Insecticide combination formula based on beet armyworm ATPase A gene dsRNA nano preparation

Abstract

The invention relates to the technical field of pesticide pest control, in particular to a pesticide combination formula based on a beet armyworm ATPase A gene dsRNA nano preparation. The invention develops a pesticide combination formula for effectively preventing and controlling agricultural pests, especially beet armyworms, through a mode of combined application of a chemical pesticide and a nano RNA pesticide, the prevention and control effect on the agricultural pests can be greatly improved, the dosage of the chemical pesticide can be effectively reduced, the resistance risk can be reduced, and the economic benefit is increased. The pollution to the environment and the chemical pesticide residue are also reduced. The invention provides a new thought for comprehensive treatment of agricultural pests.

Description

Technical Field

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

[0002] Spodoptera exigua ( Spodoptera exigua ) is a world - wide pest with a wide distribution and causes great harm to various crops and vegetables such as corn, rice, peanuts, and soybeans. Traditional pest control mainly relies on chemical pesticides, but their long - term and high - dose application poses a serious threat to the ecological environment and human health. Therefore, the research and development of green pest control technologies for crops are of great significance for the control of major pests in agricultural production.

[0003] RNA pesticides, also known as nucleic acid pesticides, are polynucleotide preparations that interfere with or inhibit the transcription of specific genes in target organisms. RNA interference (RNAi) pesticides are new - type biological pesticides developed in recent years, with outstanding advantages such as low toxicity, high targeting, low residue, long - lasting efficacy, and difficulty in generating resistance, and are the future development direction of pesticides. Currently, the application of RNAi in pest control is mainly divided into two types: (1) Plant - derived protectants, in which double - strand RNA (dsRNA) of target genes with insecticidal activity is transferred into plants through transgenic methods for pest control; (2) Non - plant - derived protectants, in which dsRNA preparations are prepared and used for pest control by spraying, root irrigation, or seed soaking.

[0004] However, on the one hand, the cycle of RNAi to play a role is relatively long, and the acceptance degree of farmers is relatively low. On the other hand, traditional dsRNA delivery systems will reduce the RNAi efficiency due to environmental factors and the internal environment of pests. At the same time, in order to further reduce the use of chemical insecticides, more and more recent research has focused on the combined application of insecticides, nanocarriers, and dsRNA, highlighting new advantages in the field of pest control.

[0005] Therefore, developing an insecticide combination formula for effective control of Spodoptera exigua has become a technical problem urgently to be solved in this field. Summary of the Invention

[0006] To solve the above - mentioned technical problems, the present invention provides an insecticide combination formula with a synergistic effect on killing agricultural pests, including a dsRNA nano - preparation of the ATPase A gene of Spodoptera exigua and emamectin benzoate + lufenuron.

[0007] First, through extensive screening of different target genes of Spodoptera exigua, the present invention found that the dsRNA molecule designed against the ATPase A gene of Spodoptera exigua can cause abnormal molting and smaller body size in Spodoptera exigua, leading to death, and has a high lethality rate against Spodoptera exigua. After preparing it into a nano - formulation and combining it with emamectin benzoate and lufenuron, it can not only significantly increase the mortality rate of agricultural pests, but also achieve the reduction of chemical pesticides and increase their efficiency.

[0008] Preferably, the dsRNA of the ATPase A gene of Spodoptera exigua is synthesized according to the nucleotide sequence shown in SEQ ID No.1.

[0009] More preferably, the dsRNA of the ATPase A gene of Spodoptera exigua is synthesized by in vitro transcription according to the nucleotide sequence shown in SEQ ID No.1.

[0010] In some embodiments, the nano - formulation includes the dsRNA of the ATPase A gene of Spodoptera exigua and a nano - carrier.

[0011] Preferably, the nano - carrier is chitosan.

[0012] Since different carriers have different loading effects and protection effects on dsRNA molecules, there are differences in RNAi efficiency after loading the same dsRNA molecule with different carriers. The present invention found that when chitosan is selected as the nano - carrier to load the dsRNA of the ATPase A gene of Spodoptera exigua, its stability and penetration ability can be greatly enhanced, thus significantly improving the RNAi efficiency.

