Preparation and application of double-component dsRNA medicament for enhancing resistance mutation risk capability of RNA pesticide to botrytis cinerea

Abstract

The invention discloses preparation and application of a double-component dsRNA (double-stranded ribonucleic acid) medicament for enhancing resistance mutation risk capability of RNA (ribonucleic acid) pesticide to botrytis cinerea, and a key pathogenic gene of botrytis cinerea is any one or two of a botrytis cinerea global regulatory factor Vel1 gene or a galacturonic acid reductase Gar2 gene. The dsRNA is any one or two of dsRNAs targeting a Vel1 gene or a Gar2 gene, and efficient prevention and control of gray mold are achieved through key pathogenic links such as spore germination inhibition, hypha colonization and infection structure formation. In addition, the effectiveness of the RNA pesticide aiming at botrytis cinerea after single target gene mutation is evaluated through cooperative use of the combination of the double-component RNA, the ds-Gar2 and the ds-Vel1, the good effect of the double-target synergistic effect in the aspect of reducing the off-target risk is proved, and the practice and popularization of a green prevention and control technology in prevention and control of gray mold are promoted.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to the preparation and application of a dsRNA agent that targets multiple pathogenic genes of Botrytis cinerea to reduce the risk of off-target effects caused by gene mutations. Background Art

[0002] Botrytis cinerea is a necrotrophic fungus that kills plant cells to obtain nutrients, causing plant tissue decay and the subsequent formation of a gray mold layer on the surface of diseased plant tissues, resulting in severe economic losses. Botrytis cinerea has a wide host range, strong invasiveness, and is prone to developing drug resistance, causing disease in many important economic crops such as tomatoes, strawberries, and grapes. The incidence of gray mold is highest in fruit and vegetable greenhouses under low temperature and high humidity conditions. This disease not only harms the stems, leaves, and fruit of the plants, but also harms the fruit during post-harvest storage, severely impacting yield and quality. Globally, economic losses due to gray mold amount to $10-100 billion annually.

[0003] As with other fungal diseases, chemical methods are currently the most commonly used to control gray mold. These methods offer high efficacy, rapid results, stable results, and simple methods. Approximately 8% of the global fungicide market is used to control this pathogen. However, Botrytis cinerea itself is highly genetically variable and susceptible to developing resistance through mutation or genetic recombination. It can spread rapidly asexually through conidia and also through sexual reproduction through genetic recombination, accelerating the spread of resistance. Long-term use of chemical pesticides not only contributes to the development and spread of resistance in gray mold, resulting in reduced or even complete loss of control effectiveness, but also exacerbates environmental pollution and harms human health. With increasing awareness of environmental protection and pollution-free practices, highly effective, low-toxic, and residue-free biopesticides are gaining attention.

[0004] In recent years, gene silencing technology has continued to advance, and the use of RNAi principles to control plant diseases has also matured. Spray-induced gene silencing (SIGS) is a new technology that is precise, easy to use, safe, and non-toxic. dsRNA targeting the target gene is synthesized in vitro and then applied to the plant surface via spraying, thereby inhibiting the expression of the target gene. Furthermore, the integrated utilization of multiple targets makes it difficult for pathogens to evade pesticides through a single mutation. Pathogens also need to accumulate multiple mutations simultaneously to develop resistance, significantly reducing the probability of resistance development. Summary of the Invention

[0005] Purpose of the invention: In response to the deficiencies in the prior art, the present invention provides a key pathogenic gene of Botrytis cinerea that is precisely targeted and inhibited. Furthermore, by specifically designing dsRNA targeted to the gene, the pathogenicity of Botrytis cinerea can be reduced, thereby preventing and controlling gray mold in crops, providing a basis for future green prevention and control of gray mold in production practices.

[0006] The present invention also provides a dsRNA agent targeting the pathogenic gene of Botrytis cinerea to reduce the off-target risk caused by gene mutation and its application.

[0007] Another objective of this invention is to investigate the effects of dsRNA target sequence mutations on SIGS by constructing synonymous substitution mutants of the Botrytis cinerea Gar2 gene. By comparing the sensitivity of wild-type and synonymous substitutions to the original target, ds-Gar2, the effects of synonymous mutations on SIGS efficiency were observed. Furthermore, ds-Gar2, which has an inhibitory effect on wild-type Botrytis cinerea, was used in conjunction with ds-Vel1 to investigate the efficacy of multi-target combination therapy in mutant strains, providing a theoretical basis for developing a multi-target screening system that can mitigate mutational risk.

[0008] Technical solution: In order to solve the above technical problems, the present invention describes a key pathogenic gene of Botrytis cinerea that is precisely targeted and inhibited. The key pathogenic gene of Botrytis cinerea is any one of the global regulatory factor Vel1 gene or the galacturonic acid reductase Gar2 gene of Botrytis cinerea, and its nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0009] Among them, by constructing a synonymous substitution mutant of the key pathogenic gene Gar2 of Botrytis cinerea, a synonymous substitution mutant strain of the Gar2 gene was obtained. The nucleotide sequence of Gar2 after synonymous replacement by the target fragment is shown in SEQ ID NO.33.

[0010] Furthermore, the present invention constructs a synonymous replacement vector for the key pathogenic gene Gar2, and then obtains a synonymous replacement fragment through the vector and transfers it into gray mold to obtain a synonymous replacement mutant, a mutant Botrytis cinerea strain.

[0011] Among them, the primers for synonymous substitution include Gar2-up-F, Gar2-up-R, Gar2-exon-F, Gar2-exon-R, Gar2-down-F, Gar2-down-R, Gar2-synonymous substitution region-F, Gar2-synonymous substitution region-R, terminal-F and terminal-R, and their nucleotide sequences are SEQ ID NO.11-SEQ ID NO.20, respectively.

[0012] The dsRNA of the present invention capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea is any one or both of the dsRNA of the Vel1 gene, a global regulatory factor of Botrytis cinerea, and the dsRNA of the galacturonate reductase Gar2 gene, and their nucleotide sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0013] Furthermore, the dsRNA capable of precisely targeting and inhibiting the Vel1 gene, a global regulatory factor of Botrytis cinerea, or the dsRNA capable of precisely targeting and inhibiting the Gar2 gene, comprises its sense strand and antisense strand, wherein the provided sequences SEQ ID NO.3 and SEQ ID NO.4 are their sense strands, and corresponding antisense strands also exist; usually, the dsRNA can be represented by its sense strand.

[0014] The present invention provides an expression vector containing the dsRNA capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea.

[0015] The method for constructing the dsRNA expression vector capable of precisely targeting and inhibiting the key pathogenic genes of Botrytis cinerea comprises the following steps:

[0016] (1) Total RNA was extracted from Botrytis cinerea and then reverse transcribed into cDNA;

[0017] (2) Using the cDNA product obtained in step (1) as a template, primers were designed to perform PCR amplification to obtain the DNA fragments of Vel1 and Gar2;

[0018] (3) the above products are connected to the vectors respectively by enzyme digestion and ligation;

[0019] (4) After correct sequencing, the expression vector is obtained.

