A siRNA targeting key genes of rice blast fungus for controlling rice blast and its application

Abstract

The present invention provides a siRNA targeting a key gene of rice blast fungus for preventing and controlling rice blast disease and its application, and relates to the field of biotechnology. MOHS1 gene (MGG_08523); the siRNA molecule can delay and inhibit the formation of appressorium of rice blast fungus, thereby reducing the pathogenicity of rice blast fungus, thereby providing a new method for preventing and controlling rice blast disease. MOHS1 The siRNA molecules of the gene are prepared into nucleic acid pesticides and sprayed on rice leaves, which effectively reduce the rice blast lesions and the area of ​​lesions on the leaves, and can be used for rice blast prevention and control, and can avoid the tedious process of cultivating genetically modified crops and the concerns of consumer groups.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and mainly relates to siRNA for preventing and controlling rice blast by targeting key genes of rice blast fungus and application thereof. Background Art

[0002] Rice ( Oryza sativa L.) is the staple food of more than half of the world's population, providing more than 20% of the calories consumed by humans, and is crucial to global food security. Rice blast, bacterial blight and sheath blight are known as the three major diseases of rice. Among them, rice blast is caused by the blast fungus Magnaporthe oryzae Rice blast fungi can infect multiple parts of rice. Depending on the time or tissue of the disease, it can be divided into seedling blast, node blast, leaf blast and ear neck blast. Among them, ear neck blast is the main cause of rice yield loss. In addition to rice, M. oryzae It can also infect more than 50 species of grasses, including economically and agriculturally important cash crops such as wheat ( Triticum spp .),barley( Hordeum vulgare ),corn( Zea mays ) and grass weeds, etc.

[0003] The infection of rice blast fungus begins with conidia. When they are scattered on the leaf surface of rice and other plants, under suitable temperature and humidity conditions, conidia germinate and grow germ tubes. The top of the germ tube swells and forms a special infection structure, namely the hemispherical appressorium. As the appressorium forms and matures, a melanin layer is formed between the cell membrane and the cell wall, and a large amount of osmotic substances (such as glycerol) accumulate inside the appressorium, causing the turgor pressure inside the appressorium to gradually increase. Under the combined action of the melanin layer and high turgor pressure, the high turgor pressure is converted into mechanical pressure to penetrate the host surface, forming an infection spike at the base of the appressorium, and penetrating the host plasma layer to form primary infection hyphae. The rice blast fungus needs the melanin layer in the appressorium to produce and maintain the high turgor pressure required for plant penetration, and the synthesis of melanin requires the participation of multiple enzymes.

[0004] In recent years, RNAi pesticides (nucleic acid pesticides) developed based on RNAi have become a hot topic of research at home and abroad. dsRNA or siRNA is sprayed on the surface of plants, and then directly or indirectly absorbed by target biological cells, causing RNAi reactions in the body, and then silencing the target gene. This technology is also called spray-induced gene silencing (SIGS). SIGS technology avoids the complex process and potential ecological risks of crop genetic modification, showing greater development potential.

[0005] So far, although there have been many attempts to use SIGS technology to control pathogenic fungi, there are very few nucleic acid molecules developed based on the key pathogenic genes of rice blast fungus, and no products have been reported, which in turn limits the application of SIGS technology in the control of rice blast fungus. Summary of the invention

[0006] In view of the above problems, the present invention uses genes related to melanin synthesis of rice blast fungus MGG_08523 ( MOHS1 ) was used as the target to design siRNA, and the obtained siRNA was analyzed for its antibacterial effect, and then applied to the leaves of rice and barley in the form of spray to inhibit the pathogenicity of rice blast fungus, thereby achieving the purpose of preventing and controlling rice blast disease.

[0007] To achieve the above object, the present invention adopts the following scheme:

[0008] In one aspect, the present invention provides a siRNA for controlling rice blast fungus, wherein the siRNA targets and silences rice blast fungus. MOHS1 Gene, the MOHS1 The gene has a nucleotide sequence as shown in SEQ ID NO: 1. siRNA targeting key genes for growth and development of rice blast fungus was screened, and it was found that the targeted silencing gene MOHS1 The siMohs-10 gene could significantly reduce the proportion of appressorium formation of rice blast fungus.

