Preparation and application of dsMpC002-coated ZIF-8 nano-composite

Abstract

The invention relates to the technical field of biology, and particularly provides preparation and application of a dsMpC002-coated ZIF-8 nano complex. According to the invention, a green peach aphid effector dsMpC002 is taken as a target, a ZIF-8 delivery system of the green peach aphid effector dsMpC002 with a stable superexcellent target is constructed, and a dsMpC002-ZIF-8 nano-composite is prepared and obtained. By observing and analyzing the MpC002 gene expression quantity, the death rate and the single female farrowing quantity of the green peach aphid after the pepper variety DYJ sprayed with the dsMpC002-coated ZIF-8 nano-complex with different concentrations is fed, the control effect of the dsMpC002-coated ZIF-8 nano-complex on the green peach aphid is determined. And a new target technology and a new idea are provided for innovating an aphid control method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the preparation and application of a dsMpC002@ZIF-8 nanocomposite. Background Art

[0002] Myzus persicae Myzus persicae (Sulzer) belongs to the order Hemiptera and the family Aphididae. It is a widely edible and dangerous pest in the world. It has nearly 285 host plants. It can not only directly suck pears, peaches, plums, plums, cherries and other Rosaceae fruit trees and peppers, cabbage, kale, radish and other vegetables, but also is the vector of 116 dangerous viruses in the world, such as cucumber mosaic virus (CMV), potato virus Y (PVY) and tobacco etch virus (TEV), and the losses caused by the transmission of viruses are more serious. The peach aphid has a short life cycle, a large number of reproduction, frequent disasters, and great difficulty in prevention and control. At present, the production of fruits and vegetables mainly relies on pesticides to prevent and control the occurrence and harm of peach aphid, but the problems of agricultural product safety, pest resistance, and ecological environment safety of the production area caused by a large number of high-frequency and unreasonable use of drugs are very prominent, which seriously restricts the healthy and sustainable development of the fruit and vegetable industry. Therefore, seeking green and effective ways to prevent and control peach aphids has become a major problem that needs to be solved in the development of the fruit and vegetable industry.

[0003] The effector factor MpC002 is the most characteristic protein identified in the saliva protein of peach aphid. It can help it manipulate host cells to promote its own infection ability and then successfully complete colonization. It plays an important role in the host affinity of peach aphid.

[0004] RNAi (RNA interference, RNAi) is a phenomenon in which dsRNA (double-strand RNA) homologous to the target gene is introduced into living organisms to cause gene silencing. Compared with other research methods, it has advantages such as specificity, safety and high efficiency, and is recognized as a promising pest control and resistance management method at home and abroad. Exploring efficient functional genes and constructing a stable double-stranded RNA (dsRNA) delivery system are of great significance to improving the application potential of RNAi technology.

[0005] by MpC002 Designing dsRNA for target control of green peach aphid has certain prospects, but it also faces some challenges. First, the stability and delivery efficiency of dsRNA are key factors affecting its control effect. Chinese patent CN114921467A provides a method based on green peach aphid effector factor. MpC002Although the dsRNA of the gene and its application in the prevention and control of peach aphids provide an RNAi technology system to construct dsRNA that can effectively prevent and control aphids, in practical applications, it is urgent to develop a more stable and efficient dsRNA delivery system to improve the efficiency of RNAi. Secondly, peach aphids may develop resistance to dsRNA, which requires further research on the resistance mechanism of peach aphids to dsRNA and the adoption of corresponding countermeasures. In addition, the safety of dsRNA is also an issue that needs to be paid attention to, and a strict safety assessment needs to be carried out to ensure that its impact on non-target organisms and the environment is minimized.

[0006] In recent years, the delivery of dsRNA through nanomaterials has become a hot research trend, which can solve the problems of poor stability and easy degradation of dsRNA itself, and achieve the purpose of effectively preventing and controlling certain plant pests. This technology has multiple advantages such as environmental friendliness, long-lasting effect, and harmlessness to crops, and can effectively prevent and control plant pests. Although RNAi nanoformulations have been studied in the prevention and control of peach aphid pests, there are few studies on the screening of highly effective fragments. Therefore, after screening the highly effective fragments, the ideas of the existing technology are effectively used to make efficient nanoformulations, and the preparations are used in the prevention and control of peach aphid pests, which is of great significance for the large-scale prevention and control of the pest.

[0007] Therefore, designing a more efficient dsRNA delivery system has become an urgent issue to be addressed. Summary of the invention

[0008] At present, aphid control relies too much on chemical control, which leads to prominent problems such as agricultural product quality safety, pest resistance, and ecological and environmental safety of production areas, as well as technical bottlenecks such as poor target effect of aphid control using gene silencing technology. In view of the situation existing in the prior art, the purpose of the present invention is to combine RNAi technology and nanomaterials to construct a stable ZIF-8 delivery system of the peach aphid effector dsMpC002 with excellent target, prepare a dsMpC002@ZIF-8 nanocomplex, clarify the control effect of the dsMpC002@ZIF-8 nanocomplex on peach aphids, and provide a new target technology and a new idea for innovating aphid control methods.

