Ribonucleic acid (RNA) molecule of targeted magnaporthe oryzae PEX3 gene

Abstract

The invention provides a target gene PEX3 which plays a role in preventing and treating the pyricularia oryzae by utilizing an SIGS technology, and an RNA molecule which can be used for preventing and treating the pyricularia oryzae is designed according to the target gene and is used for inhibiting spore germination and appressorium formation of the pyricularia oryzae, so that the pathogenicity of the pyricularia oryzae is inhibited, and a novel method for preventing and treating the pyricularia oryzae is further provided. The RNA molecule comprises siRNA and dsRNA (double-stranded ribonucleic acid). The invention proves that the siRNA molecule can coexist with the magnaporthe oryzae for more than 48 hours, effectively inhibits the germination and growth of the magnaporthe oryzae, and ensures that the siRNA can effectively prevent and control the magnaporthe oryzae within a certain time; when the dsRNA and the magnaporthe oryzae spores are simultaneously inoculated to barley in-vitro leaves, the pathogenicity of magnaporthe oryzae can be inhibited, so that the purpose of preventing and treating the magnaporthe oryzae is achieved. In conclusion, a series of siRNA and dsRNA targeting the magnaporthe oryzae PEX3 gene provided by the invention can be prepared into nucleic acid pesticides, so that the magnaporthe oryzae can be efficiently prevented and treated without pollution.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and mainly relates to an RNA molecule targeting a PEX3 gene of rice blast fungus. Background Art

[0002] Rice blast is a fungal disease caused by Magnaporthe oryzae, which is one of the reasons for the substantial reduction in rice production. In severe cases, it causes a yield loss of up to 40%-50%. Rice blast has become the main harmful factor restricting the high and stable yield of rice in my country. From 2012 to 2021, the actual yield loss of rice in my country due to rice blast reached 368,000 tons. At present, planting disease-resistant varieties and using chemical pesticides are the main measures to prevent and control rice blast, but rice stable yield still faces problems such as easy loss of rice resistance, changes in rice blast fungus populations and pesticide residues. Therefore, it is urgent to explore new and effective strategies for green prevention and control of rice blast.

[0003] The infection of rice blast fungus to plants begins when the conidia of the pathogen sense the plant leaves, and then the spores begin to germinate and extend out of the germ tube; the top of the germ tube swells to form an infection structure - the appressorium, which synthesizes and accumulates glycerol through lipid metabolism inside the appressorium, and forms a melanin layer on the cell wall. The presence of glycerol and melanin layers promotes huge turgor pressure inside the appressorium, and the latter is converted into infection spikes, which can penetrate the rice epidermis to infect host cells. In short, the formation of appressorium is a prerequisite for the infection of rice blast fungus, so interfering with the process of appressorium formation can be used as one of the ideas for preventing and controlling rice blast.

[0004] The pathogenic processes such as lipid metabolism of appressorium, melanin synthesis and overcoming reactive oxygen species (ROS) in rice after infection are closely related to peroxisomes. Peroxisomes are a cell organelle with a single-layer membrane structure, which has been shown to be essential for the infection of rice blast fungi and other plant pathogens and fungi. Peroxisomes exist in almost all eukaryotic organisms and participate in various key metabolic processes, such as fatty acid β-oxidation and the removal of reactive oxygen species. The formation of peroxisomes requires the participation of peroxisomal membrane proteins (PMPs) and matrix proteins. After being synthesized in the cytoplasm, these two types of proteins bind to signal receptors and are transported to organelles. The proteins required for peroxisome biogenesis are collectively called Peroxins (encoded by PEX genes). Currently, 37 PEX genes have been identified in fungi, plants and animals. Studies have found that PEX genes play a key role in the growth, development and infection of rice blast fungus. Knockout of PEX genes will lead to reduced or even loss of pathogenicity of rice blast fungus, such as PEX5, PEX6, PEX7 and PEX19.

