Spodoptera frugiperda chitinase A gene dsRNA and preparation and application of nano preparation thereof

Abstract

The invention relates to the technical field of agricultural pest control, in particular to preparation and application of a Spodoptera frugiperda chitinase A gene dsRNA and a nano preparation thereof. The invention provides a dsRNA molecule capable of efficiently preventing and treating spodoptera frugiperda by using an RNAi technology, and the dsRNA molecule has high specificity and high gene silencing efficiency on a target gene; the nano insecticide prepared from the nano insecticide can be used for efficiently preventing and controlling the spodoptera frugiperda, has the advantages of higher fatality rate, quick insecticidal effect and simplicity and convenience in operation, solves the environmental and health problems caused by the traditional chemical insecticide, conforms to the current agricultural sustainable development requirements, and has important practical significance on the prevention and control of the spodoptera frugiperda.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural pest control, and in particular to the preparation and application of a dsRNA of chitinase A gene of fall armyworm and a nano preparation thereof. Background Art

[0002] Fall Armyworm Spodoptera frugiperda ) is a global pest with high reproductive capacity and strong adaptability. It has a short life cycle and a fast reproduction rate, causing serious economic losses to a variety of crops, such as corn, rice, wheat, etc. Although traditional chemical pesticides can effectively control pests in the short term, long-term use brings many problems, including increased pest resistance, environmental pollution, and toxic side effects on non-target organisms. In addition, the residue problem of chemical pesticides has become a major hidden danger to public health and food safety. Therefore, the development of new, safe, efficient and environmentally friendly pesticides has become an urgent problem to be solved.

[0003] RNA interference (RNAi) is a phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). RNA pesticides developed using RNAi technology have many advantages, such as a wide range of control targets, convenient application, easy operation, green and pollution-free, no residue, and strong environmental compatibility, and have gradually become a research hotspot in the field of plant protection. RNA pesticides have many advantages, such as specific control targets, convenient target development, convenient application, green and pollution-free, no residue, and strong environmental compatibility, and are called the "third revolution" in the history of pesticides.

[0004] However, there are still some challenges in the practical application of RNAi technology, which restricts its large-scale promotion. First, the production of high-purity and stable dsRNA requires complex process flow and high costs, which limits its commercial application; second, dsRNA has poor stability in the environment and is easily degraded by nucleases, affecting its insecticidal effect and sustainability; third, existing dsRNA delivery methods (such as spraying and feeding) are difficult to ensure that it effectively enters the body of pests and exerts its effect. Therefore, reducing the production cost of dsRNA, improving its environmental stability and optimizing the delivery method are urgent issues to be solved in the field of RNA pesticides.

[0005] Nanopesticides are pesticides that are dispersed in a nanoscale (1-300 nm) state in a formulation system or dispersion system through nano-preparation technology. They have the advantages of enhanced efficacy, reduced dosage, reduced toxicity, increased stability, extended duration, fast degradation, and environmental friendliness. They are the future development direction of pesticides. Nanocarriers can protect dsRNA from degradation by nucleases in the environment and effectively deliver it to pests through spraying or soil immersion. At present, nanomaterials such as liposomes, polymer nanoparticles, and silica nanoparticles show good prospects in drug delivery. Among many nanomaterials, chitosan has gradually shown its application potential. Chitosan is a natural polymer extracted from the shell of crustaceans. It has a wide range of sources, low price, good biocompatibility and biodegradability. It has been widely used in drug delivery systems. By mediating dsRNA molecules of insect-specific genes through chitosan, the stability and delivery efficiency of dsRNA can be improved, and the insecticidal effect can be enhanced. It has great potential in pest control.

[0006] In this context, how to provide a dsRNA molecule and nanopesticide that can utilize RNAi technology to effectively control fall armyworm has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0007] First, the present invention provides a chitinase A gene of Spodoptera frugiperda, the nucleotide sequence of which is shown in SEQ ID No.1.

[0008] The present invention, through a large number of screenings of different target genes of fall armyworm, finds that a dsRNA molecule designed for the chitinase A gene of fall armyworm can efficiently silence gene expression. Experimental verification shows that a dsRNA synthesized by in vitro transcription using the gene sequence shown in SEQ ID No.1 can cause fall armyworm to die due to abnormal molting or pupation, and has a high mortality rate to fall armyworm.

[0009] Furthermore, the present invention provides dsRNA synthesized according to the gene sequence shown in SEQ ID No.1.

