DsRNA nano preparation for preventing and controlling phytophthora capsici as well as preparation method and application of dsRNA nano preparation

Abstract

The invention discloses a dsRNA nano preparation for preventing and controlling phytophthora capsici as well as a preparation method and application of the dsRNA nano preparation. The nucleotide sequence of the phytophthora capsici sugar transporter protein HXT4 gene is as shown in SEQ ID NO.1, and the ASPN-dsRNA nano preparation is obtained by mixing dsRNA as shown in SEQ ID NO.2 and amino-modified mesoporous silica. By exogenous application of the ASPN-dsRNA nano preparation, gene expression silencing of the target gene phytophthora capsici sugar transporter HXT4 is induced, so that pathogenicity of phytophthora capsici is reduced or even lost, and the purpose of protecting crops is achieved. The preparation can effectively reduce the pathogenicity of phytophthora capsici, the inhibition rate on phytophthora capsici infection can reach 72%, and the prevention and control efficiency on phytophthora capsici reaches 87%. The ASPN-dsRNA nano preparation is high in stability and obvious in prevention and control effect, and provides a new prevention and control strategy and means for developing a precise prevention and control method for crop epidemic diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a dsRNA for preventing and controlling Phytophthora capsici, and a preparation method and application thereof. Background Art

[0002] Phytophthora Phytophthora There are more than 120 species of pathogens that can harm almost all dicotyledonous plants. They are known as "plant killers". The diseases they cause are characterized by rapid spread, easy outbreaks, and difficulty in prevention and control. In production, they are called crop "blight". P. capsici ) is a type of pathogen with the widest host range among Phytophthora, which can harm more than 500 plant species in 26 families, including Solanaceae, Leguminosae, Cruciferae, Cucurbitaceae, and Rosaceae. The economic losses caused each year amount to billions of dollars, seriously affecting the quality and safety of vegetables and the ecological environment in the world. The use of fungicides is still one of the main means of disease prevention and control. However, there are relatively few types of oomycete inhibitors available, and the long-term use of single-site agents can easily lead to drug resistance in plant pathogenic oomycetes. Therefore, it is urgent to create new strategies for crop disease prevention and control that are green, safe, efficient and synergistic in production.

[0003] Nucleic acid pesticides have the advantages of strong specificity, short effective period, no residue and little impact on non-target organisms. They are known as the third revolution in the history of pesticides and are a hot spot in the field of new green pesticide creation. Spray-induced gene silencing (SIGS) is a new pest control technology that is rapidly developing. SIGS technology does not require the creation or cultivation of transgenic plants. It is applied on the surface of plants by spraying exogenous dsRNA (double-strandedRNA). In recent years, it has been reported to have good control effects on some plant pathogenic fungi. The main objects of control research are Fusarium that causes wilt, Botrytis cinerea that causes gray mold on crops and post-harvest fruits, vegetables and flowers, Sclerotinia sclerotiorum that causes stem rot, and some other important pathogenic fungi. For example, studies by Koch et al. show that in barley Hordeum vulgare Leaf surface spraying targets three Fusarium graminearum species Fusarium graminearum Ergosterol biosynthesis genes ( CYP51A , CYP51B , CYP51C ) can effectively inhibit the growth of Fusarium graminearum. However, dsRNA used in vitro is unstable in the environment, easily degraded by RNA degrading enzymes, ultraviolet light and high temperature, and difficult to effectively introduce into the target organism, which seriously limits the actual application effect of sprayable nucleic acid pesticides. Therefore, how to enhance the stability of dsRNA is an urgent problem to be solved in this field.

[0004] Using nanoparticles as delivery carriers of dsRNA can effectively improve the stability and delivery efficiency of RNA, thereby improving the biological control effect of RNAi. Nanomaterials have the characteristics of small size effect, interface effect and good biocompatibility at the nanoscale. As dsRNA carriers, they can enhance the stability of dsRNA in the environment and its ability to enter the target biological tissues or cells, thereby improving the targeted delivery efficiency. It is an effective means to solve the difficulty of exogenous application of nucleic acid pesticides. In most cases, the cationic groups of nanoparticles can bind to the phosphate groups of dsRNA through electrostatic interactions to form dsRNA / nanoparticle complexes. For example, Xu et al. used amino-functionalized mesoporous silica nanoparticles to deliver dsRNA to silence the coat protein encoding gene of potato virus Y. The experimental results showed that silica nanoparticles loaded with dsRNA can effectively silence the target gene and protect plants from virus infection for more than 14 days. Although the nanoparticle-mediated dsRNA delivery system has achieved good results in the control of other plant diseases and insect pests, there is little research on the application of this preparation in the control of pepper phytophthora.

