Bio-control fungus trichoderma source miRNA and dsRNA thereof, and application of miRNA and dsRNA in prevention and treatment of plant southern blight

Abstract

The invention belongs to the technical field of plant protection, and discloses miRNA of a biocontrol fungus trichoderma source, dsRNA of the miRNA and application of the dsRNA to prevention and treatment of plant southern blight. The sequence of the miRNA of the biocontrol bacterium trichoderma source is AAACUCUGGUGGAGGCUC, and the sequence of the miRNA of the biocontrol bacterium trichoderma source is shown in the specification. The miRNA sequence (Tri-MIR5139) disclosed by the invention can be used for effectively preventing and treating diseases caused by sclerotium rolfsii to peanuts.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protection, and more specifically, relates to a miRNA and dsRNA derived from a biocontrol fungus Trichoderma and their application in controlling plant southern blight. Background Art

[0002] Peanut (Arachis hypogaea L.) is an important oil crop and cash crop in China, with a planting area exceeding 4.6392 million hectares and a yield of over 17 million tons, accounting for half of the total output of oil crops in the country. However, peanut southern blight caused by Sclerotium rolfsii Sacc. (abbreviated as S. rolfsii, also known as the southern blight pathogen) is one of the most serious diseases during the whole growth period and after harvest of peanuts, greatly restricting the healthy development of the peanut industry. The host range of S. rolfsii is extensive, mainly harming vegetables and fruits such as legumes, solanaceous plants, and cucurbitaceae, causing serious economic losses. When peanuts are infected by S. rolfsii in hot and humid weather, it will cause the base of the stem to rot, and its main pathogenic factors include oxalic acid, cell wall degrading enzymes, secreted proteins, etc. Due to the lack of effective disease-resistant varieties, the current control of peanut southern blight mainly relies on chemical control. However, the use of chemical pesticides is not obvious in the control of peanut southern blight, and it also brings problems such as environmental pollution and food safety due to pesticide residues. Therefore, there is an urgent need to explore "efficient, green, healthy, and sustainable" control measures.

[0003] Trichoderma spp. is an important biocontrol microorganism, which has been developed into biological inoculants and biological pesticides and widely used in agricultural production. Trichoderma can control a variety of plant diseases, especially soil-borne diseases, such as fusarium wilt, sheath blight, root rot, damping-off, phytophthora blight, and southern blight, etc., with good control effects. At present, most of the research on Trichoderma in controlling plant diseases focuses on field applications, and the in-depth mechanism of its biological control still needs further exploration.

[0004] MicroRNA (miRNA) is a class of short non-coding RNAs and an important regulatory factor for post-transcriptional regulation of gene expression. RNA fungicides developed based on the characteristics of miRNA for controlling plant diseases have attracted extensive attention, that is, RNA interference (RNAi) technology is increasingly widely used in the control of crop diseases. Spray-induced gene silencing (SIGS) technology controls the expression of endogenous genes in plants or target genes of pathogens by exogenous application of dsRNA or siRNA, greatly improving the practicability of RNAi. CN117947020A discloses a method for producing dsRNA from Escherichia coli to inhibit Botrytis cinerea. Based on the Botrytis cinerea BcSAS1 gene, the obtained dsRNA can effectively reduce the infection ability of Botrytis cinerea, thereby preventing and controlling gray mold of plants and fruits. CN119162005A discloses a method for using an engineered strain Bb-dsMucin to produce dsRNA to interfere with the expression of the target gene BcMucin of Botrytis cinerea, thereby effectively preventing and controlling gray mold of tomatoes. There is no prior art technical solution in which miRNA from the biocontrol fungus Trichoderma cross-species targets and regulates the genes of Sclerotium rolfsii, and the corresponding miRNA is applied to control diseases. Summary of the Invention

[0005] The object of the present invention is to address the deficiencies of the prior art and propose a miRNA from the biocontrol fungus Trichoderma, its dsRNA, and their application in controlling Sclerotium rolfsii of plants. The miRNA sequence (Tri-MIR5139) of the present invention can effectively control the diseases caused by Sclerotium rolfsii on peanuts.

[0006] To achieve the above object, in the first aspect of the present invention, a miRNA from the biocontrol fungus Trichoderma is provided, and the sequence of the miRNA is AAACUCUGGUGGAGGCUC (SEQ ID NO: 1, 5'-3').