[0020] The primer pairs used to amplify the dsRNA of Vel1 and Gar2 are Vel1-F, Vel1-R and Gar2-F, Gar2-R, respectively, and their nucleotide sequences are shown in SEQ ID NO. 7 to SEQ ID NO. 10, respectively.

[0021] The present invention provides a recombinant bacterium containing the dsRNA capable of accurately targeting and inhibiting key pathogenic genes of Botrytis cinerea or the expression vector.

[0022] The present invention provides a method for inducing dsRNA capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea. The specific induction method comprises the following steps: coating a glycerol plate of the recombinant bacteria with an LB plate, picking a single colony and adding it to an LB liquid culture medium, inducing with IPTG, extracting total RNA, and performing post-extraction digestion to remove DNA and single-stranded RNA to obtain a pure dsRNA solution as a dsRNA agent.

[0023] The present invention provides a method for delivering dsRNA that can precisely target and inhibit key pathogenic genes of Botrytis cinerea. The delivery method is an exogenous application method, in which the dsRNA targeting the key pathogenic genes of Botrytis cinerea is applied to plants that need to be protected.

[0024] Furthermore, the configuration and application method of the dsRNA mixture or dsRNA agent in the exogenous application method of dsRNA capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea of ​​the present invention are as follows:

[0025] (1) Dissolve dsRNA in DEPC water and adjust the concentration to 50 ng / μL;

[0026] (2) Apply the dsRNA solution to the surface of the crop to be protected.

[0027] The use of any one or two combinations of the dsRNAs described in the present invention that can accurately target and inhibit key pathogenic genes of Botrytis cinerea, or any one or two combinations of the expression vectors, or any one or two combinations of the recombinant bacteria in preventing and controlling gray mold of crops or gray mold mutations of crops.

[0028] The invention relates to an application of the dsRNA in the preparation of a dsRNA agent for reducing the pathogenicity of Botrytis cinerea.

[0029] Preferably, the crops include Arabidopsis thaliana, tomato fruit and onion.

[0030] The present invention not only provides a new dsRNA and its expression vector and recombinant bacteria that can accurately target and inhibit the key pathogenic genes of Botrytis cinerea. At the same time, the present invention found that although the synonymous substitution in the CDS region of the galacturonate reductase Gar2 gene of Botrytis cinerea did not significantly change the growth characteristics of the strain, it led to a decrease in its pathogenicity and insensitivity to the original target ds-Gar2, confirming that the targeting specificity of the SIGS technology mainly depends on sequence matching. When ds-Gar2 was unable to inhibit the infection of the pathogen in the Δgar2 synonymous substitution strain, the two-component dsRNA (ds-Gar2+ds-Vel1) showed a good control effect in the synonymous substitution strain, and the leaf lesion area was reduced by 52.8% compared with the ds-Gar2 treatment group, proving that the multi-target strategy can enhance the ability of RNA pesticides to cope with the risk of resistance mutation of pathogen target genes. It provides key theoretical support and technical paths for the subsequent systematic exploration of the synergistic mechanism of multi-component RNA, the establishment of a new RNA biopesticide research and development system, and the conduct of multi-target synergistic control research.

[0031] The present invention, for the first time, synonymously replaces 400bp of the CDS region of the galacturonate reductase Gar2 gene of Botrytis cinerea to obtain a mutant gene and a mutant strain, and constructs two dsRNAs, ds-Gar2 and ds-Vel1, as well as a mixture of the two. After the synonymous replacement of the 400bp sequence of the ds-Gar2 target Gar2 gene, strict target recognition, i.e., sequence complementary pairing, is impossible, resulting in the ineffectiveness of ds-Gar2 when administered alone.

[0032] The dual-component RNA constructed by the present invention enhances the ability of RNA pesticides to cope with the risk of resistance mutation and has a synergistic effect. Currently, RNA pesticides face the problem of target gene resistance mutation during application. These mutations may be generated through adaptive evolution, significantly reducing the control effect. In order to improve the ability to cope with resistance mutation and off-target risks, the present invention evaluates the effectiveness of RNA pesticides after mutation of a single target gene of Botrytis cinerea through the synergistic use of dual-component RNA (ds-Gar2 + ds-Vel1). A target gene Gar2 that is highly effective in inhibiting gray mold that was screened in the early stage was used, and a mutant strain Δgar2 with a synonymous substitution (changing nucleotides but not amino acid sequence) was constructed. 32.5% of the nucleotides in the ds-Gar2 target region were replaced, that is, 32.5% of the nucleotides in the 400bp of the target gene were replaced. The results showed that although the growth rate and pathogenicity of the Δgar2 synonymous substitution strain were not significantly affected, the sensitivity to the original target ds-Gar2 was significantly reduced, proving that mutations in the dsRNA target gene can significantly affect the control effect of RNA pesticides. The use of two-component RNA pesticides showed a good control effect on Δgar2, and the leaf spot area was reduced by 52.8% compared with the ds-Gar2 treatment group. The above results demonstrate the effectiveness of dual-target synergy in reducing off-target risks. Furthermore, the present invention demonstrates for the first time that two-component RNA can improve the ability of RNA agriculture to cope with resistance mutations and off-target risks. The present invention constructs for the first time a synonymous codon replacement mutant strain Δgar2 of the target gene Gar2 of Botrytis cinerea. Experiments have shown that ds-Gar2 designed for the wild-type Botrytis cinerea Gar2 gene loses its inhibitory effect on the mutant strain Δgar2, indicating that dsRNA target gene mutations significantly affect the control effect of RNA pesticides. The use of two-component RNA (ds-Gar2+ds-Vel1) can effectively inhibit the infection of Δgar2, revealing that the use of multi-target composite RNA pesticides is an effective strategy to reduce the adaptive evolution of pathogens and cope with the risk of target gene mutations.

[0033] Existing studies have shown that there are significant differences in the silencing efficacy of dsRNA molecules that interfere with different target genes or different functional regions of the same gene (Gu KX, Song XS, Xiao XM, et al. Aβ(2)-tubulin dsRNA derived from Fusarium asiaticum confers plant resistance to multiple phytopathogens and reduces fungicide resistance[J]. Pesticide Biochemistry and Physiology, 2019, 153(1): 36-46.). Therefore, in the development process of RNA biopesticides, the design and screening of effective dsRNA molecules are particularly important. The present invention designs two specific corresponding dsRNAs through two specific target genes. Furthermore, the present invention protects dsRNA by constructing synonymous substitution bodies and using synonymous substitution mutants as an auxiliary.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] 1. The present invention clones a novel double-stranded RNA (dsRNA) for the first time. After incubating ds-Vel1 or ds-Gar2 with Botrytis cinerea spores, the dsRNA induces silencing of the target gene through RNAi, thereby reducing the germination rate of Botrytis cinerea spores, affecting infection pad formation and hyphal growth, and achieving high gene silencing efficiency.

