CN114958841A

CN114958841A - Key gene for inhibiting rice blast bacteria, dsRNA and preparation and application thereof

Info

Publication number: CN114958841A
Application number: CN202210302359.9A
Authority: CN
Inventors: 赵弘巍; 喻晗晞; 陈盼
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2022-08-30
Anticipated expiration: 2042-03-25
Also published as: CN114958841B

Abstract

The invention discloses a key gene for inhibiting rice blast germs, dsRNA (double-stranded ribonucleic acid) and preparation and application thereof, wherein the nucleotide sequence of the key gene is shown in any one of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, ID No.7 and ID No.9, and the dsRNA for inhibiting the rice blast germs is any one of dsRNA of MoCytb gene containing T7 promoter, dsRNA of MoRic8 gene, dsRNA of MoTrx2 gene, dsRNA of MoMBF1 gene or dsRNA of MoEnd3 gene. The invention also discloses application of the dsRNA in preparation of biological bactericide, and the dsRNA has obvious effect and is environment-friendly.

Description

Key gene for inhibiting rice blast germs, dsRNA (double-stranded ribonucleic acid) and preparation and application of dsRNA

Technical Field

The invention relates to the field of genetic engineering, in particular to a key gene for inhibiting rice blast germs, dsRNA (double-stranded ribonucleic acid) and preparation and application thereof.

Background

Rice is one of the most important crops in the world, and rice is the staple food for more than half of the world population. The rice blast caused by the rice blast fungus (magnaportheooryzae) is one of the most devastating fungal diseases of rice worldwide. The rice blast germs infect plants almost at all growth stages, so that the rice yield is reduced by 10 to 35 percent, and even in severe casesNo particles are collected. In China, the yield loss of rice caused by rice blast is hundreds of millions of kilograms since the last 90 th century, and the area of rice blast generation is 38 kilohm every year ² The above. Therefore, effective means for controlling such diseases are urgently needed to ensure the safety of global food.

In the prior art, the rice blast control mainly depends on the traditional prevention and control means such as the breeding of transgenic disease-resistant varieties and chemical pesticides, but the breeding of the disease-resistant varieties is delayed because the rice blast germs have fast variation and the variation period is far shorter than the breeding time of the disease-resistant varieties; the abuse problem of indiscriminate use of chemical pesticides is prominent, the resistance of rice blast is continuously increased, the exceeding of the standard of environmental pesticide residues is reported, and the healthy development of human beings, the environment and the industry is seriously threatened. Therefore, the development of novel prevention and control technology which is green, convenient, safe and efficient is urgent.

Small RNAs (sRNA) are a class of non-coding RNAs that are 21-24 nucleotides in length. sRNA can specifically recognize a target gene and inhibit its expression, referred to as gene silencing. sRNA and gene silencing play a variety of important roles in the interaction between host plants and pathogens, providing a variety of applicable opportunities for plant protection and disease control. Spray-induced gene silencing (SIGS) has a similar function to HIGS, but does not require the aid of transgenic organisms.

The invention content is as follows:

the purpose of the invention is as follows: aiming at the defects in the prior art, the invention provides a key gene for inhibiting rice blast germs, wherein dsRNA for inhibiting the pathogenicity of the rice blast germs has strong stability, and can inhibit spore germination, attachment cell formation and germ tube growth of the rice blast germs so as to effectively prevent and control the rice blast germs.

The invention also provides dsRNA for inhibiting rice blast bacteria key genes, a preparation method and application thereof.

The technical scheme is as follows: in order to achieve the purpose, the key gene for inhibiting the rice blast fungus is any one of MoCytb or MoRic8 or MoTrx2 or MoMBF1 or MoEnd3, and the nucleotide sequences of the key gene are respectively shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO. 9.

The specific gene for inhibiting the key gene of the rice blast germ has higher specificity in the sequence of the key gene for inhibiting the rice blast germ and avoids the sequence of an intron, and the nucleotide sequences of the specific gene are respectively shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 10.

The dsRNA for inhibiting the key genes of the rice blast germs is any one of dsRNA of MoCytb gene containing T7 promoter, dsRNA of MoRic8 gene, dsRNA of MoTrx2 gene, dsRNA of MoMBF1 gene or dsRNA of MoEnd3 gene, and the nucleotide sequence of the dsRNA is added with T7 promoter sequence TAATACGACTCACTATAGG before SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 10.

Wherein, the specific 21nt sequence in the dsRNA for inhibiting the key gene of the rice blast germ synthesizes sRNA, and 2' -O-methyl modification or Phosphorothioate (PS) skeleton modification is carried out to obtain modified enhanced ds-sRNA: the sequence of the 21nt is shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 respectively.

The invention provides an in vitro transcription synthesis method of dsRNA for inhibiting rice blast germ genes, which comprises the following steps:

(1) preparing a dsRNA template: performing PCR amplification by using the specific gene as a template and an upstream primer sequence and a downstream primer sequence containing a T7 promoter, purifying to obtain a template, and transcribing the purified template by using the kit;

(2) and (3) DNase treatment: after transcription, adding a transcriptase and uniformly mixing;

(3) purification of transcribed RNA: transferring the mixed solution into a centrifugal tube, adding the mixed solution of phenol, chloroform and isoamylol, shaking and centrifuging to obtain a supernatant; absorbing the supernatant into a new centrifugal tube, adding the chloroform-isoamylol mixed solution into the new centrifugal tube, shaking and centrifuging to obtain the supernatant; mixing the obtained supernatant with sodium acetate and isopropanol, standing at room temperature, centrifuging, removing supernatant, adding ethanol, washing precipitate, adding DEPC water to dissolve the precipitate, and storing in an ultra-low temperature refrigerator.

Preferably, the upstream and downstream primers of the dsRNA containing the T7 promoter in the step (1) are MoCytb-T7-F and MoCytb-T7-R, MoRic8-T7-F and MoRic8-T7-R, MoTrx2-T7-F and MoTrx2-T7-R, MoMBF1-T7-F and MoMBF1-T7-R, MoEnd3-T7-F and MoEnd3-T7-R respectively, and the corresponding sequences are SEQ ID NO. 17-26. Wherein the volume ratio of the phenol, the chloroform and the isoamylol in the step (3) is 25-28: 1-5.