[0005] Since the target genes in Phytophthora that can effectively inhibit the growth and development of pathogens and the infection and pathogenicity through gene silencing are still unclear, this greatly restricts the application of SIGS in crop disease prevention and control. Therefore, discovering and identifying more RNA molecules targeting different target genes is the premise and focus of RNAi pesticides and targeted prevention and control technology research. Sugar substances in plants are the most important carbon source for pathogens. Pathogens transport sugars into the body for utilization through sugar transporters on their cell membranes, such as hexose transporters and sucrose transporters. Studies have shown that sugar transporters are necessary for the normal growth, development and pathogenicity of pathogens. The preliminary research of the present invention team showed that the sugar transporter PcHXT4 (PHYCA_542873) is unique to Phytophthora, and knocking out this gene can lead to a significant decrease or even loss of the pathogenicity of Phytophthora capsici, indicating that this gene can be used as an important molecular target for the prevention and control of pepper blight. At present, there is no report on the use of dsRNA related to this gene in the prevention and control of pepper blight. Therefore, targeting this gene, designing highly effective dsRNA fragments, and combining them with amino-modified mesoporous silica nanomaterials to apply them to the prevention and control of pepper blight is of great significance for the prevention and control of the disease. Summary of the invention

[0006] The purpose of the present invention is to provide a key pathogenic gene of pepper phytophthora based on SIGS technology in view of the shortcomings of the prior art. HXT4 As a molecular target, the corresponding dsRNA was designed and synthesized. After being combined with amino-modified mesoporous silica, it was sprayed on the plant surface, which can effectively inhibit the infection and pathogenicity of pepper blight, providing a basis for future green prevention and control of pepper blight in production practices.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A sugar transporter from Phytophthora capsici HXT4 The gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0008] Phytophthora capsici sugar transporter HXT4 The dsRNA of the gene is a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO.2 and a nucleotide sequence that is reverse complementary to the nucleotide sequence shown in SEQ ID NO.2.

[0009] The method for preparing the dsRNA comprises the following steps: extracting the genomic DNA of Phytophthora capsici as a template, performing PCR amplification using a specific primer set to obtain an in vitro transcription template, and in vitro transcriptionally synthesizing dsRNA using the in vitro transcription template according to the instructions of the kit.

[0010] Furthermore, the above-mentioned specific primer set includes an upstream primer and a downstream primer, and the upstream and downstream primers contain a T7 promoter; the sequence of the upstream primer is: 5'-GGATCCTAATACGACTCACTATAGGATCGGCACGTCGTGGGTGT-3'; the sequence of the downstream primer is: 5'-GGATCCTAATACGACTCACTATAGGGATTCCGAGCATCACGAC-3'.

[0011] A product for controlling Phytophthora capsici, comprising the above-mentioned dsRNA.

[0012] An ASNP-dsRNA nano preparation for preventing and controlling pepper phytophthora is obtained by mixing the above-mentioned dsRNA with an amino-modified mesoporous silica aqueous solution and mixing by ultrasonic oscillation.

[0013] Furthermore, the concentration of the above-mentioned dsRNA is 1.0 mg / mL; the concentration of the amino-modified mesoporous silica aqueous solution is 0.5 mg / mL; and the mass ratio of dsRNA to amino-modified mesoporous silica is 1~25:1.

[0014] Application of the above ASNP-dsRNA nanoformulation in preventing and controlling pepper phytophthora.

[0015] A method for preventing and controlling pepper phytophthora, comprising the steps of spraying the above-mentioned ASNP-dsRNA nano preparation on leaves.

[0016] The beneficial effects of the present invention are: (1) This invention cloned a new double-stranded RNA (dsRNA) for the first time, which efficiently targeted the sugar transporter gene of Phytophthora capsici HXT4, the target gene was silenced by RNAi, with a silencing efficiency of 70.2%, and had no effect on the expression of non-target genes, which was highly specific. HXT4 It is a key functional gene in the pathogenic process of Phytophthora, is unique to Phytophthora, and is highly safe to non-target organisms.

[0017] (2) The nanomaterials of amino-modified mesoporous silica used in the present invention have excellent properties such as high specific surface area, good biocompatibility, adjustable pore size and high loading capacity, which can significantly improve the interference efficiency and stability of dsRNA. The results showed that compared with dsHXT4 not combined with amino-modified mesoporous silica, the antibacterial efficiency of ASNP-dsRNA was increased by 38%.