[0036] 2. After treating Arabidopsis thaliana, onion, and tomato with the Botrytis cinerea BC05.10 and ds-Vel1 and / or ds-Gar2 of the present invention, the lesion area of ​​the treated leaves was significantly reduced after 3 days. The dsRNA of the key pathogenic genes of Botrytis cinerea, namely ds-Vel1 and ds-Gar2, is expected to be developed into a new green fungicide.

[0037] 3. This study evaluated the effectiveness of RNA pesticides against single target gene mutations in Botrytis cinerea. Using dsGar2, a strain that was screened for its effectiveness in controlling gray mold, a mutant strain, Δgar2, with synonymous substitutions, was constructed for its target gene, Gar2. The effects of synonymous mutations on SIGS efficiency were investigated by comparing the sensitivity of wild-type and synonymous substitutions to the original target, ds-Gar2. ds-Gar2, which also inhibits wild-type gray mold, was used in conjunction with ds-Vel1 to explore the control efficacy of the two-component RNA pesticide in the mutant strain, providing a theoretical basis for constructing a multi-target RNA pesticide system that can resist mutation risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The key pathogenicity gene of Botrytis cinerea of ​​the present invention was verified by target fragment cloning PCR.

[0039] Figure 2 This is the inducible expression of dsRNA in the recombinant Escherichia coli of the present invention.

[0040] Figure 3 is the spore germination rate of Botrytis cinerea after treatment with the dsRNA of the present invention. a, b, c, and d are 3, 6, 9, and 12 hours after dsRNA treatment, respectively. The spore germination morphology of Botrytis cinerea is divided into three categories: Type 1 (control group): normal spore germination; Type 2: abnormal spore germination; Type 3: no spore germination.

[0041] Figure 4 Figure 2 shows the formation of infection mat structures of Botrytis cinerea after treatment with the dsRNA of the present invention, which are the numbers of infection mat structures formed by Botrytis cinerea 24 and 48 hours after treatment with the dsRNA.

[0042] Figure 5 The figures show the droplets recovered 3, 6, 9, 12, 24 and 48 hours after the spores of Botrytis cinerea were treated with the dsRNA of the present invention, with 1000 bp DNA used as a control.

[0043] Figure 6 This is the pathogenicity of Botrytis cinerea on Arabidopsis leaves after treatment with the dsRNA of the present invention.

[0044] Figure 7 Figure 2 shows the colonization of Botrytis cinerea on Arabidopsis leaves after treatment with the dsRNA of the present invention. a and b show the colonization of Botrytis cinerea on Arabidopsis leaves 24 and 48 hours after treatment with the dsRNA, respectively.

[0045] Figure 8 Figure 2 shows the pathogenicity of Botrytis cinerea on tomato fruit and onion after treatment with the dsRNA of the present invention. a and b respectively show the pathogenicity tests of the tomato fruit and onion after the dsRNA and spore suspension are premixed.

[0046] Figure 9 This is a schematic diagram of the construction of synonymous substitution mutants of key pathogenic genes of Botrytis cinerea in the present invention.

[0047] Figure 10 The invention provides a PCR process for constructing synonymous substitution mutants of key pathogenic genes of Botrytis cinerea.

[0048] Figure 11 PCR verification was constructed for the construction of synonymous substitution mutants of key pathogenic genes of Botrytis cinerea of ​​the present invention.

[0049] Figure 12q-PCR verification was constructed for the synonymous substitution mutants of the key pathogenicity genes of Botrytis cinerea of ​​the present invention.

[0050] Figure 13 The colonization of synonymous substitution mutants of key pathogenic genes of Botrytis cinerea on Arabidopsis leaves after treatment with the dsRNA of the present invention. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the accompanying drawings and examples.

[0052] The L4440 vector is a well-known vector provided by Nanjing Agricultural University. For details, see the literature: Chen Pan, Xia Shengge, Lan Ying, et al. Study on the inhibitory effect of LDH-enhanced dsRNA on rice blast [J]. Journal of Nanjing Agricultural University, 2024, 47(06): 1070-1076.

[0053] The pGKO2 vector is a well-known vector provided by Nanjing Agricultural University. For details, see the literature: Li X, Su X, Lu G, et al. VdOGDH is involved in energy metabolism and required for virulence of Verticillium dahliae [J]. Current Genetics, 2020, 66(2): 345-359, etc.

[0054] The pMD-19T plasmid was purchased from Takara.

[0055] PDB liquid culture medium: The specific preparation method is as follows: chop 200 grams of potatoes, add 1000 ml of water and boil for half an hour, filter with gauze, add 20 grams of glucose, and sterilize at 121°C for 20 minutes.

[0056] Botrytis cinerea (BC05.10) was provided by Nanjing Agricultural University.

[0057] Example 1

[0058] Method for cloning target fragments of key pathogenic genes of Botrytis cinerea and constructing dsRNA expression vectors capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea

[0059] The transcriptome of Botrytis cinerea was screened to identify Vel1 or Gar2, key genes for growth, development, or pathogenicity. Their sequences are shown in SEQ ID NOs. 1-2. Using Botrytis cinerea (BC05.10) cDNA as a template, a 400-bp target fragment of Vel1 or Gar2 was designed and ligated into the L4440 vector for expression. The nucleotide sequences corresponding to the Vel1 and Gar2 target fragments are shown in SEQ ID NOs. 5 and 6.

[0060] The specific experimental method is as follows: wait until the hyphae of Botrytis cinerea BC05.10 (provided by Nanjing Agricultural University) completely cover the entire culture medium, scrape the hyphae with a coverslip, place them in a 1.5 ml RNase centrifuge tube, pre-cool the centrifuge tube in liquid nitrogen, and then grind them into powder using a grinding rod.

[0061] TRIzol reagent (Invitrogen) was added to extract total RNA from Botrytis cinerea, and then reverse transcribed into cDNA as a template.

[0062] Using Botrytis cinerea cinerea cinerea cDNA as a template, primers for preparing a specific dsRNA expression vector were designed and constructed, as shown in Table 1, including Vel1-F, Vel1-R and Gar2-F, Gar2-R. The nucleotide sequences of the primers are shown in SEQ ID NOs. 7 to 10. PCR amplification was performed under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30-60 s / kb, for a total of 35 cycles, followed by extension at 72°C for 5 min. Vel1 and Gar2 fragments were obtained, and the PCR amplified products were subjected to electrophoresis and then recovered by gel excision. The products were purified using a DNA gel recovery kit (Axygen).

[0063] The fragment product and the L4440 vector were double-digested with SacI and XhoI (Takara). The digested product was purified using a PCR purification kit (Axygen) and ligated with the L4440 vector overnight using T4 DNA Ligase (Takara). The ligated product was transformed into Escherichia coli HT115 (DE3), and glycerol stocks were stored after the individual strains were sequenced correctly.

[0064] like Figure 1As shown, Vel1 and Gar bands of about 400 bp were amplified by the above method, which was consistent with the expected results. In this example, nucleotide fragments expressing dsRNA (Vel1 and Gar2) capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea were successfully amplified, and their DNA sequences were shown in SEQ ID NO. 5 and SEQ ID NO. 6 (i.e., Vel1-400 bp and Gar2-400 bp), respectively.