Preferably, the volume ratio of the phenol, the chloroform and the isoamyl alcohol in the step (3) is 25:24: 1. The dsRNA for inhibiting the key genes of the rice blast fungi is applied to inhibiting the rice blast.

The dsRNA for inhibiting the key genes of the rice blast fungi is applied to the preparation of an inhibiting preparation for inhibiting the rice blast fungi.

Wherein, the inhibition of the rice blast is in-vitro spray infection, the solution containing dsRNA is uniformly sprayed on rice seedlings in a three-leaf stage, and the phenotype is observed after the culture.

The principle of the invention is as follows: SIGS provide srnas by spraying, injection, or curtain coating, etc., which subsequently enter pathogen cells and inhibit the growth, development, and pathogenic ability of the pathogen. In addition to accurate inhibition of target gene expression, SIGS has great advantages over other conventional pesticides, such as shorter half-life and lower residual levels after each application period. Therefore, the dsRNA medicament with the effect of inhibiting the pathogenicity of rice blast germs is successfully developed by utilizing RNAi and SIGS.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the invention clones brand-new double-stranded ribonucleic acids dsCytb, dsRic8, dsTrx2, dsMBF1 and dsEnd3 for the first time, wherein the dsCytb effect is most obvious, the dsRNA can accurately inhibit the expression of key genes, thereby inhibiting the dsRNA of rice blast bacteria and rice blast bacteria MoCytb genes, having higher inhibition effect, obviously inhibiting the spore germination, the bud tube and the formation of attachment cells of the rice blast bacteria, and having high silencing efficiency.

2. The invention relates to rice blast fungus Guy11 and dsRNA ^MoCytb-500 After the mixed treatment of rice, the treated leaves became spotted after 5 daysThe quantity and the area are obviously less, the rice spot area, the fungal biomass and other aspects are obviously lower than those of a control group, and the rice blast bacterium target gene dsRNA ^MoCytb-500 Is expected to be developed into a novel green bactericide.

Drawings

FIG. 1 shows successful PCR verification of specific gene amplification of the target gene of Pyricularia oryzae of the present invention.

FIG. 2 shows that dsRNA of Magnaporthe grisea target gene of the invention is synthesized by in vitro transcription.

FIG. 3 shows that dsRNA of the present invention inhibits spore germination, germ tube formation and outgrowth formation of Pyricularia oryzae; a is to divide spore germination of rice blast germs into four types: type 1 (control group): normal germination of spores, germ tubes and appressorium; type 2: normal germ tube, inhibited spore germination and no attached cell; type 3: the spore germination is inhibited, the germ tube is deformed, and the anchorage cell is normal; type 4: inhibited spore germination, malformed germ tubes and attached cells; b is classification and statistics of spore growth of rice blast germs after dsRNA treatment for 6, 10 and 24 hours; c is the rate of formation of blast fungus appressorium cells (number of normal germinating spores/total number of spores) after 6, 10 and 24 hours of dsRNA treatment.

FIG. 4 sRNA structural modification of the invention enhances silencing; a is classification and statistics of spore growth of rice blast germs after sRNA treatment for 6 to 48 hours; b is the forming rate of rice blast germ attachment cells after the sRNA treatment for 6 to 48 hours; c after microscopic observation, 20. mu.l of DEPC was used to wash the slide to recover dsRNA.

FIG. 5 induced expression of dsRNA in recombinant E.coli of the invention; a is HT115 induced expression containing L4440-MoCytb and L4440-GFP (+: IPTG is added; -: IPTG is not added), and B is recovery rate of optimized bacteria induced dsRNA.

FIG. 6 dsRNA treatment of the invention can reduce the pathogenicity of Pyricularia oryzae.

Figure 7 dsRNA treatment of the invention significantly reduced fungal biomass.

FIG. 8 dsRNA treatment of the invention significantly reduces the expression of MoCytb.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention is further illustrated by the following figures and examples.

The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

All primer sequences used in the examples are shown in table 1 below.

TABLE 1

Example 1

Cloning of key genes of rice blast fungus

The key genes of MoCytb, MoRic8, MoTrx2, MoMBF1 and MoEnd3 are selected from genes for inhibiting rice blast bacteria and the like in the Gene Bank, and the sequences of the genes are shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9 through artificial synthesis.

Cloning of specific gene in key gene of rice blast bacterium

(1) After the hyphae of Pyricularia oryzae Guy11 (supplied by Nanjing university of agriculture) completely grew over the entire medium, the hyphae were scraped with a cover slip into a pre-cooled mortar and ground into powder with a grinding rod.

(2) Transferring the ground powder into a special centrifugal tube for 1.5mLRNA, adding TRIzol reagent (Invitrogen) to extract the total RNA of the rice blast germs, and then carrying out reverse transcription to form cDNA as a template. As shown in Table 1, PCR amplification was carried out using the primer sequences upstream and downstream of the dsRNA of Pyricularia oryzae gene (MoCytb-F, MoCytb-R, MoRic8-F, MoRic8-R, MoTrx2-F, MoTrx2-R, MoMBF1-F, MoMBF1-R, MoEnd3-F, MoEnd3-R) under the following conditions: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30-60s/kb for 32 cycles, extension at 72 ℃ for 5min, electrophoresis of PCR amplification products, gel cutting and product recovery, and purification of products by using a DNA gel recovery kit. As shown in figure 1, the specific gene nucleotide fragment of the key gene of Pyricularia oryzae is obtained by successful amplification, and the sequences are respectively shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 10.