[0009] In order to achieve the above object, the present invention provides the following technical solutions: The first aspect of the present invention provides a dsMpC002@ZIF-8 nanocomplex, wherein the dsMpC002@ZIF-8 nanocomplex is prepared from dsRNA and ZIF-8 nanomaterials, wherein the nucleotide sequence of the dsRNA is shown in SEQ ID NO.1.

[0010] Furthermore, the target gene of the dsRNA is MpC002Gene, the MpC002 The gene CDS sequence is shown as SEQ ID No.2.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned dsMpC002@ZIF-8 nanocomposite, and the specific steps are as follows: Step 1: synthesize dsRNA and prepare a dsRNA mother solution with a concentration of 100 mg / mL; MpC002 The gene CDS sequence is shown in SEQ ID No. 2, and the nucleotide sequence of the dsRNA is shown in SEQ ID NO. 1; Step 2: 1 g Zn(NO 3 ) 2 6H 2 O was dissolved in 10 mL of deionized water. After dissolution by magnetic stirring, 100 mg of the dsRNA stock solution obtained in step 1 was added and stirred evenly to obtain a mixed solution. Step 3: After completely dissolving 13.76 g of 2-methylimidazole in 90 mL of deionized water, add the mixture to the mixed solution of step 2, stir magnetically at room temperature for 30 min, and collect spheroids with a diameter of 200-300 nm, i.e., nanoparticles, at 10,000 g. After washing with deionized water three times, the dsMpC002@ZIF-8 nanocomposite was prepared.

[0012] Further, in step 1, the process of synthesizing dsRNA is as follows: (1) Extracting total RNA from peach aphids, performing reverse transcription using the extracted total RNA as a template to obtain cDNA; (2) Using cDNA as a template, designing specific amplification primers dsMpC002-F and dsMpC002-R carrying a T7 promoter for synthesizing dsRNA, and using the specific primers to amplify the target gene fragment; (3) Using the target gene fragment obtained by specific amplification as template DNA, dsRNA was synthesized in vitro. The reaction system was as follows: 4 μl of 5X TranscriptAid Reaction Buffer, 2 μl each of ATP solution, CTP solution, GTP solution, and UTP solution, 1 μl of template DNA, 2 μl of TranscriptAid Enzyme Mix, and DEPC-treated water to 20 μl.

[0013] Furthermore, in step (2), the nucleotide sequence of the dsMpC002-F is shown in SEQ ID NO. 3, and the nucleotide sequence of the dsMpC002-R is shown in SEQ ID NO. 4.

[0014] The third aspect of the present invention provides the use of the above dsMpC002@ZIF-8 nanocomposite in preventing and controlling green peach aphid damage on pepper plants. The method of use is: dissolving the nanocomposite with clean water to prepare a solution, and then spraying the solution onto the leaves of the pepper plants.

[0015] Furthermore, the concentration of the solution is 500-1000 ng / mL.

[0016] The present invention deeply explores the effect factors that regulate the feeding damage of peach aphid MpC002 Gene function and its metal organic framework ZIF-8 nanomaterial that significantly enhances its dsRNA efficient target function, a nano preparation of ZIF-8 encapsulated peach aphid salivary protein effector dsMpC002 is prepared, and the beneficial effects of the present invention are as follows: (1) The use of excellent nanomaterial ZIF-8 simplifies the preparation method of composite nanoformulations, making the method more convenient and easy.

[0017] (2) The combination of in vitro synthesized dsRNA and ZIF-8 nanomaterials improves the stability of dsRNA, making the prevention and treatment effect more efficient and obvious.

[0018] (3) The nanoformulation provided by the present invention can effectively inhibit the effector factors of peach aphid MpC002 Gene expression significantly increases the mortality rate of peach aphids and significantly reduces the reproductive capacity of peach aphids. The control effect is far greater than directly spraying dsRNA solution.

[0019] (4) MpC002 The gene works only when peach aphids feed on plants, and has obvious species specificity. The application of dsMpC002@ZIF-8 nanocomplex to control peach aphids has the advantages of good species specificity, safety and high efficiency, and has good market application prospects and potential economic and ecological benefits in the green control of aphids. It can provide a new target technology and new ideas for innovative peach aphid control methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Myzus persicae MpC002 Gene PCR amplification electrophoresis: M: DNA marker; 1: Myzus persicae MpC002 Gene.

