Two small rna compositions for improving the effect of termite biological control and preparation method and application thereof

Abstract

The application discloses two kinds of small RNA compositions for improving biological control effect of termites, and microRNA-like RNA (milRNA) sequences are milR-32 and milR-48 respectively. Double-stranded RNA can enhance the effect of the beetle green fungus on killing termites. The milR-32 and the milR-48 belong to the milRNA coded by the green fungus, and the gene sequences are derived from the transcriptome database of the beetle green fungus. The milR-32 and the milR-48 mimics are designed and synthesized based on the milR-32 and the milR-48 sequences respectively. After the beetle green fungus is used to infect the termites taking the milR-32 or the milR-48 mimic, the infection mortality of the termites is significantly increased. The mimic designed based on the milR-32 and the milR-48 sequences can significantly improve the biological control effect of the termites.

Description

Technical Field

[0001] This invention relates to the field of green biological control technology, specifically to a small RNA composition of milR-32 and milR-48 in Metarhizium anisopliae that enhances the biological control effect of termites, and its preparation method and application. Background Technology

[0002] Termites, a highly destructive social pest, are widely distributed in tropical and subtropical regions. They are mainly classified into wood-dwelling, soil-dwelling, and soil-wood-dwelling species. Their primary food source is trees, timber, and wood-based fiber products. Therefore, termites cause irreversible damage to buildings, water conservancy projects, cultural relics, and agriculture and forestry, resulting in significant economic losses. Termite control often relies on chemical methods, but these methods suffer from problems such as the development of pesticide resistance and environmental pollution. Therefore, there is an urgent need to develop non-toxic, pollution-free, and highly effective termite control technologies.

[0003] Metarhizium anisopliae is a relatively mature biological control technology with advantages such as low cost, wide application range, and high safety. Metarhizium anisopliae mainly invades insects through the surface, multiplies rapidly within the insect's body, and releases toxins, leading to insect death. However, termites, as social insects, possess a unique social immune mechanism that reduces fungal infection and degrades fungal toxins, severely diminishing the control effectiveness of Metarhizium anisopliae. RNAi technology is widely used in insect control. Although low concentrations of nucleic acid are unlikely to directly cause insect death, they can still significantly reduce the insect's immune, detoxification, and antioxidant functions. RNAi combined with biocontrol of fungi holds great potential in the field of insect control.

[0004] MicroRNA-like RNAs (milRNAs) are a class of non-coding single-stranded RNA molecules, approximately 20-23 nucleotides in length, encoded by fungal genomes. They bind to the 3' untranslated region (3'UTR) of target mRNAs through base pairing, guiding the RNA-induced silencing complex (RISC) to degrade the target mRNA or inhibit its translation, thereby regulating gene expression at the post-transcriptional level. Previous studies have found that *Metarhizium anisopliae* can synthesize various milRNAs, which play important regulatory roles in fungal virulence, growth, development, and reproduction through post-transcriptional regulation. Furthermore, some *Metarhizium anisopliae* milRNAs, such as milR-32 and milR-48, can cross the cell membrane into insect cells during termite infection, inducing RNAi effects (i.e., cross-border RNAi) by hijacking the insect RNAi pathway, thereby weakening insect disease defenses and increasing infection rates. This suggests that fungal milRNAs have potential applications in the development of insect immunosuppressants.

[0005] Therefore, providing a small RNA composition with two non-coding RNA sequences, milR-32- and milR-48, to enhance the termite-killing effect of Metarhizium anisopliae against termites, and to address the shortcomings of existing technologies that result in poor termite control under natural conditions, is a problem worthy of research. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for termite control using RNAi technology in conjunction with Metarhizium anisopliae. By interfering with relevant genes within termites, this method enhances the lethality of Metarhizium anisopliae against termites. This approach not only enhances the insecticidal effect of Metarhizium anisopliae but also significantly reduces the use of chemical pesticides, decreases termite resistance, and reduces environmental pollution.

[0007] The objective of this invention is achieved as follows: Two small RNA compositions for improving the biocontrol efficacy of termites, wherein the RNA sequences are milR-32- and milR-48, characterized in that the RNA sequences are derived from the Metarhizium anisopliae transcriptome database, the RNA sequence of milR-32 is SEQ ID NO:1, and the RNA sequence of milR-48 is SEQ ID NO:2.