Example 2

In vitro transcription Synthesis of dsCytb, dsRic8, dsTrx2, dsMBF1 and dsEnd3

(1) Using the specific nucleotide fragment of the key gene of Pyricularia oryzae obtained in example 1 as a template, DNAMAN was used to design a promoter containing T7, and as shown in Table 1, the sequences of the upstream and downstream primers were designed as shown in SEQ ID NO.17-SEQ ID NO.26 (MoCytb-T7-F, MoCytb-T7-R, MoRic8-T7-F, MoRic8-T7-R, MoTrx2-T7-F, MoTrx2-T7-R, MoMBF1-T7-F, MoMBF1-T7-R, MoEnd3-T7-F, MoEnd3-T7-R) to perform PCR amplification of the template, wherein the PCR conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30-60s/kb for 32 cycles, extension at 72 ℃ for 5min, electrophoresis of PCR amplification products, gel cutting, product recovery, and purification treatment with a PCR cleaning kit to obtain a DNA template containing a T7 sequence. Then, the dsRNA was synthesized by Transcription of the resulting DNA purification template containing the T7 sequence using the In vitro Transcription T7 Kit (purchased from TaKaRa), and the resulting DNA purification template was added sequentially according to the reaction solution system shown In table 2, wherein T7 RNA Polymerase and the resulting DNA template were added last to prevent the mixture from white flocculent precipitate:

TABLE 2 kit reaction System

Sucking with a pipette, mixing, quickly separating, placing in a PCR (42 deg.C, 2h) instrument, and reacting at 42 deg.C for 2 h.

(2) DNase treatment

After transcription was completed, 5. mu.L of RNase free DNase I was added and mixed well. The mixture was placed in a PCR apparatus (37 ℃ C., 30min) and reacted for 30 min.

(3) Purification of transcribed RNA

After centrifugation, if the volume of the mixture is less than 100. mu.L, RNase free ddH is used ₂ And (3) supplementing the oxygen to 100 mu L, transferring the mixed solution into a 1.5mL RNA centrifugal tube, adding 100 mu L of mixed solution of phenol, chloroform and isoamylol, wherein the pH value of the phenol is 4.5, the volume ratio of the phenol to the chloroform to the isoamylol is 25:24:1, placing the mixed solution on a vortex oscillator for fully oscillating, and centrifuging at the room temperature at 12000rpm for 2 min. And (3) sucking the supernatant into a new 1.5mL RNA centrifugal tube, adding a chloroform and isoamylol mixed solution with the same volume as that of 100 mu L supernatant, wherein the volume ratio of the chloroform to the isoamylol mixed solution is 24:1, placing the mixture on a vortex shaking instrument for fully shaking, and centrifuging at the room temperature at 12000rpm for 2 min. The supernatant was pipetted into a new 1.5mL RNA centrifuge tube, 1/10 volumes of 3M sodium acetate and an equal volume of isopropanol were added, and the mixture was shaken well. After standing at room temperature for 5 minutes, the mixture was centrifuged at 15000rpm at room temperature for 5 minutes. The supernatant was discarded and 1mL of 80% ethanol (made up of DEPC water) was added to wash the precipitate. Drying the water in the centrifugal tube in an ultraclean workbench, adding RNase free ddH ₂ Dissolving the precipitate with O, measuring the concentration of dsRNA with Nanodrop, and storing in an ultra-low temperature refrigerator at-70 deg.C for use. After the treatment, electrophoresis detection is carried out on 1% agarose gel, as shown in figure 2, dsRNA of rice blast bacteria MoCytb, MoRic8, MoTrx2, MoMBF1 and MoEnd3 genes is obtained and named as dsCytb, dsRic8, dsTrx2, dsMBF1 and dsEnd3 respectively, and the obtained dsRNA sequence containing the T7 promoter is added with a T7 promoter sequence TAATACGACTCACTATAGG before the specific gene nucleotide sequence of the rice blast bacteria key gene in the embodiment 1, namely the nucleotide sequence of dsCytb, dsRic8, dsTrx2, dsMBF1 and dsEnd 3.

Example 3

Preparation method of rice straw culture medium (SDC)

(1) Weighing 100g of common rice straw by balance and placing the rice straw in an electromagnetic oven.

(2) 1.5L of ddH was added ₂ Boiling for about 20 min, adding 0.5L ddH ₂ And O, boiling for 10 minutes.

(3) Filtering with double-layer gauze, discarding the residue, and adding ddH ₂ O constant volume is 1L, 8g of corn flour and 3g of agar powder are respectively added into each conical flask, and finally the mixture is subpackaged into 250mL conical flasks (each conical flask is filled withErlenmeyer flask plus 200 mL).

(4) Sterilizing in high pressure steam sterilizer at 120 deg.C for 20 min.

Example 4

dsRNA inhibits the growth of rice blast bacteria

(1) The rice blast fungus strain Guy11 was inoculated into the straw medium (SDC) prepared in example 3, and was cultured in an incubator at 28 ℃ while being inverted. And scraping hyphae growing on the flat plate after culturing for 3d, and then transferring to an indoor tissue culture rack for black light illumination culture for 3d to induce sporulation and collect spores.

(2) Treatment groups (dsCytb, dsRic8, dsTrx2, dsMBF1, dsEnd3 prepared in example 2, respectively) were dosed with 5. mu.L of Guy11 spore suspension (2X 10) ⁵ Spores/ml), 4. mu.L RNase ddH ₂ O and 1. mu.L of the dsRNA prepared in example 2 (111uM) were cultured on Fisher hydrophobic slides. Control (Mock) 5. mu.L of Guy11 spore suspension (2X 10) was added dropwise ⁵ Spores/ml), 5. mu.L RNase ddH ₂ And O. The slides were placed in a clear plastic box and the humidity in the container was maintained at RH > 85%. The vessel was then transferred to a 28 ℃ incubator for incubation and removed at 6, 10 and 24 hours respectively. Magnaporthe grisea was treated with 75U Micrococcus Nuclear enzyme (Thermo Scientific) at 37 ℃ for 30 minutes.