[0005] PEX3 is a docking factor involved in the early synthesis of peroxisomal membranes, and plays a role in the process of membrane protein anchoring to peroxisomes. After the membrane protein signal receptor Pex19 recognizes the membrane localization signal of PMPs, it transports PMPs to the peroxisomal membrane, and then Pex19 binds to the transmembrane domain of Pex3 to complete the docking and synergistically embed PMPs into the peroxisomal membrane. In the previous research of our research group, it was found that knocking out PEX3 by gene knockout technology completely lost the pathogenicity of rice blast fungus, slowed growth, hindered fatty acid metabolism, and abnormal peroxisomal membrane formation (Liu Maoxin, 2016), indicating that the PEX3 gene can be used as a potential target for resistance to rice blast.

[0006] RNAi (RNA interference) is a highly conservative post-transcriptional gene silencing method in eukaryotes. It uses double-stranded RNA (dsRNA) or small interfering RNA (siRNA) to specifically degrade homologous mRNA to inhibit target gene expression. In recent years, RNAi technology has made certain progress in agricultural pest control and medical research and development. The spray-induced gene silencing (SIGS) strategy is to spray dsRNA or siRNA molecules targeting the target gene of the pathogen on the surface of the plant in vitro, and interfere with gene expression after entering the pathogen to achieve disease resistance. At present, the research and development of nucleic acid pesticides based on SIGS technology has become a hot topic. Compared with chemical pesticides, the targeting and safety of nucleic acid pesticides are more in line with the concept of green development. In the context of the era of green development of agriculture, the research on green control technology of rice blast has made rapid progress. It has become a trend to establish specific targeted control technology for the key genes of rice blast development and pathogenicity.

[0007] So far, there have been no cases of RNA pesticides produced based on RNAi technology that target peroxisome formation-related genes (PEX). Therefore, creating an RNA molecule that targets the PEX3 gene of rice blast fungus provides a new method for the prevention and control of rice blast. Summary of the invention

[0008] In response to the above problems, the present invention provides an RNA molecule targeting the PEX3 gene of rice blast fungus; an RNA molecule with the peroxisome-related gene PEX3 of rice blast fungus as the target gene, and applies the designed and prepared RNA to rice blast fungus to inhibit the spore germination and appressorium formation of rice blast fungus, thereby inhibiting the pathogenicity of rice blast fungus, and further providing a new method for preventing and controlling rice blast disease.

[0009] To achieve the above object, the present invention adopts the following scheme:

[0010] In one aspect, the present invention provides an siRNA for controlling rice blast fungus, wherein the siRNA targets and silences a PEX3 gene of the rice blast fungus, wherein the PEX3 gene has a DNA sequence as shown in SEQ ID NO:1.

[0011] Peroxisomes are single-membrane-enclosed organelles that have been shown to be essential for infection by Magnaporthe grisea as well as other fungal plant pathogens. Peroxisomes are present in almost all eukaryotic organisms, and they accommodate various critical metabolic processes, such as fatty acid β-oxidation and elimination of reactive oxygen species (ROS). The proteins that make up peroxisomes, including peroxisomal membrane proteins (PMPs) and matrix proteins, are synthesized in the cytoplasm and imported into the organelles post-translationally. The genes encoding proteins involved in the import machinery of these PMPs and matrix proteins are called PEX genes. Knockout of PEX genes reduces or even abolishes the pathogenicity of Magnaporthe grisea.

[0012] In the early stage of the present invention, the PEX3 gene of the rice blast fungus was identified by bioinformatics analysis. Through gene function analysis, it was found that the PEX3 gene is indispensable to the formation of peroxisomes, biochemical metabolism, growth and development, pathogenicity and other physiological processes of the rice blast fungus, and is expected to become a new target gene for preventing and controlling the rice blast fungus. Therefore, a large number of siRNA and dsRNA molecules were designed based on the PEX3 gene, and 6 siRNA and one dsRNA molecule provided by the present invention were obtained through screening, which proved the effect of inhibiting the pathogenicity of the rice blast fungus, and is expected to be used as a nucleic acid pesticide to provide a new method for preventing and controlling the rice blast.