[0008] The preparation methods of the two small RNA compositions for improving the biological control effect of termites include the preparation of milR-32 mimics from the RNA sequence of milR-32 and the preparation of milR-48 mimics from the RNA sequence of milR-48.

[0009] The preparation of milR-32 mimics from the milR-32 RNA sequence includes the following steps: (1) Based on the milR-32 sequence SEQ ID NO:1, design the following analog templates: the sense strand SEQ ID NO:1A and its complementary strand SEQ ID NO:1B; the antisense strand SEQ ID NO:1C and its complementary strand SEQ ID NO:1D; (2) After mixing equal amounts of SEQ ID NO:1A and SEQ ID NO:1B, annealing PCR was performed to obtain the milR-32 sense strand DNA template; after mixing equal amounts of SEQ ID NO:1C and SEQ ID NO:1D, annealing PCR was performed to obtain the milR-32 antisense strand DNA template. (3) Mix equal amounts of milR-32 sense strand DNA template and antisense strand DNA template, and synthesize milR-32 mimics using the T7 in vitro transcription system.

[0010] The preparation of milR-48 mimics from the milR-48 RNA sequence includes the following steps: (1) Based on the milR-48 sequence SEQ ID NO:2, design the following analog templates: the sense strand SEQ ID NO:2A and its complementary strand SEQ ID NO:2B; the antisense strand SEQ ID NO:2C and its complementary strand SEQ ID NO:2D; (2) After mixing equal amounts of SEQ ID NO:2A and SEQ ID NO:2B, annealing PCR was performed to obtain the milR-48 sense strand DNA template; after mixing equal amounts of SEQ ID NO:2C and SEQ ID NO:2D, annealing PCR was performed to obtain the milR-48 antisense strand DNA template. (3) Mix equal amounts of milR-48 sense strand DNA template and antisense strand DNA template, and synthesize milR-48 mimics using the T7 in vitro transcription system.

[0011] The two small RNA compositions described above enhance the effectiveness of termite biological control.

[0012] The template design method for the two small RNA mimics, Metarhizium anisopliae milR-32 and milR-48, is as follows: S1. Referring to the SEQ ID NO.1 sequence of milR-32 of Metarhizium anisopliae, four template strands are involved: Referring to SEQ ID NO.1, an "enhancer" and a "T7 promoter" are added to the 5th end of the sequence, which is the positive strand template SEQ ID NO.1A, and the complementary strand of the positive strand template is SEQ ID NO.1B; Referring to the antisense strand of SEQ ID NO.1, an "enhancer" and a "T7 promoter" are added to the 5th end of the sequence, which is the antisense strand template SEQ ID NO.1C, and the complementary strand of the antisense strand template is SEQ ID NO.1D.

[0013] S2. Design the milR-48 simulant template according to the method in S1 above. The four template chains are SEQ ID NO.2A, SEQ ID NO.2B, SEQ ID NO.2C and SEQ ID NO.2D.

[0014] The synthesis methods for the Metarhizium anisopliae milR-32 and milR-48 mimic templates are as follows: S3. milR-32 mimic template: Equal volumes of SEQ ID NO.1A and SEQ ID NO.1B were mixed, and annealing PCR was performed with 10×Annealing Buffer to obtain the milR-32 positive strand template; equal volumes of SEQ ID NO.2C and SEQ ID NO.2D were mixed, and annealing PCR was performed with 10×Annealing Buffer to obtain the milR-32 antisense strand template. The annealing PCR program was: denaturation at 95℃ for 2 min; temperature decrease rate from 95℃ to 4℃ was 0.1℃ / s, and storage at 4℃ was maintained.

[0015] S4. Referring to the method in S3 above, synthesize the milR-48 positive chain template and negative chain template; The synthesis and preparation methods of the milR-32 and milR-48 mimics of *Metarhizium anisopliae* are as follows: S5. Mix equal amounts of the sense and antisense strand templates of milR-32 and add them to the T7 in vitro transcriptase system. Incubate at 37 °C for 4 h. Add enzyme-free water, 3M sodium acetate solution, water-saturated phenol solution and chloroform solution to the in vitro transcription product in sequence. After mixing, centrifuge and collect the supernatant. Add anhydrous ethanol and incubate at -20 °C overnight. Then centrifuge and collect the supernatant. Wash the precipitate with 75% alcohol and aseptically dry to obtain the milR-32 mimic.