[0007] The miRNA of the present invention is derived from Trichoderma asperellum xy021, and its identification results are as follows:

[0008] Trichoderma xy021 was inoculated on PDA, SNA, and CMD media respectively and cultured at 28 °C for 7 d.

[0009] On the PDA medium, the Trichoderma xy021 strain showed 3 concentric rings, and the green conidial masses were only distributed within the rings, and the white mycelia covered the entire plate ( Figure 5 A, D, G).

[0010] On the SNA medium, the mycelia and conidial masses were sparsely distributed at the edge of the plate, and no concentric circles were observed. ( Figure 5 B, E).

[0011] On CMD medium, no concentric circles of colonies were observed. The green sporodochia were evenly distributed on the medium, and a small amount of white mycelium adhered to the sporodochia( Figure 5 C, F).

[0012] Microscopic observation revealed that conidia were clustered at the tips of the hyphae( Figure 5 H, J), the phialidic conidia were asymmetrically distributed, and the rounded conidia were located at the tips of the conidia( Figure 5 I, K). The rounded chlamydospores were scattered in the medium( Figure 5 L).

[0013] For molecular identification, the TEF1 fragment (1052 bp) was obtained by PCR amplification. The TEF1 sequence was cloned using the Ti-18 vector. The TEF1 sequence was submitted to NCBI. The alignment results showed that the TEF1 of Trichoderma xy021 was identical to that of Trichoderma asperellum. The strain with the highest homology was selected for phylogenetic tree construction. Using MEGA11 based on the TEF1 sequence, a phylogenetic tree was generated using the maximum likelihood technique. According to the morphological and molecular identification results, Trichoderma xy021 was identified as Trichoderma asperellum( Figure 5 M).

[0014] The second aspect of the present invention provides a dsRNA, one sequence in the dsRNA is the same as the miRNA sequence, and the other sequence is complementary to the miRNA sequence.

[0015] The third aspect of the present invention provides the application of the miRNA derived from the biocontrol fungus Trichoderma or the dsRNA in the control of plant southern blight.

[0016] According to the present invention, preferably, the target gene of the miRNA is the β-1,3-glucan synthase gene of southern blight fungus.

[0017] According to the present invention, preferably, the plant is at least one of leguminous plants, solanaceous plants and cucurbitaceous plants.

[0018] According to the present invention, preferably, the leguminous plant is peanut.

[0019] According to the present invention, preferably, the control method includes:

[0020] Obtaining a dry powder of dsRNA with the same sequence as the miRNA derived from the biocontrol fungus Trichoderma;

[0021] Mixing the dsRNA dry powder with water to obtain a control solution;

[0022] Spraying the control solution on the surface of the plant plant or fruit to achieve control.

[0023] According to the present invention, preferably, the concentration of dsRNA in the control solution is 10-100 ng / μL.

[0024] The beneficial effects of the technical solution of the present invention are as follows:

[0025] The miRNA sequence of the present invention specifically targets the β-1,3-glucan synthase (SrFKS1, g.20237) gene of Sclerotium rolfsii (abbreviated as SrFKS1 gene), and inhibits the growth of Sclerotium rolfsii by destroying the cell wall of Sclerotium rolfsii. Experiments show that applying this miRNA to peanuts can effectively prevent and control the occurrence and development of Sclerotium rolfsii, thereby effectively preventing and controlling the diseases caused by Sclerotium rolfsii to peanuts.

[0026] Other features and advantages of the present invention will be described in detail in the following specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] By describing the exemplary embodiments of the present invention in more detail in conjunction with the drawings, the above and other objects, features, and advantages of the present invention will become more obvious.

[0028] Figure 1A -C shows the sequence analysis of Tri-MIR5139 and the target gene SrFKS1 provided in Example 1 of the present invention (where: Figure 1A : The secondary folding structure of Tri-MIR5139; Figure 1B : The target gene prediction result of Tri-MIR5139; Figure 1C : The gene structure and evolutionary analysis of SrFKS1).

[0029] Figure 2A -C shows the verification of the interaction relationship between Tri-MIR5139 and the target gene SrFKS1 provided in Example 2 of the present invention ( Figure 2A : GFP fluorescence verifies the targeted regulation of Tri-MIR5139 on the target gene SrFKS1; Figure 2B : qPCR detects the GFP expression level in the co-expressed tobacco leaves of 35S::Tri-MIR5139 (i.e., pBin-Pre-Tri-MIR5139) and 35S::FKS1-GFP (i.e., pBin-SrFKS1-GFP); Figure 2C : Quantitative PCR analyzes the expression levels of Tri-MIR5139 and SrFKS1).