The pictures of the treated groups and the untreated rice blast fungus are observed under an inverted fluorescence microscope (Zeiss Axio oven 3), and the pictures are found to be divided into four types (types 1-4) according to the spore germination, the germ tube formation and the attached cell morphology, which indicates that the dsRNA has influence on the growth and development of the spore of the rice blast fungus. And photographing and analyzing according to the growth and development conditions of the rice blast fungus spores so as to better distinguish and accurately quantify the inhibition effect of the dsRNA on the growth of the rice blast fungus. As shown in A in FIG. 3, spore germination of Pyricularia oryzae is classified into four types according to the spore germination, germ tube and appressorium development: type 1 (control group): normal germination of spores, germ tubes and appressorium; type 2: normal germ tube, inhibited spore germination and no attached cell; type 3: the spore germination is inhibited, the germ tube is deformed, and the anchorage cell is normal; type 4: inhibited spore germination, malformed germ tubes and attached cells.

Classification and statistics of spore growth of Pyricularia oryzae were shown in FIG. 3B after 6 hours, 10 hours and 24 hours of co-incubation of the treatment groups (dsCytb, dsRic8, dsTrx2, dsMBF1, dsEnd3) with Guy11 spore suspensions. After 6 hours of co-incubation, approximately 87.9% of the spores in the control group germinated normally (type 1), with the remaining 12.1% of the spores being developmental malformations, compared to dsCytb treatment significantly inhibited spore germination. Type 1 spores accounted for 51.2%, type 2 accounted for 13.4%, type 3 spores accounted for 12.2%, type 4 spores accounted for 23.2%, dsRic8, dsTrx2, dsMBF1, dsEnd3 also had varying degrees of inhibitory effect in the dsCytb treated samples; after 10 hours of co-incubation, most of the spores in the control group developed normally (type 1> 90%), the remaining 10% were

types

2, 3, 4, and statistics of dsCytb treatment showed 62.6% of type 1, 2.5% of type 2, 9.4% of type 3, and 25.5% of type 4; after 24 hours of co-incubation, dsCytb treatment statistics showed 80.6% of type 1, 4.6% of type 3 and 14.8% of type 4. dsRic8, dsTrx2, dsMBF1, dsEnd3 showed no significant inhibitory effect on magnaporthe oryzae spore germination, germ tube and anchorage cell formation, but dsCytb treatment significantly inhibited spore germination, germ tube and anchorage cell formation.

In order to examine whether the treatment groups (dsCytb, dsRic8, dsTrx2, dsMBF1, dsEnd3) had an effect on the formation of adherent cells, the present invention counted the rate of formation of adherent cells (number of normal forming spores of adherent cells/total number of spores) in the control group and the treatment group, as shown by C in FIG. 3, after 6 hours of co-incubation, about 88.1% of adherent cells in the control group were normally formed, and about 51.4% of adherent cells in the dsCytb-treated sample were normally formed; after 10 hours of co-incubation, approximately 95.4% of the adherent cells formed normally in the control group, while approximately 62.5% of the adherent cells formed normally in the dsCytb treated samples; after 24 hours of co-incubation, approximately 95.2% of the adherent cells formed normally in the control group, while approximately 80.6% of the adherent cells formed normally in the dsCytb treated samples. The results show that dsRic8, dsTrx2, dsMBF1, dsEnd3 did not show significant inhibition of adherent cell formation, but dsCytb treatment significantly inhibited adherent cell formation. It shows that dsCytb has an inhibitory effect on the formation rate of blast fungus (Guy11) attachment cells.

Example 5

Terminal thymine and structural modification enhance silencing effect

In order to detect whether structural modification can enhance the stability of dsRNA and enhance the inhibition effect, a 21nt sequence which does not contain introns and has high specificity is selected to synthesize sRNA, and 2' -O-methyl modification or Phosphorothioate (PS) framework modification is carried out at the same time. The invention carries out 2 '-O-methyl modification and Phosphorothioate (PS) skeleton modification on dsRNA for inhibiting rice blast fungus key gene MoCytb by utilizing the mechanism that 2' -O-methyl modification enables oligonucleotides to have relative resistance to nuclease and obviously enhances silent activity and Phosphorothioate (PS) backbone modification can protect the dsRNA from rapid degradation of nuclease in cells. Using the rice blast fungus gene dsCytb prepared in example 2 as a template, a sequence of 21nt length having no intron and strong specificity was selected, and upstream and downstream primer sequences were designed, which are shown in SEQ ID NO.11-SEQ ID NO.16 of Table 1 (ds-sCytb-F, ds-sCytb-F, ds-sCytb-Ome-F, ds-sCytb-Ome-R, ds-sCytb-SOme-F, ds-sCytb-SOme-R, which is a 21nt sequence having high specificity), and ds-sCytb, ds-sCytb-Ome and ds-sCytb-SOme were synthesized by modifying 2' -O-methyl or phosphorothioate, which were synthesized by Nanjing King Spi Biotech Co., Ltd.

The ds-sCytb, ds-sCytb-Ome, ds-sCytb-SOme synthesized as described above were used as treatment groups after incubation with Guy11 spore suspensions for 6 hours, 10 hours and 24 hours. As shown in FIG. 4 (A) for classification and statistics of spore growth of Pyricularia oryzae (Guy11), after 6h of co-incubation, about 57.5% of the spores in the ds-sCytb-treated samples germinated normally (type 1), and the remaining spores all germinated malformations (10.2% for type 2, 11.8% for type 3, and 20.5% for type 4, respectively). After 10h of co-incubation, the spore germination types in the ds-sccytb treated samples were: type 1 accounts for 74.6%, type 2 accounts for 6.5%, type 3 accounts for 3.2%, type 4 accounts for 15.7%. Whereas ds-sCytb-Ome and ds-sCytb-SOme treatments significantly inhibited spore germination compared to the control group. After 24h co-incubation, approximately 90% of the spores in the control and dsCytb treatments germinated normally, while the spores in the ds-sCytb-Ome and ds-sCytb-SOme treatments germinated normally by approximately 60%. The inhibitory effect gradually decreased over time. After 48h co-incubation, there were approximately 85% normal germinated (type 1) spores in ds-sCytb-Ome and ds-sCytb-SOme treatments, respectively, while almost 95% of spores developed normally in the control treatment. The result shows that the dsRNA modified by the framework obviously enhances the stability and the protection effect of sRNA, wherein the ds-sCytb-SOme has stronger stability and better inhibition effect.