[0013] In some embodiments, the preparation method of the dsRNA nano - formulation of the ATPase A gene of Spodoptera exigua 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, vortexing, standing again and centrifugal precipitation.

[0014] Preferably, the concentration of the chitosan solution is 0.05% - 0.5% (w / v), more preferably 0.05% - 0.2%.

[0015] Preferably, the concentration of the dsRNA solution is 1000 - 5000 ng / μL, more preferably 2000 - 4000 ng / μL.

[0016] Preferably, the chitosan solution and the dsRNA solution are mixed in a mass ratio of (0.5 - 4):1.

[0017] Preferably, the standing time is more than 2 min.

[0018] Preferably, the incubation temperature is 50 - 60 °C, preferably 52 - 58 °C.

[0019] Preferably, the incubation time is more than 1 min.

[0020] Preferably, the centrifugation time is 10 s to 50 s, more preferably 25 s to 35 s.

[0021] Preferably, the re - standing is carried out at room temperature for more than 1 h.

[0022] Preferably, the centrifugation temperature is 2 - 6 °C and the rotation speed is more than 12000 rpm.

[0023] Preferably, the centrifugation time is more than 10 min.

[0024] Furthermore, the present invention provides the application of the described combined formulation in agricultural pest management, agricultural pest control or killing agricultural pests.

[0025] Furthermore, the present invention provides a method for killing agricultural pests, comprising: successively applying the dsRNA nano - preparation of the Spodoptera exigua ATPase A gene and emamectin benzoate + lufenuron to agricultural pests.

[0026] In some embodiments, the dosage of the nano - preparation is 3 - 8 μg / head, preferably 4 - 6 μg / head.

[0027] In some embodiments, the effective net content of emamectin benzoate + lufenuron is 8% - 15% (w / w), and the dosage is 60 - 100 mg / L.

[0028] In some embodiments, first the nano - preparation is used to feed agricultural pests (preferably the 4th instar larvae of Spodoptera exigua), and 24 hours later, emamectin benzoate + lufenuron is sprayed on the agricultural pests.

[0029] Preferably, the agricultural pests are Lepidoptera pests, more preferably Spodoptera exigua.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention has developed an insecticide combined formulation for effectively controlling agricultural pests (especially Spodoptera exigua). By the combined application of a chemical insecticide and a nano - RNA pesticide, the control effect on agricultural pests can be greatly improved, the dosage of the chemical insecticide can be effectively reduced, the resistance risk can be reduced, and the environmental pollution and chemical pesticide residues can also be reduced. The present invention provides a new idea for the integrated management of agricultural pests. Description of the Drawings

[0031] Figure 1 It is the detection of the insecticidal effect of emamectin benzoate + lufenuron at different concentrations, and the death gradient curve of the treatment of Spodoptera exigua larvae with a single chemical pesticide is obtained.

[0032] Figure 2 The phenotype of Spodoptera exigua larvae 48 hours after the synergistic treatment of chemical pesticides and nano-RNA pesticides.

[0033] Figure 3 The mortality curve of Spodoptera exigua larvae after the synergistic treatment of chemical pesticides and nano-RNA pesticides.

[0034] Figure 4 The mortality curve of Spodoptera exigua larvae after feeding with chitosan / dsRNA nanoformulation alone.

[0035] Figure 5 The detection result of the silencing effect of ATPase A gene in Spodoptera exigua after synergistic treatment. Specific implementation manners

[0036] 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 in conjunction with the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0037] 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 and guiding reference books for those skilled in the art when conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, those skilled in the art can complete the corresponding experiments under the guidance of the operation manuals attached to various commercial kits or entrust professional companies to conduct them, such as gene sequencing, plasmid sequencing, and determining molecular weight, etc.

