Nanometer preparation based on beet armyworm ATP enzyme A gene dsRNA and application thereof

Abstract

The invention relates to the technical field of agricultural pest control, in particular to a nano preparation based on beet armyworm ATPase A gene dsRNA and application of the nano preparation. The invention provides a dsRNA molecule capable of efficiently preventing and treating beet armyworm by using an RNAi technology, and the dsRNA molecule has high specificity and high silencing effect on a target gene; the nano preparation prepared by the nano preparation can be used for efficiently preventing and treating beet armyworms, has the advantages of high fatality rate, good insecticidal effect, simplicity and convenience in operation and the like, meets the current agricultural sustainable development requirements, and has important practical significance on prevention and treatment of beet armyworms.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural pest control, and particularly to a nano - preparation based on dsRNA of the ATPase A gene of Spodoptera exigua and its application. Background Art

[0002] Spodoptera exigua is widespread globally and can infest a variety of plants. Its larvae are omnivorous and mainly cause damage to crops in the larval form, affecting the growth of corn, peanuts, sesame, sugar beets, and numerous vegetables. The larvae of Spodoptera exigua exhibit feigning death behavior and the habit of feeding covertly, which makes them difficult to be effectively controlled by traditional chemical insecticides. Due to the long - term reliance on chemical pesticides, Spodoptera exigua has developed resistance to a variety of insecticides, including organophosphates, pyrethroids, carbamates, and benzoylureas, etc. In addition, the excessive use of chemical insecticides may also pose threats to the environment and health. Therefore, promoting the research and application of green prevention and control technologies for crops is of extremely important value in the management of key pests in agricultural production.

[0003] RNA pesticides are pesticides developed based on RNA interference (RNAi) technology. Small interfering RNA molecules are designed to target specific pest or pathogen genes. By specifically recognizing and binding to the mRNA of the target gene, they promote mRNA degradation or translation inhibition, thus exerting the effect of pest and disease control. Due to their characteristics of high efficiency, specificity, environmental friendliness, and high R & D cost - effectiveness, RNA pesticides show great development potential in the prevention and control of agricultural pests and diseases. In agricultural applications, RNA pesticides can be implemented through various ways, including generating double - stranded RNA (dsRNA) through transgenic plants, using viruses or microorganisms to deliver dsRNA, and directly spraying exogenous dsRNA, etc.

[0004] RNA pesticides are easily decomposed in the natural environment. Although the risk of environmental pollution and residue is extremely low, meeting the requirements of green agriculture, there are indeed some challenges in practical applications that limit their large - scale promotion. First, dsRNA molecules are easily affected by various factors in the natural environment, resulting in changes in their structure or degradation. Therefore, special protection formulations need to be developed to enhance their stability in the environment. Second, achieving efficient and precise delivery of dsRNA to target pests is a major challenge. Factors such as nuclease activity and pH changes in the pest body may damage dsRNA, thus reducing the interference effect of RNA pesticides in the pest body. Finally, the production cost of dsRNA is relatively high, which affects its wide application in agriculture. Therefore, improving the delivery efficiency of dsRNA, enhancing the environmental stability of dsRNA, and optimizing the delivery method of dsRNA are urgent problems to be solved in the field of RNA pesticides.

[0005] Nanopesticides are a new type of pesticide formulation that precisely proportion the active ingredients, carriers, and auxiliary components through the application of nanotechnology and materials science to create highly efficient and superior-performing pesticide products. Nanopesticide technology has given rise to a series of innovative formulation forms, including nanoemulsions, suspensions, microcapsules, water-soluble gels, and solid dispersion systems, etc. These new formulations can significantly enhance the efficacy of pesticides, with the average pesticidal effect increasing by 30 - 50%, and the duration of pesticidal effect can be extended to 4 - 5 times the original, while also greatly reducing the residual pollution of pesticides in the environment. Nanocarriers can effectively protect dsRNA from environmental factors such as light, temperature fluctuations, and degradation by nucleases, thereby enhancing the stability of dsRNA in the environment. In addition, the application of nanocarriers helps to reduce the dependence on traditional chemical pesticides, thus reducing environmental pollution and the negative impacts on non-target organisms.

[0006] In the current research context, developing a dsRNA molecule and its corresponding nano-insecticide that can efficiently control Spodoptera exigua using RNAi technology has become an urgent technical challenge in this field. Summary of the Invention

[0007] To overcome the above technical challenges, the present invention provides a Spodoptera exigua ATPase A gene, whose nucleotide sequence is shown as SEQ ID No.1.