[0065] BMP1 in the present invention is a MAP kinase that is crucial for fungal pathogenesis. It has been reported that it can be used as a target for SIGS to reduce the pathogenicity of Botrytis cinerea. (Spada M, Pugliese C, Fambrini M, et al. Knockdown of Bmp1 and Pls1 virulence genes by exogenous application of RNAi-inducing dsRNA in Botrytis cinerea [J]. International Journal of Molecular Sciences, 2023, 24 (5): 4869.), so the present invention uses the Bmp1 gene as a positive control, and adopts the vector construction method and experimental method of the present invention to explore the inhibitory effect of Vel1 and Gar as targets of SIGS on Botrytis cinerea. The nucleotide sequence of the Bmp1 gene is shown in SEQ ID NO.31, and the corresponding dsRNA (ds-Bmp1) nucleotide sequence is shown in SEQ ID NO.32.

[0066] Table 1 Nucleotide sequences of upstream and downstream primers

[0067]

[0068]

[0069]

[0070] Example 2

[0071] Inducible expression of dsRNA for precise targeted inhibition of key pathogenicity genes Vel1 and Gar2 in Botrytis cinerea

[0072] Take 10 uL of the glycerol bacteria containing the target recombinant vector prepared in Example 1 and spread it on an LB solid plate containing ampicillin and tetracycline resistance (12.5 μg / mL Tet+, 50 μg / mL Amp+) by zigzag streak method. After 24 hours, use a sterilized pipette tip to pick a single colony and add it to 10 ml of LB liquid culture medium containing ampicillin and tetracycline resistance (12.5 μg / mL Tet+, 50 μg / mL Amp+), and place it in a 37°C constant temperature shaker at 220 rpm overnight. 10 ml of the initial culture solution was taken at a ratio of 1:10 and added to 90 ml of LB liquid culture medium containing ampicillin and tetracycline resistance (12.5 μg / mL Tet+, 50 μg / mL Amp+), placed in a 37°C constant temperature shaker, incubated at 220 rpm for 2 hours, and IPTG (0.5 mmol / L) was added to the OD 600 = 0.6-0.8, and then placed in a 37°C shaker for induction for 4-5 hours. Total RNA of Escherichia coli HT115 (DE3) was extracted using TRIzol reagent (Invitrogen). After extraction, it was digested with DNAseⅠ and RNaseA to remove DNA and single-stranded RNA. After treatment, it was detected by electrophoresis on a 1% agarose gel. Figure 2 A clear, bright band was observed at the 400-bp position of the target band, demonstrating the successful use of E. coli to induce dsRNAs that specifically inhibit the key pathogenicity genes Vel1 and Gar2 of Botrytis cinerea. These dsRNAs are ds-Vel1 and ds-Gar2, respectively. The RNA sequences of these dsRNAs are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. ds-Bmp1 was also prepared using this method.

[0073] Example 3

[0074] ds-Vel1 and ds-Gar2 inhibit spore germination of Botrytis cinerea

[0075] The spores of Botrytis cinerea (BC05.10) were suspended in PDB liquid medium and adjusted to 2×10 5 The concentrations of ds-Vel1, ds-Gar2, and ds-Bmp1 dsRNA and ds-GFP prepared in Example 2 were adjusted to 50 ng / uL. 5 μL of BC05.10 spore suspension (2×10 5CFU / mL) and 20 μL DEPC water; control group 2 (ds-GFP) was treated with 5 μL BC05.10 spore suspension and 20 μL ds-GFP; control group 3 (ds-Bmp1) was treated with 5 μL BC05.10 spore suspension and 20 μL ds-Bmp1; treatment group 1 (ds-Vel1): 5 μL BC05.10 + 20 μL ds-Vel1; treatment group 2 (ds-Gar2): 5 μL BC05.10 + 20 μL ds-Gar2. In addition, 3 μL 1000 bp DNA was added to each group as a control. The above mixed solutions were added dropwise to Fisher hydrophobic slides and incubated in a transparent plastic box with a humidity of >85%. The boxes were placed in a 28°C incubator and removed after 3, 6, 9, and 12 hours.

[0076] The morphology of Botrytis cinerea spores in the treated and control groups was observed under an inverted fluorescence microscope (Zeiss Axio observe 3). Nine photos were taken under different treatments for analysis to facilitate differentiation and accurate quantification of the inhibitory effect of dsRNA on Botrytis cinerea spore germination. Figure 3 As shown in A, the spore germination morphology of Botrytis cinerea is divided into three categories: Type 1 (control group): normal spore germination; Type 2: abnormal spore germination; Type 3: no spore germination.

[0077] Among them, the classification and statistics of Botrytis cinerea spore growth (number of normal spores germinated / total spores, number of deformed spores germinated / total spores, number of non-germinated spores / total spores) after 3, 6, 9 and 12 hours of treatment with different dsRNAs. Figure 3 As shown in (a) in B, in the control group 1 (Mock) and the control group 2 (ds-GFP), the germination rate of Botrytis cinerea spores was normal after incubation with dsRNA for 3 hours. The type 1 germination rate of the Mock group was 74%, and the type 1 germination rate of the ds-GFP group was 69%, indicating that the dsRNA that non-targetedly inhibits the key pathogenicity gene has no inhibitory effect on the germination of Botrytis cinerea spores. In contrast, the germination rate of type 1 of spores of ds-Bmp1, which targets the inhibition of the key pathogenicity gene of Botrytis cinerea, is 25%, and the non-germination rate of type 3 is 75%, indicating that ds-Bmp1 has an inhibitory effect on the germination of Botrytis cinerea spores. In the present invention, the germination rate of type 1 of spores of ds-Vel1 and ds-Gar2 is significantly inhibited like ds-Bmp1, which are 17% and 18% respectively. This indicates that ds-Vel1 and ds-Gar2 have a significant inhibitory effect on the germination of Botrytis cinerea spores after 3 hours. As shown in FIG. Figure 3As shown in (b) in B, after 6 hours of co-incubation, the type 1 in the control group 1 (Mock) was 67.0%, the type 2 was 13.2%, and the type 3 was 19.8%; the type 1 in the control group 2 (ds-GFP) was 58.8%, the type 2 was 14.8%, and the type 3 was 26.4%; the type 1 in the control group 3 (ds-Bmp1) was 19.9%, the type 2 was 22.9%, and the type 3 was 57.3%; in comparison, the type 1 in the treatment group 1 (ds-Vel1) was 7.4%, the type 2 was 15.6%, and the type 3 was 77.0%; the type 1 in the treatment group 2 (ds-Gar2) was 9.0%, the type 2 was 19.6%, and the type 3 was 80.4%; the germination rate of Botrytis cinerea spores decreased significantly, and the non-germination rate increased significantly. Figure 3 (c) and (d) in B showed the same inhibitory effect after 9 and 12 hours of co-incubation. This shows that ds-Vel1 and ds-Gar2 have the same inhibitory effect on Botrytis cinerea as ds-Bmp1, not only inhibiting the normal germination of Botrytis cinerea spores, but also inhibiting the hyphal growth of Botrytis cinerea, and exceeding that of ds-Bmp1. To test whether dsRNA is degraded during the experiment, droplet recovery was performed for groups with experimental durations of 3, 6, 9, 12, 24, and 48 hours, as shown in Figure 2. Figure 5 , there were obvious dsRNA bands, proving that dsRNA was not degraded during the experiment.