[0010] Furthermore, the present invention provides a primer pair for amplifying and obtaining the chitinase A gene of the fall armyworm, characterized in that the upstream and downstream primers are shown as SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0011] Furthermore, the present invention provides a method for synthesizing the dsRNA, comprising: extracting total RNA from fall armyworm, reverse transcribing to obtain cDNA as a template, performing PCR amplification using the primer pair shown in SEQ ID No. 2 and SEQ ID No. 3, and purifying the amplified product and using it as a template sequence to synthesize dsRNA.

[0012] Preferably, the dsRNA is synthesized by in vitro transcription.

[0013] Furthermore, the present invention provides an insecticide containing the dsRNA.

[0014] Preferably, the pesticide is a nanoformulation.

[0015] Preferably, the pesticide comprises the dsRNA and the nanocarrier.

[0016] Preferably, the nanocarrier is chitosan.

[0017] Preferably, the preparation method of the insecticide comprises: mixing chitosan, acetic acid and sodium acetate to prepare a chitosan solution; then mixing the chitosan solution with a dsRNA solution, and performing standing, incubation, vortexing, standing again and centrifugal precipitation to obtain the insecticide.

[0018] The nanopesticide prepared by the above method has good protection effect on dsRNA, high penetration efficiency, and easy operation. It can specifically silence gene expression with a gene silencing efficiency of 74.33%, which ultimately leads to the death of fall armyworm due to abnormal molting or pupation, with an abnormality rate of 85% and a mortality rate before pupation of 82.5%.

[0019] Preferably, the concentration of the chitosan solution is 0.05% to 0.5% (w / v), more preferably 0.05% to 0.2%.

[0020] Preferably, the concentration of the dsRNA solution is 100-1000 ng / μL, more preferably 200-800 ng / μL.

[0021] Preferably, the chitosan solution and the dsRNA solution are mixed in a volume ratio of (0.4-4):1.

[0022] More preferably, the chitosan solution and the dsRNA solution are mixed in a volume ratio of 1:(1.5-2.5).

[0023] Preferably, the standing time is more than 2 minutes.

[0024] Preferably, the incubation temperature is 50-60°C, preferably 52-58°C.

[0025] Preferably, the incubation time is more than 1 min.

[0026] Preferably, the vortex time is 10s to 50s, more preferably 25s to 35s.

[0027] Preferably, the further standing is standing at room temperature for more than 1 hour.

[0028] Preferably, the centrifugal temperature is 2-6°C and the rotation speed is above 12000 rpm.

[0029] Preferably, the centrifugation time is more than 10 min.

[0030] In a specific embodiment, the formulation type of the pesticide includes, but is not limited to, nanoformulation, powder, granule, aqueous emulsion, aerosol or emulsifiable concentrate.

[0031] Furthermore, the present invention provides the use of the fall armyworm chitinase A gene, the dsRNA or the insecticide in agricultural pest management, agricultural pest control or killing agricultural pests.

[0032] Preferably, the agricultural pest is a Lepidoptera pest, more preferably the fall armyworm.

[0033] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a dsRNA molecule capable of utilizing RNAi technology to effectively control fall armyworm, which has high specificity and high gene silencing efficiency for target genes; the nanopesticide prepared by the dsRNA molecule can effectively control fall armyworm, has a high mortality rate, fast insecticidal effect, and is easy to operate, thus solving the environmental and health problems caused by traditional chemical pesticides, meeting the current requirements for sustainable agricultural development, and having important practical significance for the control of fall armyworm. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the result of precipitation gel running; M is Trans2K plus DNA maker; 1 is 2ug naked dsRNA; 2 is 0.2% (w / v) chitosan; 3 is 0.5:1 chitosan and dsRNA complex; 4 is 1:1 chitosan and dsRNA complex; 5 is 2:1 chitosan and dsRNA complex; 6 is 3:1 chitosan and dsRNA complex; 7 is 4:1 chitosan and dsRNA complex.

[0035] Figure 2 is the result of supernatant gel running; M is Trans2K plus DNA maker; 1 is the supernatant of chitosan and dsRNA complex in a ratio of 0.5:1; 2 is the supernatant of chitosan and dsRNA complex in a ratio of 1:1; 3 is the supernatant of chitosan and dsRNA complex in a ratio of 2:1; 4 is the supernatant of chitosan and dsRNA complex in a ratio of 3:1; 5 is the supernatant of chitosan and dsRNA complex in a ratio of 4:1.

[0036] Figure 3is the change of chitinase A gene expression at different time points after nanoformulation treatment; β-actin is the internal reference gene, **p<0.01, ***p<0.001.