[0038] Example 1 Experiment on the killing effect of emamectin benzoate and lufenuron on Spodoptera exigua 1. Pesticide preparation Dilute the original solution of emamectin benzoate and lufenuron (the effective net content of emamectin benzoate and lufenuron is 10% (w / w)) with 20% (v / v) acetone solution to obtain working concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, and 350 mg / L.

[0039] 2. Toxicity experiment of emamectin benzoate and lufenuron against Spodoptera exigua Thirty 4th-instar Spodoptera exigua larvae on the 1st day were taken, with 10 larvae in each group, and evenly divided into 3 groups. A 50 mL spray bottle was used to evenly spray the diluted pesticide on the body surface of the larvae, as well as on their food and living environment. At the same time, 30 larvae were selected as the blank control group. The treated larvae were placed in an artificial diet in a constant temperature biochemical incubator at 28°C (light:dark time = 14h:10h, temperature 26±2°C, humidity 40%).

[0040] 3. Analysis of the virulence effect of emamectin benzoate•lufenuron against Spodoptera exigua After treatment, the living state of the larvae was observed, and the death situation of the larvae was recorded. The results showed that compared with the control group, the death rate and number of larvae treated with 50mg / L, 100mg / L, 150mg / L, 200mg / L, 250mg / L, 300mg / L, and 350mg / L insecticides gradually increased ( Figure 1 ).

[0041] Example 2 Preparation of dsRNA nanoformulation of ATPase A gene of Spodoptera exigua This example provides a dsRNA nanoformulation of the ATPase A gene of Spodoptera exigua, and its preparation method is as follows: 1. Synthesis of dsRNA of the ATPase A gene of Spodoptera exigua Using the nucleotide sequence shown in SEQ ID No.1 as a template, in vitro transcription synthesis of dsRNA was carried out using the T7 RiboMAX TM Express RNAi System (Promega) kit. Concentration determination was performed using a NaNoDrop 2000 (Thermo scientific). The dsRNA of the ATPase A gene of Spodoptera exigua was stored in an ultra-low temperature refrigerator at -80°C for later use.

[0042] 2. Preparation of chitosan / dsRNA nanoformulation 2000 ng / μl of dsRNA of the ATPase A gene of Spodoptera exigua was evenly mixed with a 0.2% chitosan solution (prepared by mixing 59 ml of 0.1 M acetic acid and 41 ml of 0.1 M sodium acetate evenly, adding 0.2 g of chitosan powder, and stirring with a magnetic stirrer at 37°C for 10 hours) in a volume ratio of 1:1, left standing for 2 minutes, incubated at 55°C for 1 minute, vortexed for 30 seconds, left standing at room temperature for 1 hour, and centrifuged at 12000 rpm at 4°C for 10 minutes. The precipitate was the chitosan / dsRNA nanoformulation.

[0043] Example 3 Synergistic effect experiment of chemical pesticides and nano-RNA pesticides against Spodoptera exigua The nano-RNA pesticide used in this example was the chitosan / dsRNA nanoformulation prepared in Example 2.

[0044] 1. Chitosan / dsRNA Nanoparticle Feeding Experiment Thirty 4-day-old Spodoptera exigua larvae were selected and divided into groups of 10, with 3 replicates for each experimental group. At the same time, thirty larvae of the same age were selected as the blank control group, and an internal reference control group was also set up. The eGFP dsRNA in the internal reference control group was dsRNA transcribed in vitro using SEQ ID No.2 as the template sequence, and the nanoparticles in the internal reference control group were prepared according to the method of Example 2. The chitosan / dsRNA nanoparticles were pipetted onto the larval diet at a dose of 5 μg dsRNA per larva. The treated larvae were placed in an artificial diet in a constant temperature biochemical incubator at 28°C for 24 h (light:dark time = 14 h:10 h, temperature 26 ± 2°C, humidity 40%).