[0021] Figure 2 Effect factors of different concentrations of dsMpC002@ZIF-8 complex on green peach aphid MpC002 Inhibitory effect on gene expression.

[0022] Figure 3 The inhibitory effect of different concentrations of dsMpC002@ZIF-8 complex on the reproductive capacity of green peach aphid.

[0023] Figure 4 The lethal effects of different concentrations of dsMpC002@ZIF-8 nanocomplex on green peach aphid.

[0024] Figure 5 The field control effect of dsMpC002@ZIF-8 nanocomplex on peach aphid. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0026] Example 1 Myzus persicae effector factor MpC002 Synthesis of dsRNA of genes Myzus persicae effector MpC002 Gene CDS sequence (SEQ ID NO. 2), primers carrying T7 promoter for synthesizing dsRNA (SEQ ID NO. 1) were designed using the primer design website E-RNAi (https: / / www.dkfz.de / signaling / e-rnai3 / idseq.php), upstream primers dsMpC002 -F (SEQ ID NO. 3) and downstream primers dsMpC002 The nucleotide sequence of -R (SEQ ID NO. 4) is as follows: dsMpC002 -F:taatacgactcactatagggGTGCACCAGGAAAAGTATGT dsMpC002 -R: taatacgactcactatagggGACGTTGTCCTCTCCAATTA.

[0027] The synthesis process was referred to the instruction manual of MEGA script RNAi Kit (Thermo Scientific, Wilmington, DE, USA).

[0028] (https: / / www.thermofisher.com / cn / zh / home / references / protocols / rnai-epigenetics-and-gene-regulation / rnai-protocol / megascript-rnai-kit.html) The method for synthesizing dsRNA specifically comprises the following steps: (1) Extracting total RNA from peach aphids, performing reverse transcription using the extracted total RNA as a template to obtain cDNA; (2) Using cDNA as a template, designing specific amplification primers dsMpC002-F and dsMpC002-R carrying a T7 promoter for synthesizing dsRNA, and using the specific primers to amplify the target gene fragment; (3) Synthesize dsRNA in vitro using the target gene fragment obtained by specific amplification as template DNA: Thoroughly mix the components of the 20µL reaction system, rotate briefly to collect all droplets, and incubate at 37°C for 2h. The reaction system is as follows: 4μl of 5XTranscriptAid Reaction Buffer, 2μL each of ATP solution, CTP solution, GTP solution, and UTP solution, 1μl of template DNA, 2μl of TranscriptAid Enzyme Mix, and add DEPC-treated water to 20μL. Add 115 μL of DEPC-treated water and 15μL of 3 M sodium acetate (pH 5.0) solution to the 20μL reaction mixture, mix thoroughly, then extract with an equal volume of 1:1 phenol (pH 4.7) / chloroform mixture, and extract twice with an equal volume of chloroform, collect the aqueous phase and transfer it to a new centrifuge tube. Then, the RNA was precipitated by adding 2 volumes of ethanol (incubated at -20°C for at least 30 min), and the precipitate was collected by centrifugation (4°C, 12000×g, 10 min), the supernatant was removed and the precipitate was washed with 500 µL of cold 70% ethanol, and finally the precipitate was collected by centrifugation (4°C, 7500×g, 5 min), the supernatant was decanted and the precipitate was dried to obtain dsRNA.

[0029] dsRNA quality and concentration detection: The obtained dsRNA was redissolved in 20 µL of DEPC-treated water. The purity and integrity of the dsRNA were detected using Nanodrop 2000 (Thermo) and agarose gel electrophoresis (voltage 130 V; current 130 mA; 20 min); its concentration was measured using a micro-UV spectrophotometer. Finally, the dsRNA was prepared into a 100 mg / mL dsRNA stock solution and stored at -20°C or -70°C for future use.

[0030] Example 2 Preparation of dsMpC002@ZIF-8 Nanocomposite

[0031] Put 1 g Zn(NO 3 ) 2 6H 2 O was dissolved in 10 mL of deionized water, and after being dissolved by magnetic stirring, 100 mg of the dsRNA mother solution obtained in step 1 was added and stirred evenly to obtain a mixed solution; 13.76 g of 2-methylimidazole was completely dissolved in 90 mL of deionized water, and then added to the mixed solution, and magnetic stirring was performed at room temperature for 30 minutes. Spheroids with a diameter of 200-300 nm, i.e., nanoparticles, were collected by centrifugation at 10,000 g, and washed with deionized water three times to obtain a dsMpC002@ZIF-8 nanocomplex.