[0016] S6. Referring to the method in S5 above, obtain the milR-48 simulant.

[0017] Positive and Beneficial Effects: This invention utilizes RNAi technology in synergistic with Metarhizium anisopliae for termite control. By interfering with relevant genes within termites, it enhances the lethal effect of Metarhizium anisopliae on termites. This method not only enhances the insecticidal effect of Metarhizium anisopliae but also significantly reduces the use of chemical pesticides, lowers termite resistance, and reduces environmental pollution. Unlike existing RNAi technologies, which target genes derived from the termite transcriptome, this invention uses two immunosuppressive-related small RNAs screened from the transcriptome of Metarhizium anisopliae, indicating a different species origin for the nucleic acid drugs. Control Effects: Feeding termites with milR-32 and milR-48 mimics significantly improved the control effect of Metarhizium anisopliae on termites. Attached Figure Description

[0018] Figure 1 This invention describes the miRNA secreted by Metarhizium anisopliae, detected by transcriptome sequencing. Figure 2 The agarose gel electrophoresis results for the two non-coding RNA mimics in Examples 2 and 4 of this invention are shown in Figure A, which shows the milR-32 mimic; and Figure B, which shows the milR-48 mimic. Figure 3This is a diagram illustrating the lethal effect of the *Metarhizium anisopliae* fungus on termites after they ingest the milR-32 simulant. Figure 4 This is a diagram illustrating the lethal effect of the *Metarhizium anisopliae* fungus on termites after they ingest the milR-48 simulant. Figure 5 This is the gene sequence listing for this invention. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments: Two small RNA compositions for enhancing the biocontrol efficacy of termites, wherein the RNA sequences are milR-32- and milR-48, characterized in that the RNA sequences are derived from the Metarhizium anisopliae transcriptome database, the RNA sequence of milR-32 is SEQ ID NO:1, and the RNA sequence of milR-48 is SEQ ID NO:2, as shown below. Figure 5 As shown.

[0020] The preparation methods of the two small RNA compositions for improving the biological control effect of termites include the preparation of milR-32 mimics from the RNA sequence of milR-32 and the preparation of milR-48 mimics from the RNA sequence of milR-48.

[0021] The template design method for the two small RNA mimics, Metarhizium anisopliae milR-32 and milR-48, is as follows: S1. Referring to the SEQ ID NO.1 sequence of milR-32 of Metarhizium anisopliae, four template strands are involved: Referring to SEQ ID NO.1, an "enhancer" and a "T7 promoter" are added to the 5th end of the sequence, which is the positive strand template SEQ ID NO.1A, and the complementary strand of the positive strand template is SEQ ID NO.1B; Referring to the antisense strand of SEQ ID NO.1, an "enhancer" and a "T7 promoter" are added to the 5th end of the sequence, which is the antisense strand template SEQ ID NO.1C, and the complementary strand of the antisense strand template is SEQ ID NO.1D.

[0022] S2. Design the milR-48 simulant template according to the method in S1 above. The four template chains are SEQ ID NO.2A, SEQ ID NO.2B, SEQ ID NO.2C and SEQ ID NO.2D.

[0023] The synthesis methods for the Metarhizium anisopliae milR-32 and milR-48 mimic templates are as follows: S3. milR-32 mimic template: Equal volumes of SEQ ID NO.1A and SEQ ID NO.1B were mixed, and annealing PCR was performed with 10×Annealing Buffer to obtain the milR-32 positive strand template; equal volumes of SEQ ID NO.2C and SEQ ID NO.2D were mixed, and annealing PCR was performed with 10×Annealing Buffer to obtain the milR-32 antisense strand template. The annealing PCR program was: denaturation at 95℃ for 2 min; temperature decrease rate from 95℃ to 4℃ was 0.1℃ / s, and storage at 4℃ was maintained.

[0024] S4. Referring to the method in S3 above, synthesize the milR-48 positive chain template and negative chain template; The synthesis and preparation methods of the milR-32 and milR-48 mimics of *Metarhizium anisopliae* are as follows: S5. Equal volumes of the sense and antisense strand templates of milR-32 were mixed and added to the T7 in vitro transcriptase system, and incubated at 37 °C for 4 h. Enzyme-free water, 3M sodium acetate solution, water-saturated phenol solution, and chloroform solution were added sequentially to the in vitro transcription product. After mixing, the supernatant was collected by centrifugation, and anhydrous ethanol was added and incubated overnight at -20 °C. The supernatant was then collected by centrifugation, and the precipitate was washed with 75% ethanol and aseptically dried to obtain the milR-32 mimic.