[0018] (3) The ASNP-dsRNA nanoformulation provided by the present invention can significantly inhibit the infection of pepper phytophthora, and is highly effective in preventing and treating the disease caused by pepper phytophthora infection, and is expected to be developed into a new type of green fungicide. It has important practical value for preventing and treating vegetable diseases caused by pepper phytophthora in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Electrophoresis detection result of in vitro synthesized dsRNA; wherein: lane M is DL 2000 DNA marker.

[0020] Figure 2 : Screening diagram for the optimal binding ratio of ASNP and dsRNA; wherein: lane M is DL 2000 DNA marker, and different lanes are the mass ratios of ASNP and dsRNA.

[0021] Figure 3 :The ASNP-dsRNA nanoformulation of the present invention treats the sugar transporter gene of pepper phytophthora HXT4 expression of.

[0022] Figure 4 : After being treated with the ASNP-dsRNA nanoformulation of the present invention, the infection of Phytophthora capsici on the detached leaves of Nicotiana benthamiana; (A) Symptoms of Phytophthora capsici infecting Nicotiana benthamiana leaves; (B) Statistical results of the lesion area.

[0023] Figure 5 : The occurrence of pepper blight on tomato seedlings after treatment with the ASNP-dsRNA nanoformulation of the present invention; (A) symptoms of disease in tomato plants; (B) statistical results of plant incidence. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it. The technical means described in the embodiments are conventional means familiar to those skilled in the art.

[0025] The sequence in the dsRNA of the present invention is derived from the sugar transporter gene of Phytophthora capsici HXT4 The cDNA coding sequence is shown in SEQ ID NO.1.

[0026] Gene identification was performed by searching the reference genome of P. capsici (Accession No. GCA_030324255.1) database using the relevant HMM prototype PF00083 in the Pfam database ( http: / / pfam.sanger.ac.uk / ) as a template through a hidden Markov model (HMM), with the E-value set to 1e-6.

[0027] SEQ ID NO. 1:

[0028] Main materials: Promega's T7 RiboMAX™ Express RNAi System kit was purchased from Promega; DNA marker was purchased from Bao Biotechnology Dalian Co., Ltd.; 2 × Phanta Max Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; the rest of the reagents were purchased from Sangon Biotech (Shanghai) Technology Co., Ltd. The primers were synthesized by Sangon Biotech (Shanghai) Technology Co., Ltd. The tomato material used was AC variety, and the pepper Phytophthora strain LT1534 was donated by the College of Plant Protection, Nanjing Agricultural University and preserved in this laboratory.

[0029] V8 liquid culture medium: Take 100 ml of V8 fruit and vegetable juice (Campbell Soup Company) and add 1 g CaCO3, stir well to mix, centrifuge at 4000 r / min for 3 min, take the supernatant and dilute it with deionized water in a ratio of 1:9 to make 10% V8 culture medium, and sterilize it under high pressure at 121℃ for 20 min.

[0030] V8 solid culture medium: Take 100 ml of V8 fruit and vegetable juice and add 1 g of CaCO3, stir well to mix, centrifuge at 4000 r / min for 3 min, take the supernatant and dilute it with deionized water at a ratio of 1:9 to make 10% V8 culture medium, and add 2% agar powder, and sterilize it under high pressure at 121℃ for 20 min.

[0031] Example 1 In vitro synthesis of dsRNA (1) Preparation of dsHXT4 transcription template: ① The pepper phytophthora strain was inoculated into V8 liquid culture medium and cultured in a constant temperature incubator at 25℃ in the dark for 3 days. The mycelium was collected and the genomic DNA of pepper phytophthora was extracted by CTAB method.

[0032] ② The extracted genomic DNA of Phytophthora capsici was used as a template for PCR amplification using dsHXT4 specific primers; the sequence of the specific primer set is as follows: Upstream primer (SEQ ID NO. 3): 5′-GGATCCTAATACGACTCACTATAGGATCGGCACGTCGTGGGTGT-3′; Downstream primer (SEQ ID NO.4): 5'-GGATCCTAATACGACTCACTATAGGGATTCCGAGCATCACGAC-3'.