[0013] Furthermore, the siRNA includes one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, and the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

[0014] On the other hand, a dsRNA for controlling rice blast fungus is characterized in that the dsRNA is transcribed from a sequence fragment of a PEX3 gene of rice blast fungus, and the PEX3 gene has a DNA sequence as shown in SEQ ID NO:1.

[0015] Furthermore, the sequence of the dsRNA has a DNA sequence as shown in SEQ ID NO:14.

[0016] On the other hand, the present invention provides a nucleic acid pesticide for controlling rice blast fungus, wherein the nucleic acid pesticide comprises one or more of siRNA and dsRNA targeting the PEX3 gene of rice blast fungus; the siRNA targeting the PEX3 gene of rice blast fungus comprises one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, wherein the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are represented by SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 11 to 12, respectively. The RNA forward and reverse sequences shown in NO:12-13 are synthesized; the dsRNA has the sequence shown in SEQ ID NO:14.

[0017] On the other hand, the present invention provides a method for controlling rice blast fungus, which comprises spraying a nucleic acid pesticide containing siRNA or dsRNA on the stems and leaves of plants to inhibit the germination of rice blast fungus spores and reduce the pathogenicity of rice blast fungus. The nucleic acid pesticide is the same as described above.

[0018] On the other hand, the present invention provides the use of siRNA for preparing a preparation for inhibiting the growth of rice blast fungus spores on a plate, wherein the siRNA includes one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, and the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

[0019] The present invention randomly selected 3 pairs of siRNA (siPEX3-90, siPEX3-251, siPEX3-546) from the above 6 pairs of siRNA to co-incubate with rice blast fungus spores on a plate. It was found that the colony diameter of the rice blast fungus treated with the above 3 pairs of siRNA was significantly reduced, indicating that the above siRNA can inhibit the germination and growth of rice blast fungus. On the other hand, it is also proved that targeted silencing of the PEX3 gene of rice blast fungus can significantly inhibit the growth of rice blast fungus, and the position of the siRNA targeting the PEX3 gene will affect the degree of inhibition.

[0020] Furthermore, the siRNA includes one or more of siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, and the siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

[0021] On the other hand, the present invention provides the use of siRNA for preparing a preparation for inhibiting the germination of rice blast spores, wherein the siRNA includes one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, and the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

[0022] The present invention selected 3 pairs (siPEX3-251, siPEX3-90, and siPEX3-546) from 6 pairs of siRNA-PEX3 fragments, and explored the effects of siRNA-PEX3 on the germination and appressorium formation of rice blast fungus. It was found that after the rice blast fungus was treated with siPEX3-251, the spore germ tubes of the rice blast fungus became elongated, morphologically abnormal appressorium was produced, melanin was reduced, and then the appressorium formation rate decreased, which indicated that siPEX3-251 had an inhibitory effect on the appressorium formation of rice blast fungus.

[0023] In another aspect, a dsRNA is used for preparing a formulation for reducing the pathogenicity of rice blast fungus, wherein the dsRNA has the sequence shown in SEQ ID NO:14.

[0024] The present invention simultaneously inoculates the dsRNA and spores of rice blast fungus into detached barley leaves, and finds that the lesions on the barley leaves treated with dsRNA are reduced, indicating that dsRNA-PEX3 has a certain inhibitory effect on the pathogenicity of rice blast fungus.

[0025] The beneficial effects of the present invention include:

[0026] 1. Provided is a target gene PEX3 that plays a role in the prevention and control of rice blast fungus, and based on this target gene, an RNA (including siRNA and dsRNA) molecule that can be used to prevent and control rice blast fungus is designed to inhibit the spore germination and appressorium formation of rice blast fungus, thereby inhibiting the pathogenicity of rice blast fungus, thereby providing a new method for preventing and controlling rice blast fungus;

[0027] 2. The RNA (including siRNA and dsRNA) molecules provided by the present invention can effectively inhibit the pathogenicity of rice blast fungus and can be prepared into nucleic acid pesticides for spraying on crops. The method for preventing and controlling rice blast avoids the cultivation of transgenic crops, saves a lot of time required for the cultivation of transgenic crops, and is more easily recognized by the public;

