Spodoptera frugiperda chitinase 2 gene dsRNA and preparation and application of nano preparation thereof
By designing the dsRNA of the chitinase 2 gene of Fallia meadow and combining with chitosan, nanopreparations were prepared, and the resistance and environmental health problems of traditional chemical pesticides were solved, and efficient control of Fallia meadow was achieved, with significant gene silencing and lethal effects.
Patent Information
- Application Number
- CN202411917379.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-04-25
AI Technical Summary
Fallia meadow is resistant to traditional chemical pesticides, and the use of chemical pesticides leads to environmental and health problems. The existing dsRNA preparation process is complex and costly, and poor environmental stability. It is difficult to ensure effective entry into the pests.
Design the dsRNA molecule of the 2 gene of Fattuynia chitinase 2 and combine it with nanocarriers such as chitosan to prepare chitosan/dsRNA nanopreparations, and improve the protection and introduction efficiency of dsRNA through steps such as stand-alone, incubation, and vortex.
It has achieved efficient lethality of fall armyworms, with a gene silencing efficiency of 87.13% and a lethality rate of 75%, solving the environmental and health problems brought by traditional chemical pesticides and having broad market application prospects.
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Figure CN119876096A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of agricultural pest control, and in particular to the preparation and application of dsRNA of chitinase 2 gene of fall armyworm and nano preparation thereof. Background Art
[0002] Fall Armyworm Spodoptera frugiperda ) is an important invasive pest that harms food production and is a major migratory pest on the global warning list of the Food and Agriculture Organization of the United Nations. It has a diverse diet and many hosts (more than 350 species), and can harm more than 80 economic crops such as corn, sugarcane, rice, peanuts and peppers. The larvae eat the leaves, stems and even ears of crops, and can also "cut" the roots to harm seedlings, causing the death of crops in the seedling stage, resulting in serious economic losses.
[0003] At present, the control of fall armyworm is still mainly based on chemical pesticides, combined with physical trapping, biological control and other means. However, with the large-scale application of chemical pesticides, its resistance is increasing. At the same time, problems such as food safety and ecological safety caused by the large-scale use of pesticides are becoming increasingly prominent. Therefore, the development of new green and low-toxic pesticides and the reduction and increase of pesticide efficiency are major needs for the effective control of fall armyworm and the sustainable development of agriculture.
[0004] RNA pesticides are new pesticides created based on RNA interference (RNA interference, RNAi) technology. They inhibit the expression of important functional genes of pests, causing their developmental stagnation or death, thereby achieving the purpose of disease and insect pest control. They have many advantages such as specific prevention and control targets, convenient target development, easy application, green and pollution-free, no residue and strong environmental compatibility, which meet the public's demand for green pesticides.
[0005] RNA interference 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. However, there are still some challenges in the practical application of RNAi technology, which restricts its large-scale promotion. The preparation of dsRNA requires complex process flow and high costs; dsRNA has poor stability in the environment and is easily degraded by nucleases, affecting its insecticidal effect and sustainability; in addition, 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.
[0006] Nanomaterials such as chitosan, liposomes and star-shaped cationic polymers (SPc) can not only protect dsRNA, but also greatly improve the efficiency of dsRNA introduction. For example, when chitosan is used to carry EcR dsRNA to feed whiteflies, the mortality rate is increased to 80% compared with feeding EcR dsRNA alone.
[0007] 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
[0008] In a first aspect, the present invention provides a chitinase 2 gene of Spodoptera frugiperda, the nucleotide sequence of which is shown in SEQ ID No.1.
[0009] After a large number of screenings of different target genes of the fall armyworm, the present invention found that the dsRNA molecules designed for the chitinase 2 gene of the fall armyworm can efficiently silence gene expression. Experimental verification shows that the dsRNA synthesized by in vitro transcription using the gene sequence shown in SEQ ID No.1 can cause the fall armyworm to die due to abnormal molting or pupation, has a high mortality rate against the fall armyworm, can effectively prevent and control the fall armyworm, and solves the environmental and health problems caused by traditional chemical pesticides.
[0010] In a second aspect, the present invention provides a dsRNA obtained by transcription of the chitinase 2 gene of Spodoptera frugiperda.