[0009] Further, the siRNA has any one or more of SEQ ID NO: 38-39, SEQ ID NO: 40-41, SEQ ID NO: 42-43, SEQ ID NO: 44-45, SEQ ID NO: 46-47, SEQ ID NO: 48-49, SEQID NO: 50-51, SEQ ID NO: 52-53, SEQ ID NO: 54-55, SEQ ID NO: 16-17, SEQ ID NO: 56-57, SEQ ID NO: 58-59, SEQ ID NO: 60-61, SEQ ID NO: 62-63, SEQ ID NO: 64-65, SEQID NO: 66-67 and SEQ ID NO: 68-69 shown in the nucleotide sequence table.

[0010] Preferably, the siRNA has any one or more of SEQ ID NOs: 48-49, SEQ ID NOs: 50-51, SEQ ID NOs: 16-17, SEQ ID NOs: 58-59 and SEQ ID NOs: 68-69 shown in the nucleotide sequence table.

[0011] Preferably, the siRNA has SEQ ID NO: 16-17 shown in the nucleotide sequence table.

[0012] After siMohs molecules were incubated with rice blast fungus for 4 h, the formation of rice blast fungus appressorium was inhibited and the morphology was smaller when siMohs-7 and siMohs-10 molecules were applied compared with siMohs-6, siMohs-12 and siMohs-17; the inhibitory effect of siMohs-10 was better than that of siMohs-7. In short, siMohs-10 molecules had the most obvious inhibitory effect on rice blast fungus.

[0013] In another aspect, the present invention provides a nucleic acid pesticide for controlling rice blast fungus, wherein the nucleic acid pesticide comprises a nucleic acid for targeting and silencing rice blast fungus. MOHS1 siRNA of the gene.

[0014] Furthermore, the working concentration range and working time of the siRNA were 6.25-25 μM and 8 h, respectively. Rice blast fungus was treated with different concentrations of siMohs-10 to explore the effects of different concentrations of siRNA on the formation of rice blast fungus appressorium. The results showed that when the siRNA concentration was 25 μM and the rice blast fungus was treated for 4 h, the inhibitory effect on the formation of rice blast fungus appressorium was the most obvious, and the inhibitory effect on the growth of rice blast fungus could last for 8 h.

[0015] Furthermore, the siRNA is wrapped by nanomaterials, and after being assembled with the nanomaterials, the inhibitory effect of siMohs-10 on rice blast fungus is prolonged.

[0016] In another aspect, the present invention provides a method for controlling rice blast fungus, the method comprising applying a siRNA solution to leaves of a plant, wherein the siRNA is as described above. In order to further explore the control effect of siMohs-10 on rice blast, siMohs-10 and spores of rice blast fungus were mixed and inoculated on rice leaves. It was found that the lesion area of ​​rice treated with siMohs-10 was smaller than that of the control, indicating that the targeted MOHS1 The siRNA of the gene can weaken the pathogenicity of rice blast fungus, further proving that siMohs-10 can be used to prevent and control rice blast.

[0017] In another aspect, the present invention provides MOHS1 The use of a gene as a target gene for controlling rice blast fungus, the MOHS1 The gene has a nucleotide sequence as shown in SEQ ID NO: 1.

[0018] In another aspect, the present invention provides the use of siRNA for preparing an agent for interfering with the development of appressorium or pathogenicity of rice blast fungus, wherein the siRNA is targeted to silence rice blast fungus. MOHS1 siRNA of the gene.

[0019] 1. This paper investigates the effects of multiple siRNA molecules targeting key genes for growth and development of rice blast fungus on the formation of appressorium of rice blast fungus. MOHS1 siRNA of the gene works best.

[0020] 2. Provides a target gene that plays a role in controlling rice blast fungus MOHS1 The optimal working concentration of the siRNA molecule designed based on this target gene was 25 μM, and it effectively inhibited the formation of appressorium of rice blast fungus within 8 hours. The packaging of nanomaterials can prolong the action time of siRNA.

[0021] 3. The siRNA molecules for resisting rice blast infection provided by the present invention can be prepared into nucleic acid pesticides and sprayed on rice leaves, avoiding the cumbersome process of cultivating transgenic crops and the concerns of consumer groups, thereby providing a new method for preventing and controlling rice blast. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 :Absorption of siRNA in vitro by rice blast fungus (fluorescence signal distribution diagram); scale bar = 20 μm

[0024] Figure 2 :The effects of siRNA targeting different genes of Magnaporthe grisea on Magnaporthe grisea; Scale bar = 20 μm, by t -test is used to analyze the difference of data. P < 0.0001; DDW, siGFP-255 and siMcherry-362 are negative controls

[0025] Figure 3 :Distribution map of different siRNA positions on the target gene sequence