[0028] 3. The present invention provides evidence that siRNA molecules can coexist with rice blast fungus spores for more than 48 hours, ensuring that siRNA can effectively prevent and control rice blast fungus within a certain period of time;

[0029] 4. The present invention confirms that the siRNA molecules targeting the PEX3 gene can inhibit the growth of spores on plates after being mixed with the spores, providing a new idea for the prevention and control of rice blast;

[0030] 5. The present invention screens a plurality of siRNA molecules targeting the PEX3 gene, wherein preferably, the siRNA-PEX3-251 has the best effect in inhibiting the formation of appressorium of rice blast fungus;

[0031] 6. The dsRNA-PEX3 provided by the present invention can reduce the area infected by rice blast fungus on barley leaves, indicating that the dsRNA inhibits the pathogenicity of rice blast fungus and can be used as a potential nucleic acid pesticide for preventing and controlling rice blast. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 :siRNA affects the plate growth of spores of rice blast fungus

[0034] Figure 2 :Inhibitory effect of siRNA-PEX3 on appressorium formation of Magnaporthe grisea

[0035] Figure 3 :Spore morphology of the siRNA-PEX3-251 treated group

[0036] Figure 4 :Schematic diagram of the region where siRNA-PEX3 (siPEX3) and dsRNA-PEX3 (dsPEX3) target the PEX3 gene

[0037] Figure 5 : Detection of purified dsRNA by agarose gel electrophoresis

[0038] Figure 6 :Effects of dsRNA-PEX3 on the pathogenicity of Magnaporthe grisea DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the strain type used is the wild type Guy11 of rice blast fungus, and the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0040] Example 1: Design and synthesis of siRNA

[0041] The target gene targeted by siRNA in the present invention is the PEX3 gene (MGG_06424, SEQ ID NO: 1), the first 19 bases of the siRNA sequence are from the PEX3 gene, and TT is added at the 3' end as an overhang to improve the stability of the siRNA, so the total length of the synthesized siRNA is 21 nt; the designed siRNA sequence is blasted in the rice genome database (https: / / www.ricedata.cn / gene / ) to exclude the possibility of matching with the rice gene, and the final siRNA is shown in Table 1.

[0042] Table 1 siRNA targeting PEX3 gene

[0043]

[0044] The siRNA double-stranded molecule of the present invention is directly synthesized by a biological company (Shanghai Jierui), and the synthesized product is a dry powder. DEPC-treated water or nuclease-free water is added for dissolution, and the final concentration is 10 μM; after dissolution, it is stored in separate tubes, and stored at -20°C for short-term use and -80°C for long-term storage; the EP tubes for subpackaging use EP tubes without nuclease (the RNA molecular experiments conducted all use RNA-specific gun tips and the experimenters wear masks and gloves to prevent pollution).

[0045] Example 2: Mixing equal volumes of spores and siRNA to measure RNA persistence

[0046] Take a plate of rice blast fungus that has grown for about 8-10 days, 2 mL of sterile water (ddH 2 O) Wash the plate, filter it with double-layer lens paper into an EP tube without nuclease, centrifuge it twice at 7,000 rpm for 5 min to ensure that no culture medium components remain; then adjust the spore concentration to 1×10 by counting with a hemocytometer. 5 spores / mL; the siPEX3-141 fragment in Example 1 was taken, mixed with an equal volume of spores, and placed on a Gelbond film (hydrophobic film) that had been wiped with 75% alcohol. The siRNA concentration was measured using a concentration instrument (Nondrop). The initial siRNA concentration was 10 μM, and the RNA concentrations at 4 h, 8 h, 12 h, 24 h and 48 h were recorded respectively. The experiments were carried out in the absence of RNase. The results are shown in Table 2.

[0047] Table 2 Measurement of RNA persistence by mixing equal volumes of spores and siRNA

[0048]

[0049] The results showed that siRNA could stably coexist with rice blast fungus spores in vitro for more than 48 hours.