[0037] Figure 4 It is the growth and development changes of fall armyworm 1-10 days after treatment with nano-agent.

[0038] Figure 5 The weight changes of Spodoptera frugiperda larvae 1-10 days after treatment with nanoformulations.

[0039] Figure 6 The weight changes of pupae of Fall Armyworm after treatment with nano-preparation. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book Molecular Cloning (J. Sambrook, EF Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for technicians in the field of molecular biology when conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, technicians in the field of molecular biology complete the corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust professional companies to conduct the experiments. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0042] Example 1 Synthesis of dsRNA of chitinase A gene of Spodoptera frugiperda 1. Primer design and synthesis: The dsRNA primers for the chitinase A gene (SEQ ID No. 4) of Spodoptera frugiperda were designed using the SnapDragon website. The upstream and downstream primers are shown in SEQ ID No. 2 and SEQ ID No. 3 (containing T7 promoter), respectively. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] 2. Synthesis of dsRNA of chitinase A gene of Spodoptera frugiperda The RNA (2 μg) of the whole insect of the third-instar fall armyworm was used as a template, and the cDNA was obtained by reverse transcription. The upstream and downstream primers SEQ ID No.2 and SEQ ID No.3 containing the T7 promoter sequence were used for PCR amplification. The PCR system was as follows: Premix Taq™ (Takara) 10 μL, upstream primer 0.5 μL (10mM), downstream primer 0.5 μL (10mM), DEPC water 7.3 μL, cDNA 1.5 μL. The PCR reaction program was as follows: 95℃, 3min; 95℃, 30s; 55℃, 30s; 72℃, 1min; 35 cycles, 72℃, 10min. A gene fragment of 489bp in length (SEQ ID No.1) was obtained by PCR amplification. The PCR product was purified by Gel Extraction Kit (Omega) and the T7 RiboMAX TM The Express RNAi System (Promega) kit was used to synthesize dsRNA by in vitro transcription. The concentration was determined using NaNoDrop 2000 (Thermo scientific). The chitinase A gene dsRNA of Spodoptera frugiperda was stored in a -80°C ultra-low temperature refrigerator for future use.

[0044] Example 2 Preparation of chitosan / dsRNA nanoformulation 1. Solvent preparation 0.1 M acetic acid, 0.1 M sodium acetate, 100 mM sodium sulfate, chitosan, and the fall armyworm chitinase A gene dsRNA obtained in Example 1; 2. Preparation of 0.1% (W / V) chitosan solution Take 59 ml of 0.1 M acetic acid and 41 ml of 0.1 M sodium acetate and mix them evenly, add 0.1 g of chitosan powder, and stir with a magnetic stirrer at 37 ° C for 10 hours.

[0045] 3. Chitosan / dsRNA nanoformulation 500 ng / μl of fall armyworm chitinase A gene dsRNA was uniformly mixed with 0.1% chitosan solution in a volume ratio of 1:1, 1:2, 1:3, 1:4, and 2:1, respectively, and allowed to stand for 2 minutes, incubated at 55°C for 1 minute, vortexed for 30 seconds, allowed to stand at room temperature for 1 hour, and centrifuged at 4°C 12000 rpm for 10 minutes. The precipitate was chitosan / dsRNA nanoformulation, which was detected by agarose gel. The results of the precipitate gel run were as follows: Figure 1 As shown, the supernatant gel results are as follows Figure 2 shown.

[0046] Example 3 Experiment on the killing effect of chitosan / dsRNA nanoformulation on fall armyworm 1. Backplane drip test Thirty 3-year-old and 2-day-old fall armyworms were selected, with 10 in each group, and the experiment was repeated three times. At the same time, 30 larvae of the same period were selected as the blank control group and the internal reference control group, and chitosan / dsRNA nanoformulation (chitosan / dsRNA nanoformulation prepared by chitinase A gene dsRNA of fall armyworm and 0.1% chitosan solution in a volume ratio of 2:1 in Example 2) was dripped on the back of the larvae at an amount of 2 μg dsRNA per larvae as the chitinase A gene treatment group, and the internal reference gene eGFP dsRNA was introduced as the internal reference control group. The internal reference control group eGFP dsRNA was a dsRNA synthesized by in vitro transcription using SEQ ID No.5 as a template sequence, and the nanoformulation of the internal reference control group was prepared according to the method of Example 2. The treated larvae were placed in a 28°C constant temperature biochemical incubator and fed with artificial diet (light: dark time = 16h:8h, temperature 26±2°C, humidity 40%).