[0033] When the fragment is amplified, the T7 promoter sequence is added to the 5' end of the two primers of PCR, and the amplified fragment automatically contains the T7 promoter sequence. The PCR reaction system (50 μL) includes: 2×Phanta mix 26 μL, upstream primer 2 μL, downstream primer 2 μL, DNA template 1 μL, nuclease-free water 19 μL. The PCR reaction program is: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 30s, 30 cycles; 72℃ extension for 5 min. After the reaction, the PCR amplification product was detected by 1.2% agarose electrophoresis, and the fragment was recovered by the DNA purification kit of Guangzhou Meiji Biotechnology Co., Ltd.

[0034] ③ Using the green fluorescent protein gene GFP double-stranded dsRNA (dsGFP) as a control, PVX-GFP plasmid as a template, and dsGFP specific primers for PCR amplification; the sequence of the specific primer set is as follows: dsGFP-F: 5'-TAATACGACTCACTATAGGGACGTAAACGGCCACAAGTTC-3'; dsGFP-R: 5'-TAATACGACTCACTATAGGGTGCTCAGGTAGTGGTTGTCG-3'.

[0035] The PCR reaction system (50 μL) included: 2×Phanta mix 26 μL, dsGFP-F 2 μL, dsGFP-R 2 μL, DNA template 1 μL, nuclease-free water 19 μL. The PCR reaction program was: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 5 min. After the reaction, 5 μL of PCR amplification product was detected by 1.2% agarose gel electrophoresis, and the fragments were recovered using the DNA purification kit of Guangzhou Meiji Biotechnology Co., Ltd.

[0036] (2) dsRNA synthesis: Using the gel-recovered electrophoresis product as a template, dsRNA was synthesized using Promega's T7 RiboMAX™ Express RNAi System transcription kit. The reaction system (20 μL) includes: RiboMAX™ Express T7 2X Buffer 10 μL, DNA template 1 μg, T7 Express Enzyme Mix 2 μL, and nuclease-free water added to 20 μL. Reaction conditions: Incubate at 37°C for 30 minutes, anneal at 70°C for 10 minutes, and then cool to room temperature. Dilute 100U / μL RNase T1 to 10U / μL with RNase T1 Dilution Buffer. For each 20 μL reaction product, add 1 μL of freshly diluted RNase T1 and 1 μL of DNase I, and incubate at 37°C for 30 minutes. Among them, dsHXT4 was sequenced as a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO.2 and a nucleotide sequence that is reverse complementary to the nucleotide sequence shown in SEQ ID NO.2.

[0037] (3) Electrophoresis detection of transcription products, such as the results Figure 1 As shown, from left to right are the detection results of marker, dsGFP, and dsHXT4, respectively, showing bands consistent with the predicted sizes, indicating that they can be transcribed to form the dsRNA of the present invention.

[0038] (4) dsRNA purification: Add 55 μL of 95% ethanol to the transcript, mix well and place on ice for 5 minutes, centrifuge at 12000 rpm for 10 minutes. Carefully aspirate the supernatant and wash the precipitate with 0.5 ml of 70% cold ethanol, and remove all ethanol after washing. Air-dry the precipitate at room temperature for 15 minutes, add 50 μL of nuclease-free water to resuspend the transcript dsRNA. Detect the A260 absorbance of the product, determine its concentration, and store at -20°C.

[0039] Example 2 Determination of the ratio of ASNP-dsRNA mixed preparation (1) Preparation of amino-modified mesoporous silica (ASNP) working solution: The nanomaterial ASNP (purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd.) was dissolved in DEPC water and mixed by ultrasonic vibration to a final concentration of 0.5 mg / mL.

[0040] (2) Combination of ASNP and dsRNA: Different masses of dsRNA were added to a constant concentration (0.5 mg / mL) of ASNP working solution to form ASNP-dsRNA mixed solutions with mass ratios (ASNP: dsRNA) of 1:10, 1:20, 1:25, 1:30, 1:40, and 1:50, respectively. The mixture was vortexed for 2-3 min and allowed to stand at room temperature for 30 min. At this time, dsRNA was adsorbed on the surface of ASNP to form a stable ASNP-dsRNA nanoformulation.

[0041] (3) Electrophoresis detection: Use 1.2% agarose gel electrophoresis to detect the unadsorbed dsRNA, and perform electrophoresis at 110 V for 25 min. Select the optimal binding ratio based on the electrophoresis results.