[0050] Example 3: siRNA targeting the PEX3 gene affects the plate growth of rice blast fungus spores

[0051] The same operation as in Example 2 was used to obtain the spore solution. The obtained spore solution (10 μL) and siRNA (10 μL) were mixed in equal volumes, cultured on a shaker at 30°C, 150 rpm for a period of time, and then spotted on agar prepared in advance. After the colonies grew for about 6 days, the colony diameters were measured. The statistical results are as follows: Figure 1As shown, DDW, DEPC and GFP are negative controls. Compared with the negative controls, the colony diameters treated with siRNA-PEX3 (siPEX3-90, siPEX3-251, siPEX3-546) are significantly smaller, indicating that targeted silencing of the PEX3 gene of rice blast fungus can significantly inhibit the growth of rice blast fungus, and the position of the siRNA targeting the PEX3 gene will affect the degree of inhibition.

[0052] Example 4: siRNA targeting the PEX3 gene affects spore germination and appressorium formation of rice blast fungus

[0053] The siRNA-PEX3 fragments (siPEX3-251, siPEX3-90, siPEX3-546) synthesized in Example 1 were selected, and the spore solution was prepared in the same manner as in Example 2; the spore solution and siRNA were mixed in equal volumes, and 20 μL of the spore solution containing RNA molecules was dripped onto the alcohol-treated hydrophobic membrane. The control treatment was DEPC-treated water or RNase-Free ddH 2 O; place the added hydrophobic membrane in a culture dish with a diameter of 150 mm, place a piece of filter paper in the culture dish, and wet it with sterile water; place it in a dark incubator at 28°C to induce attachment cells; take it out after 8 hours, and observe the growth of spores and the formation of attachment cells under an electron microscope.

[0054] Appressorium formation of rice blast fungus after siRNA treatment Figure 2 As shown in the figure, compared with the control DDW (control), the appressorium formation rate of the rice blast fungus was significantly reduced after being treated with siPEX3-251, and the spore tubes of the rice blast fungus became longer, producing abnormal appressorium morphology and reducing melanin ( Figure 3 ), indicating that siRNA-PEX3 has an inhibitory effect on the appressorium formation of Magnaporthe oryzae.

[0055] Example 5: dsRNA affects the pathogenicity of rice blast fungus

[0056] (1) Design of dsRNA and acquisition of synthetic dsRNA template: A 470 bp dsRNA (SEQ ID NO: 14) was designed according to the dsRNA design website (SnapDragon-dsRNA Design (flyrnai.org)). The positions of the dsRNA and the six pairs of siRNAs are as follows: Figure 4As shown; DNA of Guy11 strain was extracted and used as a template to amplify the target gene PEX3; the designed dsRNA region was amplified using the target gene PEX3 as a template, and the 5' ends of a pair of PCR primers for amplifying the dsRNA sequence both had a T7 promoter sequence, so as to achieve the purpose of the amplified fragment containing the T7 promoter sequence; PCR amplification reaction system (25 μL): 2×Phanta mix 13 μL, Primer-F (SEQ ID NO: 15) 2 μL, Primer-R (SEQ ID NO: 16) 2 μL, template 2 μL, nuclease-free water 6 μL; PCR reaction program: 95°C 3min, 95°C 30s—65°C 30s—72°C 30s (35 cycles), 72°C 5min; after the reaction, the PCR product was subjected to 1% agarose gel electrophoresis at 180V for 20min.

[0057] (2) dsRNA synthesis: The electrophoresis product recovered from the gel was used as a template and MEGAscript was used to synthesize the dsRNA. TM RNAi (ThermoFisher scientific) kit was used to synthesize dsRNA in vitro; the reaction system (20 μL) included 1-2 μg DNA template, 2 μL 10×T7 reaction buffer, 2 μL ATP solution, 2 μL CTP solution, 2 μL GTP solution, 2 μL UTP solution, and 2 μL T7 Enzyme Mix, and then nuclease-free water was added to 20 μL; the PCR reaction procedure was as follows: 37°C for 4 h, annealing at 75°C for 5 min, and then cooling to room temperature; the dsRNA mixture obtained by the reaction was subsequently purified according to the instructions of the above-mentioned in vitro synthesis dsRNA kit, and the subsequently purified dsRNA was detected by agarose gel electrophoresis, and the results were as follows: Figure 5 The results showed that the dsRNA (SEQ ID NO: 14) fragment targeting the PEX3 gene of Magnaporthe oryzae was successfully obtained.

