Ssrgs gene of sclerotinia sclerotiorum for regulating resistance to sclerotinia sclerotiorum of brassica napus and its inhibitor and application

Abstract

This invention belongs to the field of plant genetic engineering and biological control technology, specifically disclosing a Sclerotinia sclerotinia Ssr. gene that regulates resistance to Sclerotinia sclerotinia disease in rapeseed, its inhibitor, and its application. This invention uses ultraviolet light to induce mutations and screens weakly pathogenic mutants of Sclerotinia sclerotinia, identifying mutation sites. The key gene SsRGS affecting the growth and development of Sclerotinia sclerotinia was positively screened. Based on its coding and amino acid sequences, dsRNA-specific interference sequence primers were designed using the VIGS design website. dsRGS was obtained through vector construction and transformation into E. coli transcription. Spraying dsRGS onto detached rapeseed leaves and inoculating them with activated wild-type Sclerotinia sclerotinia confirmed that dsRGS can weaken the pathogenicity of Sclerotinia sclerotinia by silencing the SsRGS gene. This invention can significantly reduce the area of ​​Sclerotinia sclerotinia infection patches and has promising applications in the control of rapeseed Sclerotinia sclerotinia disease.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and biological control technology, specifically relating to a Sclerotinia sclerotiorum gene that regulates resistance to Sclerotinia sclerotiorum rot in rapeseed, its inhibitor, and its application. Background Technology

[0002] Rapeseed is an important oilseed crop in my country and globally, ranking among the world's top in terms of planting area and total yield. However, sclerotinia sclerotiorum, a fungus belonging to the Ascomycota, is one of the most destructive diseases affecting rapeseed production. This pathogen has a wide host range, diverse infection modes, and strong survival ability; it can survive in the soil as sclerotia for extended periods. Once environmental conditions are suitable, the sclerotia germinate and produce ascospores, which are spread by air currents and infect the stems, leaves, and petals of rapeseed, leading to wilting and lodging. This has become a key factor restricting the sustainable development of my country's rapeseed industry.

[0003] Currently, the control of rapeseed sclerotinia stem rot mainly relies on chemical pesticides, agricultural measures, and disease-resistant breeding. Traditional control methods have problems such as limited efficacy, easy development of pesticide resistance, and environmental pollution. Therefore, developing efficient, safe, and environmentally friendly new green control technologies has become a key issue that urgently needs to be addressed in the field of rapeseed sclerotinia stem rot control.

[0004] RNA interference (RNAi) is a conserved post-transcriptional gene silencing mechanism in eukaryotes. In recent years, with the continuous development of RNAi technology, spray-induced gene silencing (SIGS) technology has emerged. It induces the silencing of target genes within pathogens by exogenously applying double-stranded RNA targeting key genes, thereby inhibiting pathogenicity and enhancing plant resistance. However, the successful application of SIGS technology is highly dependent on the selection of efficient targets. Ideally, the target gene should play a crucial role in the pathogen's pathogenesis, and its silencing should significantly weaken the pathogen's infectivity while having no adverse effects on non-target organisms.

[0005] Currently, there are no reports on using SIGS technology to target the RGS gene of *Sclerotinia sclerotiorum* for the control of sclerotinia disease. Therefore, screening and validating the RGS gene in *Sclerotinia sclerotiorum* that is closely related to pathogenicity, and developing corresponding dsRNA preparations based on SIGS technology, is of great significance for overcoming existing control bottlenecks and achieving green and sustainable management of sclerotinia disease in rapeseed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes an inhibitor targeting Ssigmatae bacterium SsRGS and its application in the resistance of rapeseed to Ssigmatae bacterium rot. The aim is to positively screen for the nucleotide sequence of SsRGS, a key gene affecting the pathogenicity of Ssigmatae bacterium, and the amino acid sequence of the encoded protein. Simultaneously, based on SIGS technology, dsRNA targeting the SsRGS gene is prepared and sprayed onto rapeseed leaves to achieve RNA interference, thereby effectively inhibiting the infection of rapeseed by Ssigmatae bacterium and improving resistance to Ssigmatae bacterium rot.