[0011] In a third aspect, the present invention provides a method for synthesizing the dsRNA, comprising: extracting total RNA of 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] In a fourth aspect, the present invention provides a primer pair, wherein the primer pair is shown in SEQ ID No. 2 and SEQ ID No. 3, and the primer pair is used to amplify and obtain the chitinase 2 gene of Spodoptera frugiperda.
[0013] In a fifth aspect, the present invention provides an insecticide comprising the dsRNA.
[0014] In some embodiments, the pesticide is a nanoformulation.
[0015] In some embodiments, the pesticide comprises the dsRNA and a nanocarrier.
[0016] In some embodiments, the nanocarrier is chitosan.
[0017] In some embodiments, the pesticide is a chitosan / dsRNA nanoformulation.
[0018] In a sixth aspect, the present invention provides a method for preparing the insecticide, comprising: mixing chitosan, acetic acid and sodium acetate to prepare a chitosan solution; then mixing the chitosan solution with a dsRNA solution, and preparing the mixture by standing, incubating, vortexing, standing again and centrifuging to obtain the insecticide.
[0019] The chitosan / dsRNA nanoformulation insecticide prepared by the above method has good dsRNA protection effect, high penetration efficiency, and simple operation. It can specifically silence gene expression with a gene silencing efficiency of 87.13%, which ultimately leads to the death of fall armyworm due to abnormal molting or pupation, with an abnormality rate of 80% and a mortality rate before pupation of 75%.
[0020] In some embodiments, the concentration of the chitosan solution is 0.05% to 0.5% (w / v), more preferably 0.05% to 0.2%.
[0021] In some embodiments, the concentration of the dsRNA solution is 500-2000 ng / μl, more preferably 800-1200 ng / μl.
[0022] In some embodiments, the chitosan solution and the dsRNA solution are mixed in a volume ratio of (0.5-2):1.
[0023] Preferably, the chitosan solution and the dsRNA solution are mixed in a volume ratio of 1:(0.8-1.2).
[0024] In some embodiments, the standing time is more than 2 minutes.
[0025] In some embodiments, the incubation temperature is 50-60°C, preferably 52-58°C.
[0026] In some embodiments, the incubation time is more than 1 min.
[0027] In some embodiments, the vortex time is 10s to 50s, more preferably 25s to 35s.
[0028] In some embodiments, the step of allowing to stand again is allowing to stand at room temperature for more than 1 hour.
[0029] In some embodiments, the centrifugation temperature is 2-6°C and the rotation speed is above 12000 rpm.
[0030] In some embodiments, the centrifugation time is more than 10 min.
[0031] In some embodiments, the pesticide is in the form of a nanoformulation, a powder, a granule, an aqueous emulsion, an aerosol, or an emulsifiable concentrate.
[0032] In a seventh aspect, the present invention provides the use of the fall armyworm chitinase 2 gene, the dsRNA or the insecticide in at least one of the following aspects: (1) killing agricultural pests; (2) agricultural pest management; (3) agricultural pest control.
[0033] In some embodiments, the agricultural pest is a Lepidoptera pest, preferably Spodoptera frugiperda.
[0034] Compared with the prior art, the beneficial effects of the present invention are at least: The present invention provides a dsRNA molecule capable of utilizing RNAi technology to efficiently control fall armyworm, which has high specificity and high gene silencing efficiency for target genes, and the insecticide prepared by the dsRNA molecule can cause fall armyworm to die due to abnormal molting or abnormal pupation, has a high mortality rate to fall armyworm, has a rapid insecticidal effect, is easy to operate, can effectively control fall armyworm, solves the environmental and health problems caused by traditional chemical insecticides, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] 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.
[0036] 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.
[0037] Figure 3 is the change of chitinase 2 gene expression at different time points after nanoformulation treatment; β-actin is the internal reference gene, **p<0.01, ***p<0.001.
[0038] Figure 4 It is the growth and development changes of fall armyworm 1-10 days after treatment with nano-agent.
[0039] Figure 5 The weight changes of Spodoptera frugiperda larvae 1-10 days after treatment with nanoformulations.
