregation and can stably inherit. These homozygous lines of Brassica napus can all grow into healthy seedlings on the Basta-resistant plate.

[0078] The homozygous lines of Brassica napus with BnRLP26-CRISPR gene knockout are obtained, which can grow into healthy seedlings on the Basta-resistant plate. The sequencing results show that two mutant lines #16 and #19 respectively delete a 25 bp sequence at the target point 1 of the BnRLP26 gene and insert 1 bp at the target point 2, both of which result in the inability to normally encode functional BnRLP26 gene (B), indicating that these plants are true BnRLP26 gene CRISPR knockout plants. Figure 1 B), indicating that these plants are true BnRLP26 gene CRISPR knockout plants.

[0079] (5) Screening, identification and obtaining of Brassica napus homozygous lines with BnRLP26 gene CRISPR knockout structure against Sclerotinia sclerotiorum

[0080] The homozygous lines of Brassica napus transformed with BnRLP26 gene CRISPR knockout structure (pBSbdcas9i-BnRLP26) are used as materials to detect and analyze the resistance to Sclerotinia sclerotiorum. The inoculation method and disease resistance evaluation are as described in 6) of Example 1).

[0081] The results of the S. sclerotinum inoculation analysis of the BnRLP26-CRISPR plants constructed in the application show that the BnRLP26-CRISPR gene knockout plants are significantly 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 126.9 mm 2 , and the lesion areas of the two knockout mutant lines are 179.3 mm 2 and 178.3 mm 2 , respectively, which are significantly higher than that of the control plant Figure 2 B). It is shown that the resistance of the BnRLP26-CRISPR plants to the S. sclerotinum is significantly lower than that of the non-transgenic control plants. The inhibition of the expression of the BnRLP26 gene leads to a significant decrease in the resistance of the rape to the S. sclerotinum. The BnRLP26-CRISPR gene knockout rape is successfully obtained by constructing the BnRLP26-CRISPR gene knockout rape in the application, and the rape new material with weakened resistance to the S. sclerotinum is obtained.

[0082] In conclusion, the results of the application Figures 1-2 reveal for the first time that the rape receptor gene BnRLP26 positively regulates the resistance of the rape to the S. sclerotinum, and provide the application approaches, application techniques and examples of the BnRLP26 in the creation of the S. sclerotinum-resistant crop germplasm, and the S. sclerotinum-resistant rape is successfully obtained.

Claims

1. A type of rapeseed ( Brassica napus receptor-like genes BnRLP26 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 BnRLP26 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 BnRLP26 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) BnRLP26 Construction and acquisition of gene overexpression structures Will BnRLP26 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 BnRLP26 Obtaining Agrobacterium gene overexpression structures The built BnRLP26 Gene overexpression structures were transformed into Agrobacterium strains with strong infectivity against rapeseed using an electroporation method; (3) Overexpression BnRLP26 Creation and Acquisition of Genetically Modified Rapeseed Using Agrobacterium-mediated transformation BnRLP26 Gene overexpression structure introduced into rapeseed to obtain transgenic BnRLP26 Rapeseed with overexpression structure; (4) Overexpression BnRLP26 Obtaining homozygous lines of genetically modified rapeseed Antibiotic resistance and BnRLP26 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. BnRLP26 A genetically modified homozygous line of rapeseed; (5) Overexpression of increased resistance to sclerotinia stem rot BnRLP26 Screening, identification and acquisition of homozygous lines of transgenic rapeseed With hyperexpression BnRLP26 Using homozygous transgenic rapeseed lines as materials, resistance to sclerotinia stem rot was tested and analyzed to obtain transgenic lines with increased resistance. BnRLP26 Genetically modified rapeseed.

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