Meanwhile, the present invention also observed the formation rate of the adherent cells between the control group and the treatments of ds-sCytb, ds-sCytb-OMe and ds-sCytb-SOme, as shown in (B) in FIG. 4, after incubating for 6h, the formation rate of the adherent cells in the control group exceeded 80%, the formation rate of the adherent cells in the treatment of ds-sCytb was 56.3%, and the formation rates of the adherent cells in the treatment of ds-sCytb-OMe and ds-sCytb-SOme were 25.0% and 23.3%, respectively. However, starting from the co-incubation time of 10h, the inhibition of the formation of adherent cells by the structurally modified sRNA was more pronounced. After incubation for 10h, the formation rates of the attached cells treated by ds-sCytb, ds-sCytb-Ome and ds-sCytb-SOme were 74.5%, 70.7% and 32.7%, respectively. After 24, 36 and 48h of co-incubation, ds-sCytb and ds-sCytb-Ome no longer inhibited the formation of adherent cells. However, ds-sCytb-SOme treatment still had a significant inhibitory effect on adhesion cell formation. The results indicate that structural modification significantly enhances the inhibition of sRNA by enhancing the stability of sRNA.

After the end of the microscopic photograph, the slide was washed with 20. mu. LDEPC to recover sRNA. And detected by electrophoresis on a 1% agarose gel. As shown in FIG. 4 (C), ds-sCytb-OMe and ds-sCytb-SOMe continued until 60hpi had no significant degradation compared to ds-sCytb, and ds-sCytb-SOme treatment inhibited better, indicating that the structural modification protected sRNA from RNase.

Example 6

Induction expression of rice blast germ gene dsRNA

The rice blast fungus Guy11 RNA was extracted by the method of example 1, and then reverse-transcribed into cDNA as a template, and as shown in Table 1, the template was PCR-amplified using the upstream and downstream primer sequences (MoCytb-F-Sac I, MoCytb-R-Xho I) of dsRNA of rice blast fungus MoCytb gene under the PCR conditions: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30-60s/kb for 32 cycles, extension at 72 ℃ for 5min, electrophoresis of PCR amplification products, gel cutting and product recovery, and double enzyme digestion of purified products (GFP and MoCytb) and L4440 vector with SacI and XhoI (TaKara) respectively. The digested product was purified using a PCR purification kit (Axygen), the target gene fragment was ligated overnight with the L4440 vector using T4 DNA Ligase, and finally the ligation product was transformed into E.coli HT115(DE 3). After cloning and screening, the sequencing of a single strain is correct, and the glycerol strain is preserved.

Mixing the glycerol bacteria containing the target recombinant vector in a volume ratio of 1: 100 was added a solution containing ampicillin and tetracycline resistance (12.5. mu.g/mL Tet) ⁺ ，50μg/mL Amp ⁺ ) LB of (5) was initially incubated overnight at 37 ℃ on a shaker at 220 rpm. Sucking the primary culture liquid with a pipette gun, adding into LB liquid culture medium containing ampicillin and tetracycline resistance at a volume ratio of 4:100, placing in a constant temperature shaking table at 37 deg.C, expanding at 220rpm for about 3 hr to OD ₆₀₀ 0.6. Induction was continued for 5h with the addition of 0.5mM IPTG. Taking the induced bacterial liquid, extracting total RNA of Escherichia coli HT115, treating with DNase I at 37 deg.C for 30min, and then treating with RNase A at 37 deg.C for 30 min. After incubation, the electrophoresis was verified on a 1% agarose gel. As shown in FIG. 5 (A), dsRNA can be produced after IPTG-induced expression of the L4440 recombinant vector. dsRNA as shown in (B) in FIG. 5 ^MoCytb-500 The recovery rate can reach more than 85 percent.

Among them, dsRNA prepared in this example ^MoCytb-500 dsCytb, prepared as in example 2, is the same dsRNA, identical in sequence, but prepared in a different manner. Simultaneously, the primer SEQ ID NO.27-28 is used for amplification, and the dsRNA is obtained according to the method ^GFP ^-500 。

Example 7

dsRNA treatment for reducing pathogenicity of rice blast bacteria

Soaking rice (Nipponbare) seed in 70% ethanol for 1min for sterilization, washing the sterilized seed with sterile water for 4-6 times, placing the rice seed in a glass dish full of filter paper, and adding ddH just over the seed ₂ O, placing the seeds into a constant-temperature incubator at 28 ℃ for pregermination for about 3 days (the seeds germinate about 1cm), then planting the germinated seeds into culture pots in black soil, placing 9 seeds in each pot to ensure that the water in the pot is sufficient, and placing the pots in a constant-temperature greenhouse (at 28 ℃) with the humidity of 75 percentThe culture was performed for about 3 weeks in light for 12 hours and dark for 12 hours.

In vitro sRNA spray infection: preparation of 2X 10 ⁵ Spore/ml Guy11 spore suspension (containing 0.4% gelatin, 2.4 nMDCytb) ^MoCytb-500 Or dsRNA ^GFP-500 ) Uniformly spraying the mixture on 6 pots of rice seedlings in the three-leaf period, wherein two pots are sprayed with bacteria, two pots are sprayed with a mixed solution of the bacteria and dsGFP, two pots are sprayed with a mixed solution of the bacteria and dsCytb, each pot of plants (about 15-20 plants) is sprayed with 5mL of spore suspension, placing the plants in an infection box with the humidity of more than 80% and the temperature of 28 ℃, culturing for 24 hours in the dark, then culturing under the condition of 12 hours of dark/12 hours of light alternation, and sampling and observing the phenotype after 5 days.