[0007] To achieve the above objectives, a SsRGS gene of Sclerotinia sclerotiorum that regulates resistance to Sclerotinia sclerotiorum in rapeseed is provided, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0008] Preferably, the amino acid sequence of the protein encoded by the SsRGS gene is shown in SEQ ID No. 2.

[0009] Preferably, the inhibitor targeting the SsRGS gene is dsRNA that inhibits the expression of the SsRGS gene in *Sclerotium sclerotiorum*.

[0010] Preferably, the nucleotide sequence of the dsRNA is shown in SEQ ID No. 3.

[0011] Preferably, the primer sequences for amplifying the dsRNA from Sclerotium sclerotiorum cDNA are shown in SEQ ID No. 4 and SEQ ID No. 5.

[0012] Preferably, the method for preparing the inhibitor includes: constructing a recombinant vector L4440 based on the SsRGS gene of Sclerotinia sclerotiorum shown in SEQ ID NO.1; transforming the obtained recombinant vector into Escherichia coli HT115, inducing dsRNA expression using IPTG, and extracting and diluting total RNA from Escherichia coli HT115.

[0013] Based on a general inventive concept, the present invention also provides the application of an inhibitor in the treatment of rapeseed sclerotinia stem rot.

[0014] Preferably, the method of application is as follows: the inhibitor is applied drop by drop onto the surface of rapeseed leaves.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The effectiveness of the SsRGS gene of Sclerotinia sclerotiorum as a control target was first discovered and verified. Silencing the gene can significantly reduce the pathogenicity of Sclerotinia sclerotiorum.

[0017] (2) Using SIGS technology, the direct action of exogenous dsRNA on pathogens was realized without the need for genetic modification of rapeseed, which is environmentally friendly and easy to operate.

[0018] (3) The designed specific interference sequences and primers have good specificity and silencing efficiency. After treatment with the inhibitor, the area of ​​lesions caused by Sclerotinia sclerotiorum on rapeseed leaves was significantly reduced, and the expression level of SsRGS in plants was significantly reduced. This provides a new technical means and product for the green control of Sclerotinia sclerotiorum in rapeseed. Attached Figure Description

[0019] Figure 1 This is a diagram of the mutant screening process in Embodiment 1 of the present invention.

[0020] Figure 2 This is a comparison diagram of rapeseed leaves inoculated with the mutant B12 screened in Example 1 of this invention and the wild-type strain.

[0021] Figure 3 This is the electrophoretic verification of the target sequence cloning and recombinant vector construction in Example 3 of the present invention, wherein... Figure 3 A represents the electrophoresis results of dsRGS and dsGFP target sequence amplification; Figure 3 B is an electrophoresis diagram of PCR identification of positive clones of dsRGS and dsGFP recombinant vectors.

[0022] Figure 4 This is an electrophoresis verification diagram of IPTG-induced target gene expression in Example 3 of the present invention.

[0023] Figure 5 This refers to the result of *Sclerotinia sclerotiorum* infecting rapeseed on a PDA containing H2O, dsRGS, and dsGFP, as described in Example 4 of this invention. Figure 5 A shows the phenotypic diagram of *Sclerotinia sclerotiorum* infecting rapeseed when grown on PDA containing H2O, dsRGS, and dsGFP. Figure 5 B represents the statistical analysis of the plaque area of ​​rapeseed leaves infected with Sclerotinia sclerotiorum by PDA containing H2O, dsRGS, and dsGFP for 36 hours.