[0042] The results are as follows Figure 2 As shown, lane 1 is DNA DL2000 marker, lane 2 is untreated dsRNA, and lanes 3 to 8 are ASNP-dsRNA nanoformulations with different mass ratios. Figure 2 It can be seen that there is no obvious dsRNA band in lanes 3 to 5, indicating that no excess dsRNA is precipitated after ASNP binds to dsRNA, that is, the optimal loading ratio of ASNP to the dsRNA is 1: 25. ASNP-dsGFP and ASNP-dsHXT4 were prepared with the optimal loading ratio of ASNP to dsRNA of 1: 25.

[0043] Example 3 ASNP-dsRNA nanoformulation inhibits target genes of Phytophthora capsici HXT4 Expression detection (1) Treatment of pepper phytophthora with ASNP-dsRNA nanoformulation: Pepper phytophthora strain LT1534 was inoculated into V8 solid culture medium and cultured in a 28°C incubator in the dark for 2 days. Fresh mycelium from the edge of the colony was inoculated into 10 mL of V8 liquid culture medium, and then 10 μL of ASNP-dsGFP (200 ng / μL) and ASNP-dsHXT4 (200 ng / μL) nanoformulations were added to the culture medium. At the same time, an equal volume of ASNP and water were used as controls. The treated culture medium was cultured in a 28°C incubator. After 3 days of co-incubation, the mycelium was removed and the water was dried. The mycelium was collected and quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0044] (2) RNA extraction and detection: RNA extraction from mycelium of Phytophthora capsici was performed using the FastPure Universal PlantTotal RNA Isolation Kit (Nanjing Novozyme Biotech Co., Ltd.). The specific steps are as follows: 1) Grind the mycelium block in liquid nitrogen and add 600 μL Buffer EL. Vortex vigorously for 30 sec and centrifuge at 12000 rpm for 5 min.

[0045] 2) Take about 500 μL of the supernatant and transfer it to FastPure gDNA-Filter Columns III. Centrifuge at 12000 rpm for 30 seconds. Discard FastPure gDNA-Filter Columns III and collect the filtrate.

[0046] 3) Add 250 μL of anhydrous ethanol to the collection tube and vortex to mix for 15 sec.

[0047] 4) Transfer the above mixture to FastPure RNA Columns V, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0048] 5) Add 700 μL Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 sec, and discard the filtrate.

[0049] 6) Add 500 μL Buffer RWB to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 sec, and discard the filtrate.

[0050] 7) Repeat step 6.

[0051] 8) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm for 2 min.

[0052] 9) Transfer FastPure RNA Columns V to new RNase-free Collection Tubes 1.5mL centrifuge tubes, add 100 μL of RNase-free ddH2O preheated at 65℃ to the center of the adsorption column membrane, let it stand at room temperature for 5 minutes, and centrifuge at 12000 rpm for 1 minute.

[0053] (3) Reverse transcription of test samples: 1) Remove gDNA, the reaction system is as follows: 2) Mix by gently pipetting and incubate at 42℃ for 2 min.

[0054] 3) Reverse transcription into cDNA, the reaction system is as follows: 4) Use a pipette to gently mix. Reaction conditions: 37℃ 15min, 85℃ 15sec.

[0055] (4) qRT-PCR detection: Real-time PCR method is used to detect the target genes HXT4 and housekeeping genes Actin The relative expression level of the gene was measured and its silencing efficiency was calculated. Figure 3 As shown, compared with the control group (CK), the ASNP-dsHXT4-treated group HXT4 The expression level of Figure 3 ).

[0056] Example 4: Detection of the inhibitory effect of ASNP-dsRNA nanoformulation on Phytophthora capsici by in vitro leaf inoculation of Nicotiana benthamiana The pepper phytophthora strain LT1534 was inoculated into V8 solid culture medium and cultured in a constant temperature incubator at 28°C in the dark for 2 days. 10 μL of the ASNP-dsRNA (200 ng / μL) nanoformulation prepared in Example 2 was dripped onto the leaves of Nicotiana benthamiana, and then a freshly cultured pepper phytophthora hyphae block (5 mm in diameter) was inoculated into the droplet and placed in a 28°C incubator for moisturizing culture. The experimental results were observed after 48 hours. A total of 5 treatment groups were set up: (1) CK: clean water as the control treatment; (2) treatment with amino-modified mesoporous silica (ASNP) suspension alone; (3) ASNP-dsGFP treatment; (4) dsHXT4 treatment; (5) ASNP-dsHXT4 treatment. Five leaves were treated in each experiment and repeated 3 times. The results are shown in the figure. Figure 4 As shown, the lesion area of ​​the dsHXT4 and ASNP-dsHXT4 treatment groups was significantly smaller than that of the control treatment group, wherein the lesion area of ​​the ASNP-dsHXT4 treatment group was reduced by 72%, and the inhibitory ability was increased by 38% compared with the dsHXT4 treatment group. The results show that the ASNP-dsRNA nanoformulation of the present invention has a significant inhibitory effect on the infection of pepper phytophthora.