[0008] Through extensive screening of different target genes of Spodoptera exigua, the present invention found that the dsRNA molecule designed against the Spodoptera exigua ATPase A gene can efficiently silence gene expression. Through experimental verification, the dsRNA synthesized by in vitro transcription using the gene sequence shown as SEQ ID No.1 can cause abnormal molting and smaller body size of Spodoptera exigua, resulting in death, and has a relatively high lethality rate against Spodoptera exigua.

[0009] Furthermore, the present invention provides dsRNA synthesized from the gene sequence shown as SEQ ID No.1.

[0010] Meanwhile, the present invention provides a primer pair for amplifying the Spodoptera exigua ATPase A gene described in claim 1, and the primer pair is shown as SEQ ID No.2 and SEQ ID No.3.

[0011] Furthermore, the present invention provides a method for synthesizing the dsRNA, including: extracting the total RNA of Spodoptera exigua, reverse-transcribing to obtain cDNA as a template, performing PCR amplification using the primer pair shown as SEQ ID No.2 and SEQ ID No.3, and purifying the amplification product to be used as a template sequence to synthesize dsRNA.

[0012] In some embodiments, dsRNA is synthesized by in vitro transcription.

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

[0014] In some embodiments, the formulation type of the insecticide is a nano-formulation, powder, granule, water emulsion, aerosol or emulsifiable concentrate.

[0015] In some embodiments, the nano-formulation includes the dsRNA and a nano-carrier.

[0016] Preferably, the nano-carrier is chitosan.

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

[0018] The nano-insecticide prepared by the above method has strong protection for dsRNA and high efficient penetration ability, and the operation process is simple, and it can achieve precise inhibition of specific gene expression. 48 hours after feeding treatment, the gene silencing efficiency reaches 63%, ultimately resulting in abnormal molting, abnormal body shape and refusal to eat and death of Spodoptera exigua.

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

[0020] Preferably, the concentration of the dsRNA solution is 1000 - 5000 ng / μL, more preferably 2000 - 4000 ng / μL.

[0021] Preferably, the chitosan solution and the dsRNA solution are mixed in a mass ratio of (0.5 - 4):1.

[0022] Preferably, the standing time is more than 2 minutes.

[0023] Preferably, the incubation temperature is 50 - 60 °C, preferably 52 - 58 °C.

[0024] Preferably, the incubation time is more than 1 minute.

[0025] Preferably, the centrifugation time is 10 s - 50 s, more preferably 25 s - 35 s.

[0026] Preferably, the second standing is at room temperature for more than 1 hour.

[0027] Preferably, the temperature for centrifugation is 2-6°C and the rotation speed is above 12,000 rpm.

[0028] Preferably, the time for centrifugation is above 10 min.

[0029] Furthermore, the present invention provides the use of the Spodoptera exigua ATPase A gene, the dsRNA, the insecticide or the insecticide prepared by the preparation method in agricultural pest management, agricultural pest control or killing agricultural pests.

[0030] Preferably, the agricultural pest is a Lepidoptera pest, and more preferably Spodoptera exigua.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a dsRNA molecule capable of efficiently controlling Spodoptera exigua using RNAi technology, which exhibits high specificity for target genes and strong gene silencing ability. The nano-insecticide developed using this technology can effectively combat Spodoptera exigua, with significant lethal effects and a fast insecticidal speed. Its simple operation process not only reduces the potential risks of traditional chemical insecticides to the environment and health, but also meets the requirements of modern agriculture for sustainable development. The present invention has great practical value for the prevention and control of Spodoptera exigua. Description of the Drawings

[0032] Figure 1 is the gel electrophoresis result of the precipitate; where M is Trans2K plus DNA maker; 1 is 2 μg of naked dsRNA; 2 is 0.2% (w / v) chitosan; 3 is the chitosan and dsRNA composite precipitate with a mass ratio of 0.5:1; 4 is the chitosan and dsRNA composite precipitate with a mass ratio of 1:1; 5 is the chitosan and dsRNA composite precipitate with a mass ratio of 2:1; 6 is the chitosan and dsRNA composite precipitate with a mass ratio of 3:1; 7 is the chitosan and dsRNA composite precipitate with a mass ratio of 4:1.