[0026] Figure 4 :Comparison of the inhibitory effects of different siRNA fragments on rice blast fungus; Scale bar = 20 μm

[0027] Figure 5 :Appressorium formation rate of Magnaporthe grisea after treatment with different concentrations of siRNA for different time periods

[0028] Figure 6 :Effects of siRNA encapsulated by nanomaterials on appressorium formation of Magnaporthe grisea

[0029] Figure 7 :Effects of siRNA spraying on the pathogenicity of rice blast fungus DETAILED DESCRIPTION

[0030] 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 materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Although the Mohs in the drawings provided by the present invention and MOHS1 in the specification are inconsistent in wording, they are named by the inventor according to his habit, and the two are essentially the same and have the same meaning.

[0032] Example 1: The rice blast fungus has the ability to absorb siRNA molecules from the external environment

[0033] In order to determine whether the rice blast fungus can take up RNA molecules in the external environment, this example uses fluorescently labeled siRNA to treat the spores of the rice blast fungus (using siGFP-255 as an example, see the sequence table for the specific sequence).

[0034] Preparation of siRNA with fluorescent marker: Entrust Genema Biotechnology to synthesize a siRNA (using siGFP-255 as an example) and add a fluorescent marker to the 5' end of the sense strand. Dissolve the siRNA with fluorescent marker to a final concentration of 20μM and store for later use.

[0035] Collection of spores of rice blast fungus: Wash the culture plate of rice blast fungus (Guy11) grown for 8-10 days with sterile water, filter it into a centrifuge tube, centrifuge at 7000 rpm for 4 min, and discard the supernatant. Use a hemocytometer to adjust the spore concentration to 1×10 5 Pieces / mL.

[0036] Appressorium induction: The hydrophobic membrane was rinsed with sterile water, then wiped with 70% ethanol, and the dried hydrophobic membrane was placed in a moisturizing box. The fluorescently labeled siGFP was mixed with rice blast fungus spores and then spotted on the hydrophobic membrane. They were incubated in vitro for 4 h, 8 h, and 12 h, respectively. After incubation, the surface liquid of the hydrophobic membrane was aspirated and observed under a fluorescence microscope.

[0037] The results are as follows Figure 1 As shown in the figure, after 2 h of incubation of siRNA carrying fluorescent markers with spores in vitro, the fluorescent signal has been enriched on the germ tube of the spores, and the fluorescent signal in the germ tube can reach 40%. From 4 to 8 h, the fluorescent signal in the appressorium continues to increase, but from 8 to 12 h, the fluorescent signal begins to decrease, indicating that siRNA has a certain validity period. The results show that the spores of rice blast fungus can take up siRNA from the time they germinate from the germ tube. In addition, the fluorescent signal is mainly concentrated on the surface of the germ tube and early appressorium, with a small amount of fluorescence enrichment inside the appressorium, but no fluorescence enrichment in the spores.

[0038] Example 2: Screening of siRNA targeting key genes of rice blast fungus

[0039] Design and synthesis of siRNA: Based on the results of the above examples, this example uses the key genes that regulate the growth and development of rice blast fungus as target genes, and designs siRNAs with reference to the method of designing siRNAs on the website (http: / / siDirect2.rnai.jp). Combined with bioinformatics and gene data analysis, the possibility of targeting the rice genome and other genes of rice blast fungus is reduced, and siRNAs (siChs1-1, siChs1-3, siChs3-1, siChs3-2, siChs3-3, siChs3-4, siChs3-5, siChs3-6, siChs3-7, siChs3-8, siChs3-9, siChs3-10, siChs3-11, siChs3-12, siChs3-13, siChs3-14, siChs3-15, siChs3-16, siChs3-17, siChs3-18, siChs3-29, siChs3-30, siChs3-31, siChs3-32, siChs3-33, siChs3-34, siChs3-35 iChs3-2, siChs7-1959, siMohs-10, siBuf1-156, siBuf1-624, siMGG_04732-488, siMGG_04732-1172, siMGG_04876-381, siMGG_04876-480, siHtfg-1318, siRgs4-989, siRgs7-795, siRgs7-1595, see the sequence table for specific sequences). The siRNA double-stranded molecule of the present invention was directly synthesized by a biological company (Shanghai Jierui), and the synthesized product was dissolved in water to a final concentration of 10 μM and stored in aliquots.

[0040] The collection of rice blast fungus spores is as described in Example 1.