[0057] Example 5 Application of ASNP-dsRNA nanoformulation in the prevention and treatment of pepper blight (1) Preparation of zoospores of pepper phytophthora: Inoculate pepper phytophthora strain LT1534 into V8 solid culture medium and culture in a constant temperature incubator at 28°C in the dark for 2 days. Take fresh mycelium from the edge of the colony and inoculate it into 10 mL of V8 liquid culture medium and culture it at 28°C for 3 days. Take out the mycelium block and wash it with sterile water for 3 times until the mycelium turns white. Spread the mycelium in a culture dish and add 10 mL of sterile water. After 24 hours of strong light treatment, place the culture dish under a microscope. After a large number of sporangia are observed, place it in a 4°C refrigerator for 30 min, take it out and place it at room temperature for 30 min, filter and remove the mycelium to obtain pepper phytophthora zoospores. Use a hemocytometer to adjust the concentration of the zoospore suspension to 10 with sterile water. 4 pcs / mL, for future use.

[0058] (2) Treatment of tomato seedlings: After germination, the tomato seeds were sown and two true leaves were grown for inoculation experiments. A water control group (CK) and ASNP-dsGFP and ASNP-dsHXT4 treatment groups were set up, with 6 pots of plants in each group, and repeated 3 times. The nanoformulation containing 10 μg of dsRNA was sprayed on each leaf. After keeping moist and dark at 25°C for 12 h, the roots of the tomato seedlings were vertically scratched with a scalpel at a distance of 0.5 cm from the stems for root injury treatment. 5 mL of zoospore suspension (10 4 After 24 h of dark moisturizing treatment, and then 2 days of moisturizing under normal light (10 h light, 14 h dark) at 25℃, the diseased plants were counted and photographed.

[0059] (3) The results are as follows Figure 5 As shown in the figure, the incidence of the control group (CK) plants reached more than 93%, while the incidence of the ASNP-dsHXT4 treatment group was 12.1%, and its control efficiency reached 87%. The experimental results show that ASNP-dsRNA nanoformulation can effectively reduce the occurrence of pepper blight.

[0060] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A sugar transporter from Phytophthora capsici HXT4 A gene characterized by: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The capsici phytophthora sugar transporter according to claim 1 HXT4 A dsRNA of a gene, characterized in that: The dsRNA is a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO.2 and a nucleotide sequence that is reverse complementary to the nucleotide sequence shown in SEQ ID NO.

2.

3. The method for preparing dsRNA according to claim 2, characterized in that: The genomic DNA of Phytophthora capsici was extracted as a template, and PCR amplification was performed using a specific primer set to obtain an in vitro transcription template. The in vitro transcription template was in vitro transcribed to synthesize dsRNA according to the instructions of the kit.

4. The method for preparing dsRNA according to claim 3, characterized in that: The specific primer set includes an upstream primer and a downstream primer, and the upstream and downstream primers contain a T7 promoter; the sequence of the upstream primer is: 5'-GGATCCTAATACGACTCACTATAGGATCGGCACGTCGTGGGTGT-3'; the sequence of the downstream primer is: 5'-GGATCCTAATACGACTCACTATAGGGATTCCGAGCATCACGAC-3'.

5. A product for preventing and controlling pepper phytophthora, characterized in that : It contains the dsRNA according to claim 2.

6. An ASNP-dsRNA nanoformulation for controlling Phytophthora capsici, characterized by: According to claim 2 described The dsRNA is mixed with an amino-modified mesoporous silica aqueous solution and then uniformly mixed by ultrasonic oscillation.

7. The ASNP-dsRNA nanoformulation according to claim 6, characterized in that: The concentration of the dsRNA is 1.0 mg / mL; the concentration of the amino-modified mesoporous silica aqueous solution is 0.5 mg / mL; and the mass ratio of dsRNA to amino-modified mesoporous silica is 1-25:

1.

8. Use of the ASNP-dsRNA nanoformulation as claimed in claim 6 in preventing and controlling Phytophthora capsici.

9. A method for preventing and controlling Phytophthora capsici, characterized in that: The ASNP-dsRNA nanoformulation according to claim 6 is sprayed on leaves.

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