[0047] 2. Detection of chitinase A gene silencing in fall armyworm 24h, 48h, 72h, 96h, 120h, 144h, 168h after treatment, three larvae were collected at each time point, total RNA was extracted, reverse transcribed into first-strand cDNA, and the real-time PCR method was used to detect the changes in the expression of the chitinase A gene of Spodoptera frugiperda, with β-actin as the internal reference gene. The upstream and downstream primers of the chitinase A gene of Spodoptera frugiperda in the real-time PCR are shown in SEQ ID No.6 and SEQ ID No.7, and the upstream and downstream primers of the internal reference gene are shown in SEQ ID No.8 and SEQ ID No.9. The real-time PCR reaction system is as follows: 0.3 μL of upstream primer, 0.3 μL of downstream primer, 5 μL of 2×SYBR Green Taq HSPremix (AG Accurate Biology), 3.4 μL of DEPC water, and 1 μL of cDNA. The reaction program was as follows: 95°C, 10 s; 95°C, 15 s; 55°C, 30 s; 72°C, 30 s; 39 cycles. The gene silencing efficiency was calculated.

[0048] The results are as follows Figure 3 As shown, compared with the control group, 24 hours after injection of chitosan / dsRNA nanoformulation, the expression level of chitinase A gene in fall armyworm decreased significantly, and the gene silencing efficiency reached 74.33%, reaching an extremely significant level.

[0049] 3. Phenotypic observation of larvae after treatment with chitosan / dsRNA nanoformulation After the chitosan / dsRNA nanoparticles were dripped onto the back of the insects, all the larvae in the control group successfully molted to the next instar on the 4th day of the 3rd instar, and the adult development was good after molting. After the chitosan / dsRNA nanoparticles were treated, the larvae refused to eat, and their length and weight lagged behind. The larvae died on the 2nd day, accompanied by abnormal molting and pupation, which was manifested as the old epidermis was difficult to remove until death. Figure 4 ), body weight decreased compared with the control group ( Figure 5 ), the pupal weight of individuals that successfully pupated was lower than that of the control group ( Figure 6 ), according to statistics, the abnormality rate of fall armyworm (abnormal molting or abnormal pupation) reached 85%, and the final mortality rate before pupation reached 82.53%.

[0050] Comparative Example In this comparative example, dsRNA was designed for the chitin synthase CHS-2 gene of the fall armyworm, and the gene fragment shown in SEQ ID No. 10 was used for in vitro transcription to synthesize dsRNA with reference to the synthesis method of Example 1. Then, chitosan / dsRNA nanoformulation was prepared with reference to the preparation method of Example 2, and the insecticidal effect of the dsRNA of the comparative example was tested with reference to the experimental method of Example 3. It was found through statistics that the abnormality rate (abnormal molting or abnormal pupation) of the fall armyworm was only 4%, and the final mortality rate before pupation was only 9%.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chitinase A gene of Spodoptera frugiperda, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

1.

2. The dsRNA synthesized from the chitinase A gene of Spodoptera frugiperda according to claim 1.

3. A primer pair for amplifying the chitinase A gene of Spodoptera frugiperda according to claim 1, characterized in that: The upstream and downstream primers are shown as SEQ ID No. 2 and SEQ ID No. 3 respectively.

4. The method for synthesizing dsRNA according to claim 2, characterized in that: include: Total RNA of fall armyworm was extracted, and cDNA was obtained by reverse transcription as a template. PCR amplification was performed using the primer pair shown in SEQ ID No. 2 and SEQ ID No.

3. The amplified product was purified and used as a template sequence to synthesize dsRNA.

5. An insecticide containing the dsRNA according to claim 2.

6. The insecticide according to claim 5, characterized in that The insecticide is a nano preparation; the insecticide comprises the dsRNA according to claim 2 and a nano carrier.

7. The insecticide according to claim 6, characterized in that The nanocarrier is chitosan.

8. The method for preparing the insecticide according to claim 7, characterized in that: include: Chitosan, acetic acid and sodium acetate are mixed to prepare a chitosan solution; the chitosan solution is then mixed with a dsRNA solution, and the mixture is allowed to stand, incubated, vortexed, allowed to stand again, and centrifuged to obtain a solution.

9. The insecticide according to claim 5, characterized in that The pesticide is in the form of a nanometer formulation, a powder, a granule, an aqueous emulsion, an aerosol or an emulsifiable concentrate.

10. Use of the fall armyworm chitinase A gene according to claim 1, the dsRNA according to claim 2, or the insecticide according to any one of claims 5 to 9 in agricultural pest management, agricultural pest control, or killing agricultural pests.

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