Using the dsRNA of this example ^MoCytb-500 (2.4nM) the rice was infected by spraying with a suspension of spores of the rice blast strain Guy11 (Nipponbare). The dsRNA is paired with upstream and downstream primer sequences (Mopot2-RT-F, MoPot2-RT-R, MoCytb-RT-F, MoCytb-RT-R) respectively ^MoCytb500 And dsRNA ^GFP500 And (5) performing fluorescent quantitative PCR (polymerase chain reaction) and detecting the biomass and the gene expression level of the fungi. Control and dsRNA ^GFP-500 In contrast, as shown in FIG. 6, dsRNA ^MoCytb-500 The treated leaves were significantly less numerous and less extensive after 5 days (dpi). Fluorescent quantitative PCR assay with primer MoPot2-RT-F, MoPot2-RT-R as shown in FIG. 7 showed dsRNA ^MoCytb-500 The relative growth amount of the treated rice fungi is obviously lower than that of a control group and dsRNA ^GFP-500 Treating the growth amount of the rice; as shown in FIG. 8, the fluorescent quantitative PCR assay was performed using the primer MoCytb-RT-F, MoCytb-RT-R, and the results showed dsRNA ^MoCytb-500 The relative expression level of the key gene MoCytb of the treated rice is obviously lower than that of a control group and dsRNA ^GFP-500 Treated rice. The above results indicate that dsRNA was compared to the control group ^GFP-500 The treatment did not affect the pathogenic ability of rice blast fungus, but dsRNA ^MoCytb-500 The treatment inhibits the pathogenic capability of rice blast germs, and further proves that in vitro application of dsRNA ^MoCytb-500 Can inhibit pathogenicity of Magnaporthe grisea.

The kit can be used for synthesizing dsRNA, and can also be used for synthesizing dsRNA by in vitro induction. dsRNA extracted by the kit is purer, and the dsRNA synthesized by in vitro induction is suitable for large-scale use.

Sequence listing

<110> Nanjing university of agriculture

<120> key gene for inhibiting rice blast bacteria, dsRNA and preparation and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1182

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgagaatat taaaaagtca ttcattatta aaattagtga attcttacct tatcgatgcg 60

tcacaaccaa gtaacattag ttacttgtga aattttggtt cattattagc tgtttgttta 120

atagtacaaa ttattaccgg tattacatta gctatgcatt atagtcctag tgtaatggaa 180

gcttttaact caatagagca tataatgaga gatgttaata acgggtgatt agttcgttat 240

ctacatagta atacagcttc tgctttcttt ttcttagtgt atttacacat aggaagaggt 300

atatattacg gatcatatag agctcctcgt actttagttt gagctattgg tactgttata 360

ttaatattaa tgatggctat cggtttccta ggttatgttt taccttatgg acagatgtca 420

ttatgaggtg ctacagttat tactaatctt attagtgcta taccttgaat agggcaagat 480

attgttgaat tcatttgagg tggtttttct gttaataatg ccactttaaa cagatttttt 540

gcattacatt ttgtattgcc ttttgtatta gctgctttag ttttaatgca cttaattgca 600

cttcatgata ctgctggttc aagcaatcct cttggtgttt caggtaatta cgatagaatt 660

acatttgctc catatttttt atttaaagat ttaattacta tttttatatt tatttttgta 720

ttaagtgctt ttgtattctt tatgcctaat gttttagggg atagtgataa ttatattatg 780

gctaatccta tgcaaactcc tgctgctatt gtacctgaat gatacttatt acctttctat 840

gctattttaa gatctatacc taataaatta ttaggtgtta tagcgatgtt tagtgctatt 900

ttagctatta tgttattacc tgttacagat ttaggtagat ctagaggttt acaatttaga 960

ccatttagta aaatagcttt ctgagttttt gttgctaatt tcttagtttt aatgcaatta 1020

ggtgctaaac acgttgaaga tccatttata ttattaggtc aattaagtac tgtattatac 1080

tttagttatt ttgttgctat attaccttta gctagttact tagataatag tttaactgat 1140

ttatctaata aatctgaatt atttttaaat aaaactaact aa 1182

<210> 2

<211> 320

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgagaatat taaaaagtca ttcattatta aaattagtga attcttacct tatcgatgcg 60

tcacaaccaa gtaacattag ttacttgtga aattttggtt cattattagc tgtttgttta 120

atagtacaaa ttattaccgg tattacatta gctatgcatt atagtcctag tgtaatggaa 180

gcttttaact caatagagca tataatgaga gatgttaata acgggtgatt agttcgttat 240

ctacatagta atacagcttc tgctttcttt ttcttagtgt atttacacat aggaagaggt 300

atatattacg gatcatatag 320

<210> 3

<211> 1431

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgttgaata ccaatacact gaagggcaca gcgaagctat ctgctgtaaa gggcttgcta 60