[0024] Figure 6 This is the expression level of SsRGS in different treatment groups of Sclerotinia sclerotiorum in the quantitative real-time PCR method of Example 4 of the present invention. Detailed Implementation

[0025] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0026] Example 1

[0027] Screening and identification of weak pathogenic mutants of Sclerotinia sclerotiorum and mutation sites

[0028] 1. Ultraviolet light-induced mutation of wild-type Sclerotium sclerotiorum strains

[0029] Wild-type *Sclerotinia sclerotiorum* strain 1980 was preserved in potato dextrose agar (PDA). The sclerotia of the strain germinated to produce ascospores. The ascospores were wrapped in aluminum foil and stored in a desiccator at 4°C in the dark. The ascospores were resuspended and diluted to 10-1 / mL with sterile PDB medium (purchased from Shanghai Baiwei Biotechnology Co., Ltd.). 4 1 spore. Spread 300 μl of spore suspension evenly onto a 90 mm diameter PDA plate. After drying, irradiate with ultraviolet light (TL-2000 UV irradiator) for 15 seconds at an energy intensity of 9000 mJ / cm². Incubate the petri dishes at room temperature for 2 days until fungal colonies are visible. Then, use a sterile toothpick to pick up colonies and transfer them to 96-well plates. Add 150 μL of PDA medium to each well.

[0030] 2. Mutant screening

[0031] Approximately 7 days later, by comparing with the wild type, mutants exhibiting abnormal mycelial growth and slower growth rate were transferred to fresh PDA medium and cultured for another 2 days. Marginal mycelia were then inoculated onto lettuce leaves using a 2mm punch, with the mycelial portion adhering tightly to the leaf surface. The mixture was cultured for 36 hours under stable conditions of 22℃ and approximately 85% relative humidity. The pathogenicity of the induced mutant population was then assessed on tobacco leaves, and the infection area was observed. One mutant, B12, with a significant pathogenicity defect was identified. Figure 1 As shown in the figure. The selected B12 and wild-type mycelia were inoculated onto the same tobacco leaf for comparison, and the results are as follows. Figure 2 As shown, the infection area of ​​B12 is significantly smaller than that of the wild type.

[0032] 3. Mutation site identification

[0033] The weakly pathogenic mutant strain B12, selected above, was inoculated onto fresh, cellophane-lined PDA medium. After two days of culture, it was inoculated into 50 ml of PDB medium. Active fungal mycelia cultured in PDB medium for 5-7 days were collected and ground into a fine powder in liquid nitrogen. Genomic DNA was extracted from the mutant using the cetyltrimethylammonium bromide (CTAB) method. The crude DNA was then purified using the DNeasy Plant Maxi kit, and the samples were sent to Novogene Biotechnology Co., Ltd. for whole-genome next-generation sequencing (NGS). The NGS sequencing data were aligned to the wild-type strain's reference genome using BWA-MEM, and mutation sites were identified using SAMtools. False and invalid mutations in repetitive sequences were manually removed.

[0034] Example 2

[0035] SsRGS, a key gene regulating the growth and development of Sclerotinia sclerotiorum, was identified through positive screening.

[0036] According to Example 1, a total of 47 B12-specific SNPs and INDELs were screened, as shown in Table 1. Of these, only 7 SNPs were located in exon regions. Synonymous mutations, intron mutations, and intergenic mutations were excluded because they were unlikely to significantly affect the encoded protein. The remaining 3 SNP mutations leading to non-synonymous mutations in the B12 genome became the main candidate mutations and were retained for subsequent analysis. Quantitative real-time PCR showed that only one of these 3 SNPs exhibited a different expression level during the *Sclerotinia sclerotiorum* infection stage, while the other two showed no significant change. Analysis of its domains revealed that the gene contains two DEP domains and one RGS domain; therefore, it was named SsRGS, and its nucleotide sequence is shown in SEQ ID NO. 1.