[0078] Example 4

[0079] ds-Vel1 and ds-Gar2 inhibit the formation of Botrytis cinerea infection pad structures

[0080] The control and treatment groups co-cultured on hydrophobic glass slides in Example 3 were removed after 24 and 48 hours, and the number of Botrytis cinerea infection pads in the treated and control groups was observed under an inverted fluorescence microscope (Zeiss Axio observe 3). Nine photographs were taken for each treatment and analyzed to facilitate differentiation and accurate quantification of the inhibitory effect of dsRNA on the formation of Botrytis cinerea infection pad structure.

[0081] like Figure 4As shown, in control group 1 (Mock) and control group 2 (ds-GFP), the formation of the infection pad structure of Botrytis cinerea spores after 24 hours of co-incubation with dsRNA was normal. The number of infection pads in the Mock group was 73, and the number of infection pads in the ds-GFP group was 62, indicating that the dsRNA that non-targetedly inhibits the key pathogenicity genes has no inhibitory effect on the formation of Botrytis cinerea infection pads. In contrast, the number of infection pads of ds-Bmp1, which targets the inhibition of the key pathogenicity genes of Botrytis cinerea, was 47, and the number of infection pads of ds-Gar2 was 50, indicating that ds-Bmp1 has an inhibitory effect on the formation of the infection pad structure of Botrytis cinerea. The number of infection pads of ds-Vel1 in the present invention was significantly suppressed to 13, and the inhibitory effect of ds-Vel1 significantly exceeded that of ds-Bmp1, with the strongest inhibitory effect. After 48 hours, the same trend was shown, indicating that the inhibitory effects of ds-Vel1 and ds-Gar2 on Botrytis cinerea were the same as those of ds-Bmp1, and they had a significant inhibitory effect on the formation of Botrytis cinerea infection mats, among which the inhibitory effect of ds-Vel1 was similar to that of ds-Bmp1.

[0082] Example 5

[0083] ds-Vel1 and ds-Gar2 reduce the pathogenicity of Botrytis cinerea on Arabidopsis leaves

[0084] Evenly spread Columbia 0 type Arabidopsis seeds (provided by Nanjing Agricultural University) on a seedling pot filled with vermiculite, cover with tin foil and place in a 4-degree refrigerator to avoid light. Low-temperature vernalization treatment for 2 days, remove the tin foil after 2 days and place in a 23-degree greenhouse. After germination in about a week, move the seedlings to a new pot. After growing for about four weeks, cut off the leaves of uniform size for later use.

[0085] The dsRNA is delivered to Arabidopsis thaliana by exogenous application, and the specific steps are as follows:

[0086] (1) Dissolve the dsRNA prepared in Example 2 in DEPC water and adjust the concentration to 50 ng / μL;

[0087] (2) Use DEPC water to scrape the mixture of Botrytis cinerea spores (BC05.10) and mycelium from the plate, filter it with lens paper, and adjust the concentration to 2×10 5 CFU / mL.

[0088] (3) The dsRNA solution was mixed with the Botrytis cinerea spore suspension at a volume ratio of 1:5 to prepare a mixed solution. The control group 1 (Mock): 5uL BC05.10 + 20uL DEPC water; the control group 2 (ds-GFP): 5uL BC05.10 + 20uL ds-GFP; the control group 3 (ds-Bmp1): 5μL BC05.10 + 20uL ds-Bmp1; the treatment group 1 (ds-Vel1): 5uL BC05.10 + 20uL ds-Vel1; the treatment group 2 (ds-Gar2): 5uL BC05.10 + 20uL ds-Gar2;

[0089] (4) Add 25 μL of the above mixed solution to the center of the Arabidopsis leaf respectively, place it in a storage box with a wet filter paper on the bottom, and place the container in a 25°C incubator for moisturizing culture. The humidity in the container is RH>85%. Take pictures and observe after three days.

[0090] like Figure 6 As shown, in vitro pathogenicity of Arabidopsis leaves showed that the lesion area of ​​treatment group 1 (ds-Vel1) and treatment group 2 (ds-Gar2) was significantly smaller than that of control group 1 (Mock), control group 2 (ds-GFP), and control group 3 (ds-Bmp1). Quantitative statistics of lesion area showed the same trend. This indicates that ds-Bmp1 has a certain inhibitory effect on Botrytis cinerea pathogenicity. The ds-Vel1 and ds-Gar2 of the present invention have the same significant inhibitory effect on Botrytis cinerea pathogenicity as ds-Bmp1, and the lesion area of ​​treatment group ds-Vel1 is significantly smaller than that of the other groups.

[0091] Example 6

[0092] ds-Vel1 and ds-Gar2 reduce the colonization of Botrytis cinerea in Arabidopsis leaves

[0093] Arabidopsis leaves treated by the exogenous application method described in Example 5 were dropwise added with a mixture of a Botrytis cinerea spore suspension and dsRNA (ds-Vel1, ds-Gar2). The leaves were then subjected to a high humidity control (RH>85%). Samples were taken on day 1 and day 2, and the mixture was removed from the leaves using trypan blue staining. The leaves were then placed in a 10 ml EP tube, 5 ml of staining solution was added, and the tubes were waterbathed at 100°C for 2 minutes. The tubes were then allowed to stand overnight at room temperature for staining. The leaves were then washed three times with a 2.5 g / mL chloral hydrate solution on a decolorizing shaker. The colonization of Botrytis cinerea in the Arabidopsis leaves was observed under a microscope.

[0094] The detached leaves of Arabidopsis thaliana infected with Botrytis cinerea were sampled 1-2 days later and the colonization of Botrytis cinerea was observed using trypan blue staining. Figure 7(a) shows that after 24 hours of treatment with dsRNA (ds-Vel1, ds-Gar2), it can be clearly seen that the colonization of treatment group 1 (ds-Vel1) and treatment group 2 (ds-Gar2) was significantly inhibited compared with control group 1 (Mock) and control group 2 (ds-GFP), and the colonization was not much different from that of control group 3 (ds-Bmp1). Among them, the hyphae of Botrytis cinerea stained with trypan blue were significantly reduced, indicating that dsRNA (ds-Vel1, ds-Gar2) can inhibit the colonization of Botrytis cinerea in Arabidopsis leaves within 24 hours. Figure 7 As shown in (b), after 48 hours of treatment with dsRNA (ds-Vel1, ds-Gar2), it can be seen that the colonization of treatment group 1 (ds-Vel1) and treatment group 2 (ds-Gar2) was significantly inhibited compared with control group 1 (Mock) and control group 2 (ds-GFP), and the colonization was not much different from that of control group 3 (ds-Bmp1). Among them, the number of Botrytis cinerea hyphae stained with trypan blue was significantly reduced, indicating that ds-Vel1 and ds-Gar2 of the present invention can inhibit the colonization of Botrytis cinerea in Arabidopsis leaves within 48 hours.