gataaactca ctctcgactt gaaggaacac aatctctcct cgcaagagcg tgacgcggcg 120

ctcgagcagt tgaaagttta tgggagagac cctcgtgatg cagagcccat ctttacaaag 180

gaggggatat cgacgctggc cgcgcactcc ttcgatggat cgtcggacac aacatccaga 240

aatgcgctcc gctgcctggc gaatgcaatg cttcttcaac ccagctcccg ccaagagctc 300

gttgatcttg gatatcacat caaggcatgt gctaagcttg caagtgatag ctgggacgac 360

gagttcctgg tttcacgggt gctgatgctg gcagcaaatg ccagaaacat caatcacgag 420

atattgatta cacaacatgg ccttgcagag cttattgctg caaggttaga gagtcatatg 480

cgctttttac cagatgagga ctcaagcccg aacaccatgg agggtatggc gctcagcgaa 540

actctcaaaa tgatgttcgc cgtcctacgg ttttgtccgg gacagagtac gagattcgct 600

ggagccgcag gccacattct aacactgata tgtcagcgac cactacctgc tgaatccgtc 660

cttgatcctc ctttcggtcc cttgatcaat gcgttatgca ccctcgatct cgaggacgag 720

gctatcacga atactttgtt cccggagagg gatccaagcc tcgtaacaag caggctgatt 780

agcatcctgg acagggcgtt gagtgagttc aaaggcaatg gcatggacga cccaacaatg 840

acagtgcttg ccggctcgtt gtgcattatt tttgaggccg cacccgagcc cgttcgcata 900

tccatgcagg cccaactcct acctaccgac gaagacagga aggaggttct tggcagcacg 960

gagagtctgc catcaaagct cctgaagatg atgactaacg ccatggctcc gaaatcccga 1020

aaggccatat cgcatctact ttttggtctt tcggacaggg acgctggtaa gtttgtggag 1080

aaagtcggat atgggtatgc ctctggcttt ctgttcgaag aaaacatccc agtaccacag 1140

tctgcgctgg ccagcgaagg tgcttcgggt tcgacaatgc aaggaagagc tgtgaatccc 1200

ataactgggc agttcctgga cgcggagaag ctgtcggata ttcctgaaat gtcacaggaa 1260

gagaaggaga aggaggcgga gaggttgttc gttttgtttg agagactcaa gaagactggc 1320

gttgttgacg tacagaatcc ggttgagaag gcattccaag aaggccgcgt tgaagagctt 1380

gaagacgatg aagatggtga cgagaataac aagaaagtgg agcaaaagta g 1431

<210> 4

<211> 400

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcgctcgagc agttgaaagt ttatgggaga gaccctcgtg atgcagagcc catctttaca 60

aaggagggga tatcgacgct ggccgcgcac tccttcgatg gatcgtcgga cacaacatcc 120

agaaatgcgc tccgctgcct ggcgaatgca atgcttcttc aacccagctc ccgccaagag 180

ctcgttgatc ttggatatca catcaaggca tgtgctaagc ttgcaagtga tagctgggac 240

gacgagttcc tggtttcacg ggtgctgatg ctggcagcaa atgccagaaa catcaatcac 300

gagatattga ttacacaaca tggccttgca gagcttattg ctgcaaggtt agagagtcat 360

atgcgctttt taccagatga ggactcaagc ccgaacacca 400

<210> 5

<211> 516

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtcttgca tcagcagaag ccgcgcttta tttcaaatcc ggtcgcttgc atcatcgtcg 60

cacttgcccc tcccaaagct gcgcccctca cccgccatct acaaaagcgc aaacaagacc 120

gcgttcaacg catcgccgtc cacgtcgcaa tcagcaacaa ccaccagagg attcagatcg 180

accgccgcaa aaatgactgt ccacaacctt accaacgccc aggatttcaa ggacgccctc 240

aagtcccaca agttcgtcct cgtcgacttt ttcgccacat ggtgtggacc ttgcagggcc 300

atcgccccca agatcgccga gtggtccgat gcgttcccca acatccacta cgtcaaggtc 360

gacgtcgacg aggtccctga tgtcgcccag gagtacaacg tccgcgccat gccgacattc 420

ctgctcttca aggatggaga gaaggtggac gaggttgttg gcgccaaccc acccaagctg 480

caggccctca tcagcgcaaa ccacccgtcg tcatag 516

<210> 6

<211> 400

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cctcacccgc catctacaaa agcgcaaaca agaccgcgtt caacgcatcg ccgtccacgt 60

cgcaatcagc aacaaccacc agaggattca gatcgaccgc cgcaaaaatg actgtccaca 120

accttaccaa cgcccaggat ttcaaggacg ccctcaagtc ccacaagttc gtcctcgtcg 180

actttttcgc cacatggtgt ggaccttgca gggccatcgc ccccaagatc gccgagtggt 240

ccgatgcgtt ccccaacatc cactacgtca aggtcgacgt cgacgaggtc cctgatgtcg 300

cccaggagta caacgtccgc gccatgccga cattcctgct cttcaaggat ggagagaagg 360

tggacgaggt tgttggcgcc aacccaccca agctgcaggc 400

<210> 7

<211> 486

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggccgacg actgggacac cgttaccaag atcggaagca gggtcggcgg cggtggtggc 60

ggcggccctc gcttgaccac catcaagaac aagagccagc ttaatgctgc tcagcgcgca 120

ggcggtatcg tgggaacgga gaagaagtac ggcactgcca actctagcag atcagaggcc 180

ggctccggac aattcttgac caaggttgac cgatctgacg acattgtcaa gcccaagact 240

ggtgataagg aattgggcat gtacataatg cagaaccgtg aacagaagaa gttggggaac 300

cggcttgagt tcggaaagaa agtcggcata aacgagaagg atcttgcaag gattgagaaa 360

ggcgaggtcc cgatcacaca agatcaggtc aacaggatcg agaggggctt ggagatgttc 420

atcagaggcg ttaagaaggg tgagccaaag gtcaagacct ttaagagggc tgctgataag 480

aaatag 486

<210> 8

<211> 284

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caagaacaag agccagctta atgctgctca gcgcgcaggc ggtatcgtgg gaacggagaa 60

gaagtacggc actgccaact ctagcagatc agaggccggc tccggacaat tcttgaccaa 120

ggttgaccga tctgacgaca ttgtcaagcc caagactggt gataaggaat tgggcatgta 180

cataatgcag aaccgtgaac agaagaagtt ggggaaccgg cttgagttcg gaaagaaagt 240

cggcataaac gagaaggatc ttgcaaggat tgagaaaggc gagg 284

<210> 9

<211> 1191

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggcacctc gcatagaggc tcaggagatt gagacgtact ggaacatctt cagcgcgagg 60