[0041] Co-incubation of siRNA and rice blast fungus: The hydrophobic membrane that induced the formation of appressorium was cleaned in advance, then wiped with 70% ethanol, and the wiped hydrophobic membrane was placed in a moisturizing box. siRNA was mixed with the spore solution of rice blast fungus and then spotted on the center of the treated hydrophobic membrane. The final concentration of siRNA was 5 μM. After incubation for 4 h, the formation of appressorium was observed and recorded.

[0042] The results are as follows Figure 2 As shown, compared with negative controls (DDW, siGFP-255, and siMcherry-362) and siRNAs targeting other genes, siRNAs targeting P. oryzae MOHS1 The siRNA of the gene (siMohs-10) can significantly reduce the proportion of rice blast fungus appressorium formation, and the rate of rice blast fungus appressorium formation decreased by about 25%. At the same time, the spore morphology of rice blast fungus was observed 4 hours after treatment. Compared with the control, the germ tube of rice blast fungus treated with siMohs-10 was elongated, the appressorium morphology was smaller, and the melanin layer of the appressorium was lighter. The results of this example show that the exogenously added targeted rice blast fungus MOHS1 The siMohs-10 gene can inhibit the morphological construction of appressorium of rice blast fungus.

[0043] Example 3: Targeting MOHS1 siRNA design for genes

[0044] According to the results of the above implementation 2, it is shown that MOHS1 Gene is a target gene for effective control of rice blast fungus. Therefore, in order to select targeted silencing MOHS1 The siRNA with the best gene effect is targeted at MOHS1 siRNA was designed for the target silencing region present in the full length of the gene (the specific sequence is shown in the sequence table, the position is shown in Figure 3 As shown), a total of 17 pairs of siRNAs were obtained (including the siRNA siMohs-10 involved in Example 2). The design principles of siRNAs are as described in Example 2.

[0045] Example 4: Targeting MOHS1 Comparison of the inhibitory effects of siRNAs with different genes on rice blast fungus

[0046] Four siRNAs designed in Example 3 (siMohs-6, siMohs-7, siMohs-12 and siMohs-17) were randomly selected to compare their antibacterial effects with the siRNA siMohs-10 in Example 2. The effects on the rice blast fungus were observed and recorded for 4 hours. Figure 4 As shown. At 4 h, compared with other siRNAs, the formation of rice blast fungus appressorium was inhibited by applying siMohs-7 and siMohs-10 molecules, and the appressorium morphology was smaller; however, the inhibitory effect of siMohs-10 was better than that of siMohs-7. The results of this example show that siMohs-10 is a target MOHS1 The siRNA molecule with the best gene effect.

[0047] Example 5: Effects of different concentrations of siRNA on appressorium formation of rice blast fungus.

[0048] Combined with the results of Example 3 and Example 4, this example selected siMohs-6, siMohs-7, siMohs-10, siMohs-12 and siMohs-17 as research objects to explore the effects of different concentrations of siRNA on the formation of rice blast appressorium. The dissolution and storage methods of siRNA molecules are the same as those of Example 1.

[0049] In this example, siRNA was first prepared into mother solutions with initial concentrations of 50 μM, 25 μM, and 12.5 μM, and then mixed with the adjusted concentration of rice blast fungus spore solution in Example 3 to form siRNA with final concentrations of 25 μM, 12.5 μM, and 6.25 μM. The siRNA was then dotted onto the center of the hydrophobic membrane and incubated for 4 h, 8 h, and 24 h, respectively, to observe and record the formation of appressorium. The results are shown in FIG. Figure 5 As shown. The formation rate of appressorium of rice blast fungus co-incubated with siMohs-10 for 4 h was significantly lower than that of the control, and as the concentration of siMohs-10 increased, the formation rate of appressorium decreased, that is, the treatment with 25 μM siMohs-10 had the best effect; after siMohs-10 and rice blast fungus were co-incubated for 8 h, siRNA could still effectively inhibit the formation of appressorium of rice blast fungus compared with the control, but the inhibitory effect of siRNA of different concentrations was relatively consistent. By 24 h, appressorium of rice blast fungus gradually formed, and the inhibitory effect of siMohs-10 gradually weakened. The results of this example show that the inhibitory effect of 25 μM siMohs-10 treatment for 4 h on the formation of appressorium of rice blast fungus is the most obvious; the inhibitory effect of siMohs-10 on the growth of rice blast fungus can last for 8 h. The above experiment was also performed on siMohs-6, siMohs-7, siMohs-12 and siMohs-17, but the results were not as good as siMohs-10.