[0095] Example 7

[0096] ds-Vel1 and ds-Gar2 reduce the pathogenicity of Botrytis cinerea on tomato fruits and onions

[0097] After cleaning the purchased onions, wipe the surface with 75% alcohol and cut them into uniform sizes for later use. After cleaning the purchased tomatoes, wipe the surface with 75% alcohol and use a 1ml syringe to make a 5mm deep wound for later use. The control group (Mock, ds-GFP, ds-Bmp1) and the treatment group (ds-Vel1, ds-Gar2) used the exogenous application method described in Example 5 to deliver dsRNA. The only difference was that the Arabidopsis leaves were replaced with onions and tomatoes, and the other processes remained unchanged. 25uL of the mixed solution was added dropwise to the wound of the tomato fruit and the center of the inner epidermis of the onion, respectively, and placed in a storage box with soaked filter paper on the bottom layer. The container was placed in a 25°C incubator for highly moisturizing culture, and photographed and observed after three days.

[0098] Tomato fruit and onion pathogenicity showed, e.g. Figure 8 As shown in the figure, in comparison, the lesion area of ​​treatment group 1 (ds-Vel1) and treatment group 2 (ds-Gar2) was significantly smaller than that of control group 1 (Mock) and control group 2 (ds-GFP), and there was no significant difference compared with control group 3 (ds-Bmp1). Quantitative statistics of the lesion area showed the same trend, indicating that ds-Vel1 and ds-Gar2 have the same significant inhibitory effect on the pathogenicity of Botrytis cinerea on various plants as ds-Bmp1.

[0099] Example 8

[0100] Method for constructing synonymous substitution mutants of key pathogenicity genes of Botrytis cinerea

[0101] The CDS region of Gar2 was divided into two parts: the first part was the 400bp sequence of the dsRNA target, and the second part was the remaining CDS region except the target sequence. The first part of the nucleotide sequence was synonymously replaced according to the standard codon table. The obtained sequence was analyzed by the codon adaptation index (CAI) with the help of bioinformatics analysis tools and confirmed to be correct. It was then synthesized by General Bio (Anhui) Co., Ltd. The second part was obtained by PCR amplification. Figure 9 As shown in the schematic diagram, a synonymous substitution mutant of the key Botrytis cinerea pathogenic gene, galacturonate reductase Gar2, was constructed. Using Botrytis cinerea (BC05.10) cDNA as a template, upstream and downstream homology arms approximately 1500 base pairs long were designed centered around the CDS region of Gar2. Synonymous substitution in the targeted 400-bp fragment of Gar2 altered the nucleotide sequence while maintaining the expressed protein. This was then linked to the remaining CDS region. The upstream and downstream homology arms were then linked to the PGKO2 vector, and the expressed fragments were then transformed into Botrytis cinerea to generate the synonymous substitution mutant of Gar2. The nucleotide sequence of Gar2 after synonymous replacement by the target fragment was synthesized by General Corporation, and its nucleotide sequence is shown in SEQ ID NO.33. In order to minimize the impact of synonymous replacement on gene expression, the synthesized fragment also includes a 66-bp intron sequence present in the target region, and its nucleotide sequence is shown in SEQ ID NO.34. The nucleotide sequences of the remaining CDS region, upstream and downstream homology arms up and down, and the terminator on the pMD19T plasmid are shown in SEQ ID NO.35-SEQ ID NO.38.

[0102] The specific experimental method is as follows: wait until the hyphae of Botrytis cinerea BC05.10 (provided by Nanjing Agricultural University) completely cover the entire culture medium, scrape the hyphae with a coverslip, place them in a 1.5 ml RNase centrifuge tube, pre-cool the centrifuge tube in liquid nitrogen, and then grind them into powder using a grinding rod.

[0103] TRIzol reagent (Invitrogen) was added to extract total RNA from Botrytis cinerea, and then reverse transcribed into cDNA as a template.

[0104] Upstream and downstream homology arms (up and down) approximately 1500 bp long were selected centered around the CDS region of Gar2. Using Botrytis cinerea cDNA as a template, primers for the upstream and downstream homology arms and the remaining CDS region excluding the target sequence were designed, as shown in Table 1. These primers include homologous recombination primers Gar2-up-F, Gar2-up-R, Gar2-exon-F, Gar2-exon-R, Gar2-down-F, and Gar2-down-R, with nucleotide sequences shown in SEQ ID NOs. 11 to 16. Using the plasmid synthesized by the company with the Gar2-synonymous replacement region as a template, the homologous recombination primers Gar2-synonymous replacement region-F and Gar2-synonymous replacement region-R of the GAR2 synonymous replacement mutant were designed and constructed, and the nucleotide sequences are shown in SEQ ID NO.17-SEQ ID NO.18; using the pMD19T plasmid as a template, the terminator region was selected to design primers Terminal-F and Terminal-R, and the nucleotide sequences are shown in SEQ ID NO.19-SEQID NO.20.

[0105] The above fragments were amplified by PCR using the following reaction conditions: 35 cycles of denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30-60 seconds / kb, followed by 5 minutes of extension at 72°C. The resulting fragments were Gar2-up, Gar2-exon, Gar2-down, Gar2-synonymous substitution region, and terminator. The PCR amplification products were subjected to electrophoresis and then recovered by gel extraction and purification using a DNA gel extraction kit (Axygen).

[0106] The pGKO2 vector was double-digested with SalI and KPNI (Takara). The digested product was purified using a PCR purification kit (Axygen) and ligated with the pGKO2 vector using the ClonExpress II One Step Cloning Kit (Vazyme) at 37°C for 30 min. The ligation product was transformed into Escherichia coli DH5α, and glycerol stocks were stored after the individual strains were sequenced correctly.

[0107] The pGKO2 vector, into which the GAR2-down fragment had been ligated, was double-digested with SmaI and StuI (Takara). The digested product was purified using a PCR purification kit (Axygen) and ligated with the pGKO2 vector into which the GAR2-down fragment had been ligated using the ClonExpress II One Step Cloning Kit (Vazyme). The ligated product was transformed into Escherichia coli DH5α, and glycerol stocks were stored after the individual strains were sequenced correctly.

[0108] Overlap PCR was used to connect the Gar2-Exon and terminator fragments to obtain Gar2-Exon-Terminal. Subsequently, overlap PCR was used to connect the Gar2-synonymous substitution region and Gar2-Exon-Terminal to obtain Gar2-synonymous substitution fragment-Exon-Terminal. This fragment was used as a template for reamplification using homologous recombination primers. The nucleotide sequences of the primers are shown in SEQ ID NO. 21 and SEQ ID NO. 22.

[0109] The pGKO2 vector, into which the Gar2-up and Gar2-down fragments had been ligated, was digested with StuI (Takara). The digested product was purified using a PCR purification kit (Axygen). The purified product (GAR2-synonymous substitution fragment-Exon-Terminal fragment) was homologously ligated with the pGKO2 vector into which the Gar2-up and Gar2-down fragments had been ligated using the ClonExpress II One Step Cloning Kit (Vazyme). The ligated product was transformed into Escherichia coli DH5α, and glycerol stocks were stored after the individual strains were sequenced correctly.