[0037] Table 1: Mutations of 47 SNPs and INDELs

[0038]

[0039]

[0040]

[0041] Example 3

[0042] SsRGS gene dsRNA synthesis

[0043] 1. SIGS primer design

[0044] The coding and amino acid sequences of SsRGS were extracted from the Scleraotinia sclerotiorum genome database (http: / / www.broadinstitute.org / annotation / genome / Scleraotinia sclerotiorum / Uenomeslndex.html), as shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. Specific interference sequences were selected using VIGS design (https: / / vigs.solgenomics.net / ), and the target site sequence is shown in SEQ ID No. 3.

[0045] 2. Construction and transformation of prokaryotic expression dsRNA vector

[0046] The target sequence fragments dsRGS and dsGFP were amplified from the cDNA of wild-type Sclerotium sclerotiorum 1980 and the plasmid pCambia1300-GFP containing the GFP sequence, respectively, using primers SsRGS-L4-F / SsRGS-L4-R (sequences shown in SEQ ID No. 4 and SEQ ID No. 5) and GFP-L4-F / GFP-L4-R (sequences shown in SEQ ID No. 6 and SEQ ID No. 7). Figure 3 A. The obtained PCR product was recovered by gel electrophoresis and homologously recombined into the L4440 vector, which was linearized by double digestion with XbaI and HindIII. The L4440 construct was then transformed into E. coli HT115 via heat shock transformation. The results of bacterial PCR and electrophoresis are as follows: Figure 3 As shown in B, a positive clone has been successfully obtained. L4440, containing the GFP target sequence, was used as a negative control for SIGS.

[0047] Table 2: Primers used for vector construction

[0048]

[0049] 3. IPTG-induced expression of dsRNA

[0050] Positive transformants were picked and placed in 25 mL of LB medium containing 50 mg / L ampicillin. After induction with 0.2 mM / L IPTG at 37°C and 220 rpm for 5 h, total RNA was extracted and detected by agarose gel electrophoresis. The results are as follows: Figure 4 As shown, compared with the control group without IPTG, the IPTG-induced group showed RNA bands of the corresponding size.

[0051] Example 4

[0052] Application of dsRGS in the control of sclerotinia stem rot in rapeseed

[0053] 1. Prepare rapeseed test materials

[0054] After disinfection and vernalization, rapeseed seeds were inoculated onto sterilized nutrient soil and cultured for 6 weeks under 16 hours of light, 8 hours of darkness, and at 22°C.

[0055] 2. Acquisition and Pretreatment of dsRNA

[0056] Total RNA was extracted from successfully constructed and induced *E. coli* HT115. 50 μL of RNA was extracted, analyzed by 1% agarose gel electrophoresis, and diluted to a concentration of 500 ng / μL. 80 μL of 500 ng / μL dsRNA solution and ddH2O were added dropwise to isolated rapeseed leaves, with three replicates per group.

[0057] 2. Inoculate with Sclerotinia sclerotiorum.

[0058] The specific inoculation steps are as follows: Wild-type Sclerotinia sclerotiorum strain 1980 is inoculated into PDA medium and cultured at 20°C for 1 day. Then, edge mycelia are taken using a 2 mm diameter punch and inoculated into PDA medium, and cultured at 20°C for 2 days. Edge mycelia grown for 2 days are taken using a 2 mm diameter punch for inoculation. The prepared 2 mm mycelial blocks are inoculated onto detached rapeseed leaves pretreated with dsRNA in the previous step, with the mycelial side in close contact with the leaf. The inoculated leaves are placed in a temperature and humidity controlled environment, with the temperature set at 22°C and the relative humidity maintained at approximately 85%.

[0059] 3. Virulence identification of Sclerotinia sclerotiorum

[0060] Forty-eight hours after inoculation, the area of ​​lesions was statistically analyzed using ImageJ software, and SPSS software was used for statistical analysis. The experiment was repeated three times, with 3-5 leaves each time. The results are as follows: Figure 5 A and Figure 5 B shows that, using ImageJ software, the leaf lesion area under ddH2O, dsGFP, and dsRGS treatments was 7.529 cm². 2 7.338 cm 2 and 4.055 cm 2 Compared with the control group ddH2O, dsRGS from Escherichia coli HT115 reduced the area of ​​lesions on rapeseed leaves by Sclerotinia sclerotiorum by 46.1%.