[0050] Example 6: Nanomaterials can prolong the duration of siRNA's inhibitory effect on rice blast fungus

[0051] Combined with the results of Example 5 above, this example uses nanomaterials (liposomes) to encapsulate and prolong the inhibitory effect of five siMohs molecules on the growth of rice blast fungus. First, the siRNA material is mixed with the nanomaterial, and then the mixture is diluted to 1 mL for storage. At this time, the concentration of the siRNA mother solution is 5 μM; the siRNA encapsulated by the nanomaterial is co-incubated with the rice blast fungus spores for 4 h and 8 h, and the formation of appressorium is observed and recorded. The results are shown in Figure 2. Figure 6As shown in the figure, compared with the control, the formation of appressorium was significantly inhibited after the nanomaterial-encapsulated siMohs-10 was treated with rice blast fungus for 4 h and 8 h; the germ tube of rice blast fungus after 8 h treatment was elongated, the appressorium was small in morphology, and the melanin layer was not formed normally. The experimental results show that nanomaterials can prolong the action time of siMohs-10. The above experiments were also carried out on siMohs-6, siMohs-7, siMohs-12 and siMohs-17, and similar conclusions were obtained.

[0052] Example 7: Control effect of siRNA spray on rice blast

[0053] In this example, the effect of siMohs on rice blast fungus was further explored by spray inoculation.

[0054] Planting of rice: soak and germinate the rice seeds, select rice seeds with consistent growth status, sow 22-25 seeds in each pot, wait for the rice to grow to the stage of three leaves and one heart, use a fresh-keeping bag to cover the rice plants to keep moisture and prevent liquid from overflowing during spraying.

[0055] Collection and acquisition of rice blast fungus spore liquid: Select a rice blast fungus (Guy11) plate grown for 10 days, use sterile water and a disposable coating stick to scrape the surface mycelium, filter the obtained liquid, and the filtrate is the spore liquid. Then use a hemocytometer to adjust the spore liquid concentration to 2×10 5 Then, 0.3% gelatin solution was used to dilute it to 1×10 5 Pieces / mL.

[0056] Rice leaf pathogenicity experiment: In this example, siRNA molecules were mixed with rice blast fungus spores for spray inoculation. siRNA and rice blast fungus spores were mixed in equal volumes. The final concentration of siRNA was 5 μM. Each pot of rice was sprayed with 2 mL of siRNA and spore mixture. Then, the rice was placed in an incubator and cultured in the dark at 22°C for 2 days, and then at 25°C with light:dark = 10 h:12 h. The disease condition of rice leaves was observed, and leaves were cut to count the disease results.

[0057] The results are as follows Figure 7 As shown in the figure, compared with the control, the size of the lesions on the rice leaves was significantly reduced after being treated with siMohs-10, indicating that the pathogenicity of the spores of the rice blast fungus on the rice leaves was significantly reduced; siMohs-6, siMohs-7, siMohs-12 and siMohs-17 were also subjected to the above experiment, but the antibacterial effect of siMohs-10 was the best. The results of this example show that targeting the key gene of the rice blast fungus MOHS1The siMohs-10 can control the rice blast fungus. The results provide a certain theoretical and experimental basis for SIGS to control the rice blast fungus.

[0058] 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.

Claims

1. A siRNA for controlling rice blast fungus, characterized in that: The siRNA targeted silencing of Magnaporthe oryzae MOHS1 Gene, MOHS1 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO: 1, and the nucleotide sequence of the siRNA is shown in the sequence listing SEQ ID NO: 16-17.

2. Use of siRNA for preparing an agent that interferes with the development of appressorium or pathogenicity of rice blast fungus, characterized in that: The siRNA is targeted to silence rice blast fungus MOHS1 The specific sequence of the siRNA of the gene is shown in SEQ ID NO: 16-17 in the sequence table.

3. A nucleic acid pesticide for controlling rice blast fungus, characterized in that: Comprising the siRNA as described in claim 1.

4. The nucleic acid pesticide according to claim 3, characterized in that The working concentration range and working time of the siRNA are 6.25-25 μM and 8 h, respectively.

5. The nucleic acid pesticide according to claim 3, characterized in that The siRNA is encapsulated by nanomaterials.

6. A method for preventing and controlling rice blast fungus, characterized in that: The solution containing the siRNA according to any one of claims 1 to 2 or the nucleic acid pesticide according to any one of claims 3 to 5 is sprayed on rice plants or leaves.

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