[0110] like Figure 10 As shown, the synonymous substitution mutant nucleotide fragment of the key pathogenic gene Gar2 of Botrytis cinerea was successfully amplified by the above method.

[0111] Example 9

[0112] Transformation and verification of synonymous substitution mutants of key pathogenicity genes of Botrytis cinerea

[0113] Using a synonymous replacement vector of the Gar2 gene containing a synonymous replacement mutant sequence as a template, PCR amplification was performed using primers Gar2-up-F and Gar2-down-R, the nucleotide sequences of which are shown in SEQ ID NO.11 and SEQ ID NO.16. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30-60 s / kb, for a total of 35 cycles, and extension at 72°C for 5 min. A Δgar2 fragment of approximately 7 kb was obtained, containing upstream and downstream homology arms, the remaining CDS region of the synonymous replacement region, and the terminal. The PCR amplification product was subjected to electrophoresis and then gel-cleaved to recover the product. The product was purified using a DNA gel recovery kit (Axygen). The purified product was used for subsequent protoplast transformation of Botrytis cinerea. The specific steps are as follows:

[0114] (1) Strain activation: Inoculate the ultra-low temperature-stored Botrytis cinerea B05.10 onto PDA medium and culture at 22°C for 3-5 days;

[0115] (2) Mycelial culture: rinse the spores with sterile water, transfer the spore suspension to PDB liquid medium, and culture with shaking at 28°C for 36 hours until the mycelium grows vigorously;

[0116] (3) Preparation of protoplasts: Collect fresh mycelium by filtration and rinse with sterile water to remove residual culture medium. Add 0.5% enzymatic hydrolysate (prepared and used immediately) dissolved in 20 mL KCl buffer in the dark, and perform enzymatic hydrolysis at 120 rpm at 25°C for 2.5 hours. Filter the protoplasts obtained by lysis, centrifuge the filtrate at 2200 rpm for 10 minutes, discard the supernatant and collect the protoplasts. Wash twice with 5 ml of KCl buffer solution pre-cooled at 4°C, centrifuge at 2200 rpm for 10 minutes, and discard the supernatant. Resuspend the precipitate with 10 mL STC solution, centrifuge at 1500 rpm for 8 minutes, and discard the supernatant. Resuspend the precipitate with 800 μL STC solution and add 200 μL STC solution dropwise.

[0117] (4) Protoplast transformation: Take 100 μl of the resuspension, add 5 μl of 50 mM spermidine and 100 μl of the target DNA fragment, mix gently, place on ice in the dark for 30 min, then add 1 ml of SPTC and let it stand at room temperature for 30 min;

[0118] (5) Protoplast regeneration: Transfer the transformation mixture into RM regeneration medium and culture overnight at 25°C and 100 rpm. Pour the recovered mycelium onto PDA plates with hygromycin added at a moderate temperature. Pick the transformants after 4-5 days.

[0119] (6) Select the transformants that can grow normally on the hygromycin PDA plate and inoculate them on a new PDA plate. After culturing at 22°C for about a week, perform single spore isolation: After the transformants produce spores, prepare a spore suspension and adjust its concentration to 10 2 CFU / ml, aspirate 100 μL and spread onto a PDA plate. Re-cultivate for 1-3 days, pick the mycelium that germinated from a single spore and inoculate it onto a new PDA plate for culture.

[0120] Approximately 10 transformants with normal growth were selected and their mycelia or spores were inoculated into 20 ml of PDB medium. After shaking culture at 28°C for 16-24 hours, the culture pellets were filtered with lens paper and pre-cooled in liquid nitrogen. The pellets were then ground into powder using a grinding rod and divided into two portions. TRIzol reagent (Invitrogen) was added to each portion to extract total DNA and total RNA from Botrytis cinerea.

[0121] Using the DNA of the selected synonymous substitution transformants of Botrytis cinerea as templates, verification primers HPH-F, HPH-R and synonymous substitution verification-F, synonymous substitution verification-R were designed. The nucleotide sequences of the primers are shown in SEQ ID NO.23-SEQ ID NO.26. PCR amplification of a hygromycin fragment of about 1000 bp and a synonymous substitution region fragment of about 500 bp was performed to verify whether the mutant was successfully constructed. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 30-60 s / kb, for a total of 35 cycles, and extension at 72°C for 5 min. The results are shown in FIG. Figure 11 As shown, (a) is the verification of the HPH results. Transformants No. 1-13 and the positive control all have a band of about 1 kb, and WT as a blank control has no corresponding band, proving that hygromycin has been successfully transferred; (b) is the verification of the synonymous replacement region results. Transformants No. 1-13 and the positive control all have a band of about 500 bp, and WT and the negative control have no corresponding band, proving that the synonymous replacement region has been successfully transferred.

[0122] The RNA of the selected synonymous substitution transformants of Botrytis cinerea was reverse transcribed into cDNA as a template, and the expression levels of the remaining exon regions and the original target region of the Gar2 gene in the transformants were detected respectively. Quantitative primers were designed to include the remaining exon regions (exon), namely exon-qF, exon-qR and Gar2-qF, Gar2-qR. The nucleotide sequences of the primers are shown in SEQ ID NO.27-SEQ ID NO.30. The reverse transcribed cDNA was diluted 10 times and used as a template for qPCR reaction. The reaction procedure was 95°C for 5 minutes, 95°C for 10s, 60°C for 30s, and 40 cycles. The relative expression level of the target gene was analyzed using the 2^-ΔΔCt method. The reaction system was prepared as follows:

[0123]

[0124] like Figure 12 The figure shows the changes in gene expression in some transformants. qPCR was used to detect gene expression in the remaining exon region (Exon) and the original target region of the transformants. The results showed that the expression level of the remaining exon region of transformant No. 2 was least affected by the synonymous substitution, indicating that its mRNA stability and translation efficiency may be higher. At the same time, transformants No. 1, 2, and 4 all showed significant downregulation of gene expression in the original target region. Therefore, we believe that transformant No. 2 is the synonymous substitution mutant closest to the wild type and will be used for subsequent mechanism analysis and functional studies.

[0125] Example 10

[0126] Application of ds-Vel1 alone or a mixture of ds-Vel1 and ds-Gar2 reduces the pathogenicity of the Botrytis cinerea Δgar2 synonymous substitution mutant on Arabidopsis leaves

[0127] Evenly spread Columbia 0 type Arabidopsis seeds (provided by Nanjing Agricultural University) on a seedling pot filled with vermiculite, cover with tin foil and place in a 4-degree refrigerator to avoid light. Low-temperature vernalization treatment for 2 days, remove the tin foil after 2 days and place in a 23-degree greenhouse. After germination in about a week, move the seedlings to a new pot. After growing for about four weeks, cut off the leaves of uniform size for later use.