[0045] 2. Chemical Pesticide Treatment After the above larvae were treated for 24 h, the LC 30 concentration of emamectin benzoate·lufenuron solution at 83.817 mg / L was evenly sprayed on the larval body surface, food, and living environment. Then the treated larvae were placed in an artificial diet in a constant temperature biochemical incubator at 28°C (light:dark time = 14 h:10 h, temperature 26 ± 2°C, humidity 40%).

[0046] 3. Observation of Larval Phenotype and Detection of Survival Rate after Synergistic Treatment After treatment with chitosan / dsRNA nanoparticles, the larvae in the blank control group grew well, while most of the larvae in the experimental group (Spodoptera exigua ATPase A gene dsRNA nanoparticles) showed anorexia 24 h after treatment. After subsequent spraying with emamectin benzoate·lufenuron, the physiological activity of the larvae in the experimental group decreased, they showed anorexia, and their bodies were lying flat ( Figure 2 ), and then they began to lose vital signs. The mortality rate reached over 63% at 96 h (4 days), over 85% at 120 h (5 days), and 100% at 144 h (6 days) ( Figure 3 ), indicating a significant insecticidal effect.

[0047] In addition, in this example, the mortality rate of larvae after feeding with chitosan / dsRNA nanoparticles alone was also detected according to the above feeding experiment. The test results are as Figure 4 shown. By comparison, it can be seen that the mortality rate after synergistic treatment increased from 23.33% after treatment with nanoparticles alone to 100%.

[0048] 4. Detection of the Silencing Effect of the Spodoptera exigua ATPase A Gene after Synergistic Treatment After 48 hours of treatment, 3 larvae were randomly selected, total RNA was extracted, reverse transcribed into the first-strand cDNA, and the Real-time PCR method was used to detect the change in the expression level of the ATPase A gene in Spodoptera exigua, and the gene silencing efficiency was calculated. Using β-actin as the internal reference gene, the upstream and downstream primers of the ATPase A gene in Spodoptera exigua in Real-time PCR are shown in SEQ ID No.3 and SEQ ID No.4, and the upstream and downstream primers of the internal reference gene are shown in SEQ ID No.5 and SEQ ID No.6. 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 (AG AccurateBiology), 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.

[0049] The results showed that, compared with the internal reference control group, after 48 hours of treatment with the experimental group (dsRNA nanopreparation of the ATPase A gene in Spodoptera exigua), the expression level of the ATPase A gene in Spodoptera exigua decreased significantly by nearly 3 times ( Figure 5 ).

[0050] Finally, it should be noted that the above embodiments 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 embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A pesticide combination formulation, characterized in that: Including beet armyworm ATPase A gene dsRNA nano preparation and chlorfenapyr.

2. The combined formulation according to claim 1, characterized in that: The dsRNA of the Spodoptera exigua ATPase A gene is synthesized according to the nucleotide sequence shown in SEQ ID No.

1.

3. The combined formulation according to claim 2, characterized in that: The nano preparation comprises dsRNA of Spodoptera exigua ATPase A gene and a nano carrier.

4. The combined formulation according to claim 3, characterized in that: The nanocarrier is chitosan.

5. The combined formulation according to claim 4, characterized in that: The preparation method of the beet armyworm ATPase A gene dsRNA nano preparation 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 preparation.

6. Use of the combined formulation according to any one of claims 1 to 5 in agricultural pest management, agricultural pest control or killing agricultural pests.

7. A method for killing agricultural pests, characterized in that: include: The beet armyworm ATPase A gene dsRNA nano preparation and chlorfenapyr-lufenuron described in claim 1 are sequentially applied to agricultural pests.

8. The method according to claim 7, characterized in that The usage amount of the nano preparation is 3-8 μg / head; and / or the effective net content of emamectin benzoate and lufenuron is 8%-15% (w / w), and the usage amount is 60-100 mg / L.

9. The method according to claim 7, characterized in that: The nano preparation is firstly used to feed agricultural pests, and then 24 hours later, emamectin-lufenuron is sprayed on the agricultural pests.

10. The method according to any one of claims 7 to 9, characterized in that: The agricultural pest is a Lepidoptera pest, more preferably Spodoptera exigua.

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