[0030] Figure 3A -B shows dsTri-MIR5139 provided in Example 3 of the present invention FAMSituation of sclerotium and mycelium of Sclerotium rolfsii( Figure 3A : Flow chart of FAM fluorescence observation; Figure 3B : FAM fluorescence observation of dsTri-MIR5139 FAM Result diagram of sclerotium and mycelium of Sclerotium rolfsii).

[0031] Figure 4A -B shows the effect of dsTri-MIR5139 provided in Example 4 of the present invention in controlling southern blight of peanut( Figure 4A : Growth and expansion of Sclerotium rolfsii on peanut fruits; Figure 4B : Column chart of incidence area of peanut fruit).

[0032] Figure 5 Shows the morphological and molecular biological identification of Trichoderma asperellum xyo21. Specific embodiments

[0033] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0034] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0035] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources unless otherwise specified.

[0036] Example 1 Sequence analysis of Tri-MIR5139 and its target gene

[0037] Using the online software RNAfold to analyze the precursor sequence of Tri-MIR5139, it is found that the precursor structure of Tri-MIR5139 conforms to the configuration of the stem-loop structure, and the mature body of Tri-MIR5139 is located on the stem of the stem-loop structure, as Figure 1A shown.

[0038] Further using psRNATarget to online predict the target gene of Tri-MIR5139 in the genome of Sclerotium rolfsii, and the result shows that Tri-MIR5139 may target and regulate the β-1,3-glucan synthase (SrFKS1) of Sclerotium rolfsii, as Figure 1B shown.

[0039] AsFigure 1C As shown in Figure 1C , the FKS1 gene structure analysis indicated that the FKS1 of Sclerotium rolfsii was similar to the domains of other species. Phylogenetic tree analysis showed that the FKS1 of S. rolfsii and P. crispa clustered on the same branch, suggesting that the target gene FKS1 of Tri-MIR5139 played an important role in the cell wall synthesis of Sclerotium rolfsii.

[0040] Example 2 Tri-MIR5139 Targetedly Regulates the SrFKS1 Gene

[0041] The precursor of Tri-MIR5139 was cloned into the pBin-GFP vector using BamH I and Hind III restriction endonucleases to obtain the recombinant plasmid pBin-Pre-Tri-MIR5139;

[0042] The target gene SrFKS1 was cloned into the pBin-GFP vector using Kpn I and BamH I restriction endonucleases, namely obtaining the recombinant plasmid pBin-SrFKS1-GFP;

[0043] The above recombinant plasmid pBin-Pre-Tri-MIR5139 and recombinant plasmid pBI121-SrFKS1-GFP were respectively introduced into Agrobacterium tumefaciens GV3101 to obtain Agrobacterium suspensions.

[0044] Numbers were marked on the tobacco leaves. Using a 1 mL syringe, the Agrobacterium suspensions were injected into the tobacco leaves from the back of the leaves under pressure for co-expression in tobacco. Then the leaves were sprayed with water, covered with a fresh-keeping bag, and placed in the dark overnight.

[0045] The next day, the fresh-keeping bag was opened. Samples were taken 2 days after injection, and GFP fluorescence observation and photography were carried out using a fluorescence microscope.

[0046] Four groups were set up in the experiment, namely:

[0047] OD 600 = 0 of pBin-Pre-Tri-MIR5139 and OD 600 = 0.1 of pBin-SrFKS1-GFP,

[0048] OD 600 = 0.3 of pBin-Pre-Tri-MIR5139 and OD 600 = 0.1 of pBin-SrFKS1-GFP,

[0049] OD 600 = 0.6 of pBin-Pre-Tri-MIR5139 and OD 600 = 0.1 of pBin-SrFKS1-GFP,

[0050] OD 600 pBin-Pre-Tri-MIR5139 with OD = 0.9 and OD 600 pBin-SrFKS1-GFP with OD = 0.1.

[0051] According to the fluorescence intensity analysis of the GFP reporter gene, the effect of Tri-MIR5139 on the expression of the target gene SrFKS1 was analyzed to judge the targeting regulatory relationship between the two.