[0025] S6. Referring to the method in S5 above, obtain the milR-48 simulant.

[0026] Example 1: Screening process for milR-32 and milR-48 Metarhizium anisopliae was cultured in potato dextrose agar (PDA) for 2-3 weeks at 25°C. The medium was then washed with 0.1% Tween 80, and a spore suspension was collected. This suspension was then added to animal-derived SDY medium for further culture. A suitable amount of the culture was used for transcriptome sequencing. The sequencing results are shown below. Figure 1 As shown, a total of 50 miRNAs secreted by Metarhizium anisopliae were obtained, and two miRNAs, milR-32 and milR-48, were screened out from them.

[0027] Example 2: Preparation process of milR-32 simulant Based on the sequence SEQ ID NO: 1 of milR-32 secreted by *Metarhizium anisopliae*, a mimic template was designed, consisting of: SEQ ID NO: 1A of the sense strand and its complementary strand SEQ ID NO: 1B; and SEQ ID NO: 1C of the antisense strand and its complementary strand SEQ ID NO: 1D. Equal volumes of SEQ ID NO: 1A (2 μL) and SEQ ID NO: 1B (2 μL) were mixed and annealed by PCR to obtain the milR-32 sense strand DNA template. Equal volumes of SEQ ID NO: 1C (2 μL) and SEQ ID NO: 1D (2 μL) were mixed and annealed by PCR to obtain the milR-32 antisense strand DNA template. Subsequently, 2.5 μL of the above milR-32 sense strand DNA template was mixed with 2.5 μL of the milR-32 antisense strand DNA template, and the milR-32 mimic was synthesized using a 20 μL T7 in vitro transcription system. The original solution was diluted and detected by gel electrophoresis. Figure 2 A).

[0028] Example 3: Effect of milR-32 simulant on mortality rate of termites infected with fungi 50 μg of milR-32 simulant was dissolved in 60 μL of enzyme-free water, then dropped onto a 1.8 cm diameter circular filter paper disc, thoroughly wetting it. This disc was then placed in a 3.5 cm diameter circular petri dish as food, and 12 *Reticulitermes lanceolatum* were reared as the treatment group (3 replicates per treatment group, totaling 36 termites). 50 μg... GFP siRNA was dissolved in 60 μL of enzyme-free water and then dropped onto a 1.8 cm diameter circular filter paper disc, thoroughly wetting it. This disc was then placed in a 3.5 cm diameter circular petri dish as food for 12 *Reticulitermes lanceolatum* termites (3 replicates, total 36 termites). Two days after feeding, the termites were treated with a suspension of *Metarhizium anisopliae* spores (concentration 10...). 6 Soak the fed termites with a concentration of spores per ml, then quickly blot off excess moisture with filter paper and return them to a 3.5 cm petri dish for 10 days of further culture. Record the number of dead termites in both the treatment and control groups daily for a total of 10 days. Figure 3 As shown, the mortality rate of termites infected with the fungus was significantly higher in the treatment group (χ²). 2 =12.069, P<0.01 (Kaplan–Meier method), indicating that the milR-32 mimic can enhance the ability of biocontrol bacteria to kill termites.

[0029] Example 4: Preparation process of milR-48 simulant Based on the sequence SEQ ID NO: 2 of milR-48 secreted in vitro by *Metarhizium anisopliae*, a mimic template was designed, consisting of: SEQ ID NO: 2A of the positive strand and its complementary strand SEQ ID NO: 2B; and SEQ ID NO: 2C of the negative strand and its complementary strand SEQ ID NO: 2D. Equal volumes of SEQ ID NO: 2A and SEQ ID NO: 2B (100 μM) were mixed and annealed by PCR to obtain the milR-32 positive strand DNA template. Equal volumes of SEQ ID NO: 2C and SEQ ID NO: 2D (100 μM) were mixed and annealed by PCR to obtain the milR-48 negative strand DNA template. Subsequently, 2.5 μL of the above milR-48 positive strand DNA template was mixed with 2.5 μL of milR-48 negative strand DNA template, and the milR-48 mimic was synthesized using a 20 μL T7 in vitro transcription system. The original solution was diluted and detected by gel electrophoresis. Figure 2 B).