[0058] (3) Pathogenicity determination: The effect of dsRNA on the pathogenicity of the pathogen was determined by artificial inoculation of detached barley leaves. First, barley leaves with the same growth status were cut and placed in a 150 mm culture dish containing filter paper (each treatment was repeated three times). After the filter paper strips were attached to both ends of the leaves, they were wetted with sterilized water (moisturizing treatment); then the spores of the rice blast fungus Guy11 were mixed with dsRNA in a volume ratio of 1:1 (the final concentration of dsRNA was 100 ng / μL), 20 μL was dropped on the surface of the leaves, and the inoculated leaves were placed in a culture dish wiped with 75% alcohol. The leaves were cultured in an incubator at 26°C, first in dark culture for 24 hours, and then in a 12h:12h light and dark alternating cycle. After 4 days, the disease was observed and photographed. The results are as follows Figure 6 As shown, compared with the control ddH 2 Compared with O, the lesion area on the barley leaves treated with dsRNA was reduced, indicating that dsRNA-PEX3 had a certain inhibitory effect on the pathogenicity of rice blast fungus and could be used as a potential nucleic acid pesticide for the treatment of rice blast.

[0059] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

[0060] Sequence Listing

[0061]

[0062]

Claims

1. A siRNA for controlling rice blast fungus, characterized in that: The PEX3 gene of rice blast fungus is targeted for silencing, wherein the PEX3 gene has a DNA sequence as shown in SEQ ID NO:

1.

2. The siRNA according to claim 1, wherein It includes one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, wherein the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

3. A dsRNA for controlling rice blast fungus, characterized in that: The dsRNA is transcribed from a sequence fragment of the PEX3 gene of the rice blast fungus, and the PEX3 gene has a DNA sequence as shown in SEQ ID NO:

1.

4. The dsRNA according to claim 3, characterized in that The sequence of the dsRNA has the DNA sequence shown in SEQ ID NO:

14.

5. A nucleic acid pesticide for controlling rice blast fungus, characterized in that: The invention comprises one or more of siRNA and dsRNA targeting PEX3 gene of rice blast fungus; the siRNA targeting PEX3 gene of rice blast fungus comprises one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546 and siRNA-PEX3-251; the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546 and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2-3, SEQ ID NOs: 4-5, SEQ ID NOs: 6-7, SEQ ID NOs: 8-9, SEQ ID NOs: 10-11 and SEQ ID NOs: 12-13, respectively; the dsRNA has the sequence shown in SEQ ID NO:

14.

6. A method for preventing and controlling rice blast fungus, characterized in that: A nucleic acid pesticide containing siRNA or dsRNA is sprayed on the stems and leaves of plants to inhibit the germination of rice blast fungus spores and reduce the pathogenicity of rice blast fungus. The nucleic acid pesticide is as described in claim 5.

7. Use of siRNA for preparing a preparation for inhibiting the growth of rice blast fungus spores on plate, characterized in that: The siRNA includes one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, and the siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

8. The use according to claim 7, characterized in that The siRNA includes one or more of siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251, and the siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

9. Use of siRNA for preparing a preparation for inhibiting spore germination of rice blast fungus, characterized in that: The siRNA includes one or more of siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251. The siRNA-PEX3-652, siRNA-PEX3-141, siRNA-PEX3-1286, siRNA-PEX3-90, siRNA-PEX3-546, and siRNA-PEX3-251 are synthesized from the RNA forward and reverse sequences shown in SEQ ID NOs: 2 to 3, SEQ ID NOs: 4 to 5, SEQ ID NOs: 6 to 7, SEQ ID NOs: 8 to 9, SEQ ID NOs: 10 to 11, and SEQ ID NOs: 12 to 13, respectively.

10. Use of dsRNA for preparing a preparation for reducing the pathogenicity of rice blast fungus, wherein the dsRNA has the sequence shown in SEQ ID NO: 14.

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