[0052] The results are as follows:

[0053] As Figure 2A shown, the tobacco co-expression system was used to verify the targeting regulatory relationship of Tri-MIR5139 to the target gene SrFKS1. It was found that as the concentration of Tri-MIR5139 in the bacterial liquid increased, the fluorescence intensity of the leaves decreased (wherein, on Figure 2A and 2B , 35S::Tri-MIR5139 is pBin-Pre-Tri-MIR5139, and 35S::FKS1-GFP is pBin-SrFKS1-GFP);

[0054] As Figure 2B shown, after collecting tobacco leaves under different treatment conditions, RNA was extracted, and the expression of the GFP gene was analyzed by qPCR. It was found that the expression of the GFP gene was significantly inhibited, and as the concentration of Tri-MIR5139 in the bacterial liquid increased, the inhibitory effect was greater.

[0055] As Figure 2C shown, qPCR analysis found that there was a negative regulatory relationship between the expression levels of Tri-MIR5139 and the target gene SrFKS1 during the antagonism of Trichoderma asperellum xy021 against Sclerotium rolfsii.

[0056] Example 3 Tri-MIR5139 is absorbed by Sclerotium rolfsii

[0057] As Figure 3A shown, the mature sequence of Tri-MIR5139 was synthesized into dsTri-MIR5139 with a FAM fluorophore by RNA synthesis technology FAM (FAM modification at the 3' end). By adding 5 μL of in vitro synthesized dsTri-MIR5139 FAM (100 ng / μL) and co-culturing with the sclerotia (Sclerotium) and mycelia (Mycelium) of Sclerotium rolfsii, green fluorescence was observed in the sclerotia and mycelia of Sclerotium rolfsii, indicating that Tri-MIR5139 can enter Sclerotium rolfsii ( Figure 3B ).

[0058] Example 4 Role of Tri-MIR5139 in Controlling Southern Blight of Peanut

[0059] Fresh and uniformly sized peanut fruits (Huayu 20 variety from Shandong Peanut Research Institute) were collected. A 9-mm mycelial disc of Sclerotium rolfsii was inoculated on the surface of each peanut fruit. Subsequently, 500 μL of in vitro synthesized dsTri-MIR5139 (50 ng / μL) was evenly sprayed on one of the peanut fruits, and the fruits were kept moist for 24 h. The incidence of southern blight was investigated on the 3rd day after inoculation. The results are as Figure 4A and 4B shown.

[0060] As can be seen from Figure 4A and 4B , dsTri-MIR5139 had a significant inhibitory effect on the growth and spread of Sclerotium rolfsii on the surface of peanut fruits. Compared with the control group (S. rolfsii) without dsTri-MIR5139 application, the control effect reached 57.13%. These results demonstrated the effect of in vitro synthesized dsTri-MIR5139 in controlling southern blight of peanut. In addition, Figure 4A the CK group in

[0061] showed that the mycelia on the peanut fruits were caused by the externally inoculated mycelial discs, rather than the fungi carried by the peanuts themselves. The above has described the embodiments of the present invention. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A miRNA derived from the biocontrol fungus Trichoderma, characterized in that The sequence of the miRNA is AAACUCUGGUGGAGGCUC.

2. A dsRNA, characterized in that One sequence in the dsRNA is identical to the miRNA sequence described in claim 1, and the other sequence is complementary to the miRNA sequence described in claim 1.

3. Use of the miRNA derived from the biocontrol fungus Trichoderma according to claim 1 or the dsRNA according to claim 2 in controlling plant southern blight.

4. The use according to claim 3, wherein: The target gene of the miRNA is the β-1,3-glucan synthase gene of Sclerotium rolfsii.

5. The use according to claim 3, wherein: The plant is at least one of a leguminous plant, a solanaceous plant and a cucurbitaceous plant.

6. The use according to claim 5, wherein: The legume plant is peanut.

7. The use according to claim 3, wherein: Methods of prevention and treatment include: Obtaining dsRNA dry powder having the same sequence as the miRNA from the biocontrol fungus Trichoderma; Mixing the dsRNA dry powder with water to obtain a control solution; The control liquid is sprayed on the surface of the plant or fruit to achieve control.

8. The use according to claim 7, wherein: The concentration of dsRNA in the control solution is 10-100 ng / μL.

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