[0032] Example 3 Control effect of dsMpC002@ZIF-8 nanocomplex on green peach aphid

[0033] With clean water, ZIF-8 and dsMpC002 as controls, ZIF-8, dsMpC002 and the dsMpC002@ZIF-8 nanocomplex obtained in Example 2 were dissolved in clean water and set to 100 ng / mL, 200 ng / mL, 300 ng / mL, 500 ng / mL and 1000 ng / mL, respectively, and then wingless female adult peach aphids of the same developmental period and size were inoculated on the upper leaves of pepper variety DYJ plants that had been transplanted for 30 days and sprayed with different concentrations of ZIF-8, dsMpC002 and dsMpC002@ZIF-8 nanocomplexes. The number of live peach aphids and the number of offspring per female were observed and counted at 1d, 2d, 4d and 8d after inoculation, and the live insects were collected for total RNA extraction of peach aphids, and then the peach aphid effector factor was analyzed. MpC002 qPCR analysis of gene expression ( Figure 1 ). Each plant was inoculated with 6 aphid leaves, each leaf was inoculated with 30 aphids, and each concentration treatment was set up with 3 replicates. The results showed that ZIF-8500 ng / mL had a significant effect on the growth of green peach aphid. MpC002 The 500 ng / mL and 1000 ng / mL dsMpC002@ZIF-8 nanocomplexes had the best effect, not only significantly inhibiting the growth of green peach aphids, but also significantly inhibiting the growth of green peach aphids. MpC002 Gene expression ( Figure 2 ), and can significantly reduce the reproductive capacity of green peach aphid ( Figure 3 ), significantly increasing the mortality rate of peach ( Figure 4 and Figure 5 ), the control effect is far greater than directly spraying dsRNA solution.

[0034] In summary, this example confirms that the dsMpC002@ZIF-8 nanocomplex has a good control effect on peach aphids, provides a direct scientific basis for the wide application of the dsMpC002@ZIF-8 nanocomplex in the control of peach aphids, and provides a new target technology and new ideas for innovating peach aphid control methods.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dsMpC002@ZIF-8 nanocomposite, characterized in that: The dsMpC002@ZIF-8 nanocomplex is prepared from dsRNA and ZIF-8 nanomaterials, wherein the nucleotide sequence of dsRNA is shown in SEQ ID NO.

1.

2. The dsMpC002@ZIF-8 nanocomposite according to claim 1, characterized in that: The target gene of the dsRNA is MpC002 Gene, MpC002 The gene CDS sequence is shown in SEQ ID No.

2.

3. A method for preparing a dsMpC002@ZIF-8 nanocomposite according to claim 2, characterized in that: The specific steps are as follows: Step 1: synthesize dsRNA and prepare a dsRNA mother solution with a concentration of 100 mg / mL; MpC002 The gene CDS sequence is shown in SEQ ID No. 2, and the nucleotide sequence of the dsRNA is shown in SEQ ID NO. 1; Step 2: Dissolve 1 g Zn(NO3)2·6H2O in 10 mL deionized water, stir under magnetic stirring until dissolved, add 100 mg dsRNA stock solution obtained in step 1, and stir evenly to obtain a mixed solution; Step 3: After completely dissolving 13.76 g of 2-methylimidazole in 90 mL of deionized water, add the mixture to the mixed solution of step 2, stir magnetically at room temperature for 30 min, and collect spheroids with a diameter of 200-300 nm, i.e., nanoparticles, at 10,000 g. After washing with deionized water three times, the dsMpC002@ZIF-8 nanocomposite was prepared.

4. The method according to claim 3, characterized in that In step 1, the process of synthesizing dsRNA is as follows: (1) Extracting total RNA from peach aphids, performing reverse transcription using the extracted total RNA as a template to obtain cDNA; (2) Using cDNA as a template, designing specific amplification primers dsMpC002-F and dsMpC002-R carrying a T7 promoter for synthesizing dsRNA, and using the specific primers to amplify the target gene fragment; Wherein, the nucleotide sequence of the dsMpC002-F is shown in SEQ ID NO. 3, and the nucleotide sequence of the dsMpC002-R is shown in SEQ ID NO. 4; (3) Using the target gene fragment obtained by specific amplification as the template DNA, dsRNA was synthesized in vitro. The reaction system was as follows: 4 μl of 5XTranscriptAid Reaction Buffer, 2 μl each of ATP solution, CTP solution, GTP solution, and UTP solution, 1 μl of template DNA, 2 μl of TranscriptAid Enzyme Mix, and DEPC-treated water to 20 μl.

5. Use of a dsMpC002@ZIF-8 nanocomposite according to claim 1 or 2 in preventing and controlling green peach aphid damage on pepper plants, characterized in that: The method for using the nanocomposite is as follows: dissolving the nanocomposite with clean water to prepare a solution, and then spraying the solution onto leaves of pepper plants.

6. The method for controlling green peach aphid damage on pepper according to claim 5, characterized in that: The concentration of the solution is 500-1000 ng / mL.

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