[0033] Figure 2 is the gel electrophoresis result of the supernatant; where M is Trans2K plus DNA maker; 1 is 2 μg of naked dsRNA; 2 is 0.2% (w / v) chitosan; 3 is the supernatant of the chitosan and dsRNA complex with a mass ratio of 0.5:1; 4 is the supernatant of the chitosan and dsRNA complex with a mass ratio of 1:1; 5 is the supernatant of the chitosan and dsRNA complex with a mass ratio of 2:1; 6 is the supernatant of the chitosan and dsRNA complex with a mass ratio of 3:1; 7 is the supernatant of the chitosan and dsRNA complex with a mass ratio of 4:1.

[0034] Figure 3Changes in the expression level of ATPase A gene at different time points after treatment with nano - preparations; among them, β - actin is the reference gene, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and ns represents p > 0.05.

[0035] Figure 4 Are the growth and development changes of Spodoptera exigua after treatment with nano - preparations for 1 - 5 days.

[0036] Figure 5 Are the weight changes of Spodoptera exigua larvae after treatment with nano - preparations for 1 - 5 days.

[0037] Figure 6 Is the survival rate graph of Spodoptera exigua larvae after treatment with nano - preparations. Detailed implementation mode

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book "Molecular Cloning" (written by J. Sambrook, E.F. Fritsch, and T. Maniatis, published by Science Press in 1994). This book and its subsequent published versions are the most commonly used guiding reference books for those skilled in the art when conducting experiments related to molecular biology. In addition, according to different experimental purposes, those skilled in the art complete corresponding experiments under the guidance of the operation manuals attached to various commercial kits (Kit) or entrust professional companies to do so. Reagents or instruments without indicating the manufacturer can be obtained as conventional products through regular channels.

[0040] Example 1 Synthesis of dsRNA of ATPase A gene of Spodoptera exigua 1. Primer design and synthesis: The dsRNA primers of the ATPase A gene of Spodoptera exigua (SEQ ID No.4) were designed using the SnapDragon website. The upstream and downstream primers are shown in SEQ ID No.2 and SEQ ID No.3 respectively (containing the T7 promoter). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] 2. Synthesis of dsRNA of ATPase A gene of Spodoptera exigua Using the total RNA (2 μg) of 4th instar Spodoptera exigua larvae as a template, cDNA was reverse transcribed. PCR amplification was carried out using the upstream and downstream primers SEQ ID No.2 and SEQ ID No.3 containing the T7 promoter sequence. The PCR system was as follows: Premix Taq™ (Takara) 10 μL, upstream primer 0.5 μL (10 mM), downstream primer 0.5 μL (10 mM), DEPC water 7.3 μL, cDNA 1.5 μL. The PCR reaction program was as follows: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; 35 cycles, 72°C for 10 min. A gene fragment with a length of 300 bp (SEQ ID No.1) was obtained by PCR amplification. The PCR product was purified using the Gel Extraction Kit (Omega), and in vitro transcription was performed to synthesize dsRNA according to the T7 RiboMAX TM Express RNAi System (Promega) kit. The concentration was measured using a NaNoDrop 2000 (Thermo scientific). The dsRNA of the Spodoptera exigua ATPase A gene was stored in an ultra-low temperature freezer at -80°C for standby.

[0042] Example 2 Preparation of chitosan / dsRNA nanoparticles 1. Solvent preparation 0.1 M acetic acid, 0.1 M sodium acetate, chitosan, dsRNA of the Spodoptera exigua ATPase A gene obtained in Example 1; 2. Preparation of 0.2% (W / V) chitosan solution Mix 59 ml of 0.1 M acetic acid and 41 ml of 0.1 M sodium acetate evenly, add 0.2 g of chitosan powder, and stir at 37°C for 10 hours using a magnetic stirrer.

[0043] 3. Chitosan / dsRNA nanoparticles The dsRNA of the Spodoptera exigua ATPase A gene at 2000 ng / μl and the 0.2% (w / v) chitosan solution were evenly mixed according to mass ratios of 1:0.5, 1:1, 1:2, 1:3, and 1:4 respectively, allowed to stand for 2 minutes, incubated at 55°C for 1 minute, vortexed and mixed evenly for 30 seconds, allowed to stand at room temperature for 1 hour, centrifuged at 12000 rpm at 4°C for 10 minutes. The precipitate was the chitosan / dsRNA nanoparticles, which were detected by agarose gel electrophoresis. The gel electrophoresis results of the precipitate are as Figure 1 shown, and the gel electrophoresis results of the supernatant are as Figure 2 shown.