[0128] Among them, ds-Vel1, ds-Gar2, and ds-Vel1+ds-Gar2 were delivered to Arabidopsis thaliana using the exogenous administration method. The specific steps are as follows:

[0129] (1) Dissolve dsRNA in DEPC water and adjust the concentration to 50 ng / μL;

[0130] (2) Use DEPC water to scrape the spore and mycelial mixture of the synonymous substitution mutant of Botrytis cinerea Δgar2 from the plate, filter with lens paper, and adjust the concentration to 2×10 5 CFU / mL.

[0131] (3) The dsRNA solution and the Botrytis cinerea spore suspension were mixed at a ratio of 1:5 to prepare a mixed solution.

[0132] Control group 1 (ds-GFP): 5 μL Δgar2 / 5 μL BC05.10 + 20 μL ds-GFP;

[0133] Treatment group 1 (ds-Gar2): 5 μL Δgar2 / 5 μL BC05.10 + 20 μL ds-Gar2;

[0134] Treatment group 2 (ds-Vel1): 5 μL Δgar2 / 5 μL BC05.10 + 20 μL ds-Vel1;

[0135] Treatment group 3 (ds-Vel1+ds-Gar2): 5μLΔgar2 / 5μL BC05.10+20μL (ds-Vel1+ds-Gar2);

[0136] (4) Add 25 μL of the above mixture to the center of each Arabidopsis leaf, place it in a container with a wet filter paper on the bottom, and place the container in a 25°C incubator to maintain moisture and culture. The humidity in the container should be greater than 85%. Take photos and observe after three days.

[0137] To evaluate the effect of target gene sequence mutation on SIGS, we verified whether dsRNA treatment still had an inhibitory effect on the mutant. In vitro infection experiments were performed with ds-GFP, ds-Gar2, ds-Vel1, and ds-Gar2 + ds-Vel1 (1:1 mixture) and the lesion area of ​​the infected leaves was counted ( Figure 13 ).

[0138] In the wild-type strain WT, treatment with ds-Gar2, ds-Vel1, and their combination significantly inhibited pathogenicity, with lesion area decreasing by over 69% compared to the ds-GFP control, a highly significant difference. This result is consistent with the previous section, demonstrating that the ds-Gar2 and ds-Vel1 used in this experiment exerted an inhibitory effect. However, the combination of ds-Gar2 and ds-Vel1 showed no significant difference from the single treatments. This may be related to the ratio, type, and amount of dsRNAs used in the combined application, or because the single dsRNA treatment was already highly effective, resulting in a combination treatment with only a similar effect. Notably, the lesion area of ​​the Δgar2 mutant in the ds-GFP control was similar to that of the WT, indicating that the synonymous substitution in the GAR2 gene did not affect pathogenicity. This suggests that the reduced lesion size was caused by the dsRNA, rather than the synonymous substitution in the target gene, leading to decreased pathogenicity.

[0139] In Δgar2, the lesion area (54.84 mm 2 ) showed no significant difference from the control group, but was highly significant compared to the WT ds-Gar2-treated group. Due to a synonymous substitution within the 400-bp target sequence of the GAR2 gene in Δgar2, ds-Gar2 was unable to effectively bind to the target RNA, resulting in gene silencing failure. This result validates the targeting specificity of SIGS.

[0140] In addition, although ds-Gar2 had no effect when applied alone in the synonymous substitution strain, the combination of ds-Gar2 and ds-Vel1 still had a significant inhibitory effect, reducing the lesion area to 25.90 mm 2 , which was reduced by 52.8% compared with the mutant ds-Gar2 group. There were extremely significant differences between the mixed treatment group and the ds-GFP and ds-Gar2 groups, proving that ds-Vel1 is still playing a continuous role in target silencing.

[0141] In summary, the highly effective inhibition of ds-Gar2 in WT strains and its ineffectiveness in the Δgar2 strain confirm the targeting specificity of SIGS and demonstrate that multi-target RNAi can overcome resistance caused by single gene mutations. This further demonstrates that the present invention, using dsRNA designed based on multiple target genes, can effectively reduce off-target effects caused by single mutations in target genes.

Claims

1. A key pathogenic gene of Botrytis cinerea that is precisely targeted and inhibited, characterized in that: The key pathogenicity gene of Botrytis cinerea is any one or both of the global regulatory factor Vel1 gene and the galacturonic acid reductase Gar2 gene of Botrytis cinerea, and the nucleotide sequences thereof are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. The precisely targeted inhibition of the key pathogenic gene Gar2 of Botrytis cinerea according to claim 1, characterized in that: Preferably, a synonymous substitution mutant of the key pathogenic gene Gar2 of Botrytis cinerea is constructed to obtain a synonymous substitution mutant strain of the Gar2 gene. The nucleotide sequence of Gar2 after synonymous substitution by the target fragment is shown in SEQ ID NO.

33.

3. A dsRNA capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea according to claim 1, characterized in that: The dsRNA is any one or both of the dsRNA of the Botrytis cinerea global regulatory factor Vel1 gene or the dsRNA of the galacturonate reductase Gar2 gene, and the nucleotide sequences thereof are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. An expression vector for preparing the dsRNA according to claim 3 that can precisely target and inhibit key pathogenic genes of Botrytis cinerea.

5. A method for constructing a dsRNA expression vector capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea according to claim 4, characterized in that: The steps include: (1) Total RNA was extracted from Botrytis cinerea and then reverse transcribed into cDNA; (2) Using the cDNA product obtained in step (1) as a template, primers were designed to perform PCR amplification to obtain the DNA fragments of Vel1 and Gar2; (3) the above products are connected to the vectors respectively by enzyme digestion and ligation; (4) After correct sequencing, the expression vector is obtained.

6. The method for constructing a dsRNA expression vector according to claim 5, characterized in that: The primer pairs for amplifying the DNA fragments of Vel1 and Gar2 are Vel1-F, Vel1-R and Gar2-F, Gar2-R, respectively, and their nucleotide sequences are shown in SEQ ID NO.7 to SEQ ID NO.10, respectively.

7. A recombinant bacterium containing the dsRNA according to claim 3 that can accurately target and inhibit the key pathogenic genes of Botrytis cinerea or the expression vector according to claim 4.

8. A method for inducing dsRNA that can precisely target and inhibit key pathogenic genes of Botrytis cinerea, characterized in that: The specific method of the induction method is: taking the glycerol bacteria of the recombinant bacteria of claim 7 and coating an LB plate, picking a single colony and adding it to the LB liquid culture medium, using IPTG to induce and extract total RNA, and digesting and removing DNA and single-stranded RNA after extraction to obtain a pure dsRNA solution as a dsRNA agent.

9. A method for delivering dsRNA capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea, characterized in that: The delivery method is an exogenous application method, which involves applying dsRNA targeting the key pathogenicity gene of Botrytis cinerea to plants that need to be protected.

10. Use of any one or a combination of two of the dsRNAs capable of precisely targeting and inhibiting key pathogenic genes of Botrytis cinerea according to claim 3, any one or a combination of two of the expression vectors according to claim 4, or any one or a combination of two of the recombinant bacteria according to claim 7 in preventing and controlling gray mold of crops or gray mold mutations of crops.

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