[0040] Figure 6 The weight changes of pupae of Fall Armyworm after treatment with nano-preparation. DETAILED DESCRIPTION
[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. 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.
[0042] The reagents or instruments used in the following examples without indicating the manufacturer are all conventional products that can be purchased through regular channels.
[0043] 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 those skilled in the art to guide the experimental operations related to molecular biology. In addition, according to different experimental purposes, those skilled in the art complete the corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust specialized companies to perform the experiments.
[0044] Example 1 Synthesis of dsRNA of chitinase 2 gene of Spodoptera frugiperda 1. Primer design and synthesis: The dsRNA primers for the chitinase 2 gene of Spodoptera frugiperda (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 (containing T7 promoter), respectively. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0045] 2. Synthesis of dsRNA of chitinase 2 gene of fall armyworm The RNA of the whole insect of the third-instar fall armyworm was used as a template, and 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 323bp 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 dsRNA of chitinase 2 gene of Spodoptera frugiperda was stored in a -80°C ultra-low temperature refrigerator for future use.
[0046] Example 2 Preparation of chitosan / dsRNA nanoformulation 1. Solvent preparation 0.1 M acetic acid, 0.1 M sodium acetate, 100 mM sodium sulfate, 0.1% chitosan, and the chitinase 2 gene dsRNA of Spodoptera frugiperda 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.
[0047] 3. Chitosan / dsRNA nanoformulation 1000 ng / μl of fall armyworm chitinase 2 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, centrifuged at 4°C 12000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was the chitosan / dsRNA nanoformulation, and the agarose gel was used for detection. The results of the precipitate and supernatant were shown in Figure 2. Figure 1 , Figure 2 shown.
[0048] Example 3 Experiment on the killing effect of chitosan / dsRNA nanoformulation on fall armyworm 1. Backplane drip test 30 3-year-old and 2-day-old fall armyworms were selected, with 10 in each group, and the experiment was repeated 3 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 the chitosan / dsRNA nanoformulation prepared in Example 2 was dripped on the back plate of the larvae at an amount of 2 μg dsRNA per larvae as the chitinase 2 gene treatment group, and the dsRNA introduced into the internal reference gene eGFP was used as the internal reference control group. The dsRNA of the internal reference gene eGFP was a dsRNA synthesized by in vitro transcription using SEQ ID No.5 as the template sequence, and the nanoformulation of the internal reference control group was prepared according to the method of Example 2. After the nanoformulation was completely absorbed, 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%).
[0049] 2. Detection of chitinase 2 gene silencing in fall armyworm At 24h, 48h, 72h, 96h, 120h, 144h, and 168h after treatment, three larvae were collected at each time point, and total RNA was extracted by Trizol method, reverse transcribed into first-strand cDNA, and the expression of chitinase 2 gene was detected by Real-time PCR method, with β-actin as the internal reference gene. The upstream and downstream primers of chitinase 2 gene in Real-time PCR are shown in SEQ ID No.6 and SEQID 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 (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.
[0050] 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 2 gene in fall armyworm decreased significantly, and the gene silencing efficiency reached 87.13%, reaching an extremely significant level.
[0051] 3. Phenotypic observation of larvae after treatment with chitosan / dsRNA nanoformulation After the chitosan / dsRNA nanoparticles were dripped onto the back of the insect, all the larvae in the internal reference control group successfully molted to the next instar on the fourth day of the third 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 third 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 80%, and the final mortality rate before pupation reached 75%.
[0052] 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%.
[0053] 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 2 gene from Spodoptera frugiperda, characterized in that: Its nucleotide sequence is shown in SEQ ID No.
1.
2. A dsRNA, characterized in that It is obtained by transcribing the chitinase 2 gene of the fall armyworm as claimed in claim 1.
3. 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.
4. A primer pair, characterized in that The primer pair is shown as SEQ ID No.2 and SEQ ID No.
3.
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 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 2 gene according to claim 1, the dsRNA according to claim 2, or the insecticide according to any one of claims 5 to 9 in at least one of the following aspects: (1) Lethal agricultural pests; (2) Agricultural pest management; (3) Agricultural pest control.