[0044] Example 3 Experiment on the killing effect of chitosan / dsRNA nanoparticles on Spodoptera exigua 1. Feeding experiment Thirty 4-day-old Spodoptera exigua larvae were selected and divided into groups of 10. The experiment was set up with 3 replicates. At the same time, 30 larvae of the same period were selected as the blank control group and the internal reference control group. According to the amount of 2 μg dsRNA per larva, the chitosan / dsRNA nanopreparation (the chitosan / dsRNA nanopreparation prepared by mixing the dsRNA of the Spodoptera exigua ATPase A gene and a 0.2% chitosan solution in a volume ratio of 1:1 as described in Example 2) was dropped onto the cut 1 cm × 1 cm feed as the ATPase A gene treatment group, and the internal reference control group was transfected with eGFP dsRNA. The eGFP dsRNA of the internal reference control group was synthesized by in vitro transcription using SEQ ID No.5 as the template sequence, and the nanopreparation of the internal reference control group was prepared according to the method of Example 2. The treated larvae were fed with artificial feed in a constant temperature biochemical incubator at 28 °C (light:dark time = 14 h:10 h, temperature 28 ± 2 °C, humidity 40%).

[0045] 2. Detection of the silencing of the Spodoptera exigua ATPase A gene At 24 h, 48 h, 72 h, 96 h, and 120 h after treatment, 3 larvae were randomly selected at each time point, and total RNA was extracted and reverse transcribed into the first-strand cDNA. The change in the expression level of the Spodoptera exigua ATPase A gene was detected by Real-time PCR using β-actin as the internal reference gene. The upstream and downstream primers of the Spodoptera exigua ATPase A gene in 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 HS Premix (AGAccurate Biology), 3.4 μL of DEPC water, and 1 μL of cDNA. The reaction procedure is 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.

[0046] The results are as Figure 3 shown. Compared with the control group, after feeding with the chitosan / dsRNA nanopreparation for 24 h, the expression level of the Spodoptera exigua ATPase A gene decreased significantly by 51%, and after feeding for 48 h, the expression level decreased by 63%, both reaching a highly significant level, indicating a high gene silencing efficiency.

[0047] 3. Observation of the phenotypes of larvae after treatment with chitosan / dsRNA nanopreparations After feeding with chitosan / dsRNA nanoparticles, all the larvae in the control group successfully molted to the next instar on the 4th day of the 4th instar, and the adult development was in good condition after molting. After treatment with chitosan / dsRNA nanoparticles, the larvae showed anorexia, abnormal body shape and retarded weight development. Death occurred on the 2nd day after treatment, and was accompanied by abnormal molting, manifested as difficulty in shedding the old cuticle until death ( Figure 4 ), and the weight decreased significantly compared with the control group ( Figure 5 ), and the mortality rate reached 23.33% on the fifth day ( Figure 6 ).

[0048] Comparative Example In this comparative example, dsRNA was designed against the chitin synthase 7 gene of Spodoptera exigua. Referring to the synthesis method of Example 1, dsRNA was synthesized by in vitro transcription using the gene fragment shown in SEQ ID No. 10. Then, referring to the preparation method of Example 2, chitosan / dsRNA nanoparticles were prepared, and referring to the experimental method of Example 3, the insecticidal effect of the dsRNA in this comparative example was detected. After statistics, it was found that the abnormality rate and mortality rate of Spodoptera exigua were relatively low, and the mortality rate was only 10%.

[0049] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention in each example.

Claims

1. A beet armyworm ATPase A gene, characterized in that Its nucleotide sequence is shown in SEQ ID No.

1.

2. dsRNA synthesized from the beet armyworm ATPase A gene according to claim 1.

3. A primer pair for amplifying the ATPase A gene of Spodoptera exigua according to claim 1, characterized in that: The primer pair is shown as SEQ ID No.2 and SEQ ID No.

3.

4. The method for synthesizing dsRNA according to claim 2, characterized in that: include: Total RNA of Spodoptera exigua 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, characterized in that: It contains the dsRNA according to claim 2.

6. 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.

7. The insecticide according to claim 6, characterized in that The nanoformulation comprises the dsRNA according to claim 2 and a nanocarrier.

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

9. The method for preparing the insecticide according to claim 8, 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, mixed, allowed to stand again and centrifuged to obtain the product.

10. Use of the beet armyworm ATPase A gene according to claim 1, the dsRNA according to claim 2, the insecticide according to any one of claims 5 to 8, or the insecticide prepared by the preparation method according to claim 9 in agricultural pest management, agricultural pest control or killing agricultural pests.

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