acgaacggct ccaagttcct cacgggagag caggcggcac ccgtgctcaa gaactcgggg 120

ctccgtgatg atcagctcga gagggtctgg gaccttgccg acgtcgacaa tgatggaaac 180

ctggactttg aggagttttg cgtggccatg cgcgtcatct ttgacatact gaacggggaa 240

cacgccgacg tcccgtctac actcccggat tggctcgttc cagaatccaa ggcgcacctg 300

gtccaagcca accgagctct gacaggaaag caagtacaat tcgagcgcgt ggatgatgac 360

cccgactcac cgggactcaa ggatggattc gactggtata tgagcccggc ggataagtcc 420

aagtacgaat ctatatacca ggagaatagg gacatgcggg gagaagtgtc gttcggcgcc 480

ctcgaggacc tttatgaatc actcgatgtc ccagatacag acattagatc ggcctggaat 540

ctgatcaacc cttcagctgg gcctacgatc aacaaagacg cctgcctcgc ctttctccac 600

attctcaact accgtcacga aggctaccgg atccccagga cggtccctgc atctctccgc 660

gcttccttcg agcgcaacaa gattgactac caggttgaca agcaggcggc ctcgcggtgg 720

gcaaccaagg ccgacgatga gacctctaca ggccgcaagg ccaagtttgg tgaccagtac 780

ctgacccgtc tggggcgcgg cagcttcaag acttctggaa ccgacttctc aagtgctcag 840

actgattctg aatgggagga ggtgcggctg aagaagcagc tcgctgagct ggatgccaag 900

atggcgagcg tagaggctga tgccgagagg cggaagggtg gaaagcggga ttcgaagccg 960

gcgcttgtca aacgcgaact ggagcaactt ctcgactaca agcggaagca actgcgagag 1020

atagaagagg gcaaaaccaa gggtcagggt ggtggaagcc ttaaggggat acaggatgat 1080

ttgcagactg ttcgtgagca gacggatggc ctagcgtcgc atctgaggtc gaggcaacaa 1140

cacttggaag aattgcgtcg gcaaatcgag gacgagaagg ccggcaggtg a 1191

<210> 10

<211> 400

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaacacgccg acgtcccgtc tacactcccg gattggctcg ttccagaatc caaggcgcac 60

ctggtccaag ccaaccgagc tctgacagga aagcaagtac aattcgagcg cgtggatgat 120

gaccccgact caccgggact caaggatgga ttcgactggt atatgagccc ggcggataag 180

tccaagtacg aatctatata ccaggagaat agggacatgc ggggagaagt gtcgttcggc 240

gccctcgagg acctttatga atcactcgat gtcccagata cagacattag atcggcctgg 300

aatctgatca acccttcagc tgggcctacg atcaacaaag acgcctgcct cgcctttctc 360

cacattctca actaccgtca cgaaggctac cggatcccca 400

Claims

1. The key gene for inhibiting the rice blast germs is any one of MoCytb, MoRic8, MoTrx2, MoMBF1 or MoEnd3, wherein the nucleotide sequences are respectively shown as SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7 and SEQ ID No. 9.

2. The specific gene for inhibiting the key gene of rice blast fungus as claimed in claim 1, wherein the specific gene is a specific gene with an intron of the key gene of rice blast fungus removed, and the nucleotide sequences of the specific gene are respectively shown as SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 and SEQ ID No. 10.

3. The dsRNA for inhibiting the key genes of rice blast fungus as claimed in claim 1, wherein the dsRNA for inhibiting the key genes of rice blast fungus is any one of dsRNA of MoCytb gene containing T7 promoter, dsRNA of MoRic8 gene, dsRNA of MoTrx2 gene, dsRNA of MoMBF1 gene or dsRNA of MoEnd3 gene, and the nucleotide sequence of the dsRNA is added with T7 promoter sequence TAATACGACTCACTATAGG before the sequences of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 10.

4. The dsRNA for inhibiting key genes of rice blast fungus according to claim 3, wherein a specific 21nt sequence in the dsRNA for inhibiting key genes of rice blast fungus is synthesized into sRNA, and 2' -O-methyl modification or Phosphorothioate (PS) skeleton modification is carried out to obtain modified enhanced ds-sRNA: ds-sCytb-Ome and ds-sCytb-SOme, and the 21nt sequences are respectively shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.

5. An in vitro transcription synthesis method according to claim 3, characterized in that the synthesis steps are as follows:

(1) preparing a dsRNA template: performing PCR amplification by using the specific gene as a template and upstream and downstream primer sequences containing a T7 promoter, purifying to obtain a template, and transcribing the purified template by using a kit;

(2) and (3) DNase treatment: after the transcription of the step (1), adding a transcriptase and uniformly mixing;

(3) purification of transcribed RNA: transferring the mixed solution into a centrifugal tube, adding the mixed solution of phenol, chloroform and isoamylol, shaking and centrifuging to obtain a supernatant; sucking the supernatant into a new centrifugal tube, adding a mixed solution of chloroform and isoamylol into the new centrifugal tube, shaking and centrifuging the mixed solution, and taking the supernatant; mixing the obtained supernatant with sodium acetate and isopropanol, standing at room temperature, centrifuging, removing supernatant, adding ethanol, washing, precipitating, adding sterile water to dissolve the precipitate, and storing in an ultra-low temperature refrigerator.

6. The method for synthesizing dsRNA of claim 5, wherein the upstream and downstream primers of the dsRNA containing T7 promoter of step (1) are MoCytb-T7-F and MoCytb-T7-R, MoRic8-T7-F and MoRic8-T7-R, MoTrx2-T7-F and MoTrx2-T7-R, MoMBF1-T7-F and MoMBF1-T7-R, MoEnd3-T7-F and MoEnd3-T7-R, respectively, and their corresponding sequences are SEQ ID NO. 17-26.

7. The method for synthesizing dsRNA of claim 4, wherein the volume ratio of phenol, chloroform and isoamyl alcohol in step (3) is 25-28: 1-5.

8. Use of the dsRNA for inhibiting a key gene of Pyricularia oryzae according to claim 3 or claim 4 for inhibiting Pyricularia oryzae.

9. Use of the dsRNA for inhibiting a key gene of Pyricularia oryzae according to claim 3 or claim 4 for the production of an inhibitory agent for inhibiting Pyricularia oryzae.

10. The use of claim 8, wherein the inhibition of rice blast is achieved by in vitro spray application, wherein a solution containing rice blast and dsRNA is sprayed uniformly onto young rice plants in the trefoil stage, and the phenotype is observed after cultivation.