[0030] Example 5: Effect of milR-48 simulant on mortality rate of termites infected with fungi. 50 μg of milR-48 simulant was dissolved in 60 μL of enzyme-free water, then dropped onto a 1.8 cm diameter circular filter paper disc, thoroughly wetting it. This disc was then placed in a 3.5 cm diameter circular petri dish as food, and 12 *Reticulitermes lanceolatum* were reared as the treatment group (3 replicates per treatment group, totaling 36 termites). 50 μg... GFP siRNA was dissolved in 60 μL of enzyme-free water and then dropped onto a 1.8 cm diameter circular filter paper disc, thoroughly wetting it. This disc was then placed in a 3.5 cm diameter circular petri dish as food for 12 *Reticulitermes lanceolatum* termites (3 replicates, total 36 termites). Two days after feeding, the termites were treated with a suspension of *Metarhizium anisopliae* spores (concentration 10...). 6 Soak the fed termites with a concentration of spores per ml, then quickly blot off excess moisture with filter paper and return them to a 3.5 cm petri dish for 10 days of further culture. Record the number of dead termites in both the treatment and control groups daily for a total of 10 days. Figure 4 As shown, the mortality rate of termites infected with the fungus was significantly increased in the treatment group (χ²). 2 =6.988, P<0.01 (Kaplan–Meier method), indicating that the milR-48 mimic can significantly improve the ability of biocontrol bacteria to kill termites.

[0031] Therefore, the milR-32 and milR-48 mimics synthesized from the sequences of the two non-coding RNAs milR-32 and milR-48 provided in this invention enhance the lethality of Metarhizium anisopliae against termites, resulting in a significant increase in the lethality of Metarhizium anisopliae against termites, and providing a new and effective strategy for the biological control of termites by "using fungi to control insects".

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Two small RNA compositions for enhancing the biocontrol efficacy of termites, wherein the RNA sequences are milR-32- and milR-48, characterized in that, The RNA sequences were obtained from the Metarhizium anisopliae transcriptome database, with the RNA sequence of milR-32 being SEQ ID NO:1 and the RNA sequence of milR-48 being SEQ ID NO:

2.

2. The method for preparing the two small RNA compositions for improving the biological control effect of termites as described in claim 1, characterized in that: This includes the preparation of milR-32 mimics using milR-32 RNA sequences and the preparation of milR-48 mimics using milR-48 RNA sequences.

3. The method for preparing the two small RNA compositions for improving the biological control effect of termites according to claim 1, characterized in that, The preparation of milR-32 mimics from the milR-32 RNA sequence includes the following steps: (1) Based on the milR-32 sequence SEQ ID NO:1, design the following analog templates: the sense strand SEQ ID NO:1A and its complementary strand SEQ ID NO:1B; the antisense strand SEQ ID NO:1C and its complementary strand SEQ ID NO:1D; (2) After mixing equal amounts of SEQ ID NO:1A and SEQ ID NO:1B, annealing PCR was performed to obtain the milR-32 sense strand DNA template; after mixing equal amounts of SEQ ID NO:1C and SEQ ID NO:1D, annealing PCR was performed to obtain the milR-32 antisense strand DNA template. (3) Mix equal amounts of milR-32 sense strand DNA template and antisense strand DNA template, and synthesize milR-32 mimics using the T7 in vitro transcription system.

4. The method for preparing the two small RNA compositions for improving the biological control effect of termites according to claim 1, characterized in that, The preparation of milR-48 mimics from the milR-48 RNA sequence includes the following steps: (1) Based on the milR-48 sequence SEQ ID NO:2, design the following analog templates: the sense strand SEQ ID NO:2A and its complementary strand SEQ ID NO:2B; the antisense strand SEQ ID NO:2C and its complementary strand SEQ ID NO:2D; (2) After mixing equal amounts of SEQ ID NO:2A and SEQ ID NO:2B, annealing PCR was performed to obtain the milR-48 sense strand DNA template; after mixing equal amounts of SEQ ID NO:2C and SEQ ID NO:2D, annealing PCR was performed to obtain the milR-48 antisense strand DNA template. (3) Mix equal amounts of milR-48 sense strand DNA template and antisense strand DNA template, and synthesize milR-48 mimics using the T7 in vitro transcription system.

5. The two small RNA compositions for enhancing the biological control of termites as described in any one of claims 1-4 are used to enhance the biological control of termites.

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