[0061] 4. Detection of SsRGS expression level in Sclerotinia sclerotiorum by real-time PCR

[0062] (1) Extraction of RNA from rapeseed leaves: Wild-type Sclerotinia sclerotiorum strain 1980 was inoculated on leaves treated with dsRNA. After 48 hours of infection, the images were taken and the area of ​​the infected spots was counted by ImageJ. 1g of sample was taken from the lesion and ground into powder with liquid nitrogen. RNA was extracted according to the Promega Total RNA Kit (LS1040).

[0063] (2) Reverse transcription: The RNA extracted in the previous step was reverse transcribed at a total amount of 1000 ng per sample. The specific steps were performed according to the Promega RNA Reverse Transcription Kit (A5001) for cDNA reverse transcription. The fluorescent quantitative primers and PCR program are shown in Tables 3-4.

[0064] Table 3: Primers for Quantitative Fluorescence

[0065]

[0066] Table 4: Quantitative Real-Time PCR Procedure

[0067]

[0068] (3) Quantitative Real-Time PCR Detection: RT-PCR was performed using primers SsRGS-RT-F and SsRGS-RT-R to detect the expression level of SsRGS. SsRGS-RT-F / SsRGS-RT-R are shown in SEQ ID No. 8 and SEQ ID No. 9, while Tublin1-RT-F / Tublin1-RT-R served as internal control primers, with sequences shown in SEQ ID No. 10 and SEQ ID No. 11. Results are as follows: Figure 6 The results showed that after 48 hours of treatment with ddH2O, dsGFP, and dsRGS, RNA was extracted from the lesion area and reversed to cDNA for fluorescence quantification. The expression levels of SsRGS were 1.001, 1.033, and 0.618, respectively. It was found that the expression level of SsRGS in tobacco leaves of the dsRGS treatment group decreased by 38.3%, indicating that the dsRGS transcribed by Escherichia coli silenced SsRGS and inhibited the infection of Rapeseed leaves by Sclerotinia sclerotiorum.

[0069] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A Sclerotinia sclerotiorum SsRGS gene that regulates resistance to Sclerotinia sclerotiorum in rapeseed, characterized in that, The nucleotide sequence of the SsRGS gene is shown in SEQ ID No.

1.

2. The SsRGS gene of *Sclerotinia sclerotiorum* according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the SsRGS gene is shown in SEQ ID No.

2.

3. An inhibitor targeting the SsRGS gene of *Sclerotinia sclerotiorum* as described in claim 1, characterized in that... The inhibitor is a dsRNA that inhibits the expression of the SsRGS gene in *Sclerotium sclerotiorum*.

4. The inhibitor according to claim 3, characterized in that, The nucleotide sequence of the dsRNA is shown in SEQ ID No.

3.

5. The inhibitor according to claim 3, characterized in that, The amplification primer sequences for the dsRNA are shown in SEQ ID No. 4 and SEQ ID No.

5.

6. The inhibitor according to claim 3, characterized in that, The method for preparing the inhibitor includes: constructing a recombinant vector L4440 based on the SsRGS gene of Sclerotinia sclerotiorum shown in SEQ ID NO.1; transforming the obtained recombinant vector into Escherichia coli HT115, inducing dsRNA expression using IPTG, extracting total RNA from Escherichia coli HT115 and diluting it to obtain the inhibitor.

7. The use of the SsRGS gene of *Sclerotinia sclerotiorum* as described in any one of claims 1-2 or the inhibitor as described in any one of claims 3-5 in regulating resistance to *Sclerotinia sclerotiorum* in rapeseed.

8. The application according to claim 7, characterized in that, The method of application is as follows: the inhibitor is applied to the surface of rapeseed leaves.

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