Preparration And Biological Activity Of Nanopesticide Targeting The Chlorantraniliprole Resistance Gene Of Chilo Suppressalis

Pesticides are important materials in agricultural production,but unscientific and unreasonable use makes the resistance of pests evolve rapidly,and the management of resistance to pests has become an important issue.Silencing genes related to pest metabolic enzymes by RNAi can reduce the metabolic resistance to insecticides.It can be used as a potential tool for resistance management by using nanocarriers to co-deliver dsRNA for resistance gene and pesticide molecules.Mesoporous organosilica nanoparticle(MON)is a class of inorganic nanomaterials with high specific surface area and easy modification property.The construction of a co-delivery system based on MON can carry dsRNA and pesticide molecules into insects at the same time.The co-delivery system can deliver dsRNA to silence resistance-related genes and release pesticide molecules to achieve the resistance management of pests.This study intends to take Chilo suppressalis as the research object,the gene related to C.suppressalis resistance as the target,and construct an efficient co-delivery system of pesticide molecules and dsRNA based on mesoporous organosilica nanoparticles to achieve precise and efficient C.suppressalis resistant management.The main results of this study are as follows:(1)A synthetic method for efficiently inducing and expressing CsCYP321F3 and CsUGT40AL1 related to the resistance of C.suppressalis was constructed based on L4440 plasmid and Escherichia coli HT115(DE3)strain by using molecular biology technology.The dsRNA bands produced by inducible expression are clear and correct.(2)Mesoporous organic silica nanoparticles(MON)with a particle size of about 80 nm was synthesized by a hydrothermal method using cetyltrimethylammonium bromide and sodium salicylate as co-templates,tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-tetrasulfide as silicon source.MON was modified by carboxylation with silane coupling agent 3-(ethoxysilyl)propyl succinic anhydride and then loaded chlorantraniliprole.Under the catalysis of EDS and NHS,PEI was bound to the surface of MON by amide reaction,and finally the loading of dsRNA was completed under the electrostatic effect,and the co-delivery nanocarrier dsRNA/Chl@MON-PEI was successfully constructed.The co-delivery nanocarriers were analyzed by a number of characterization techniques,and the results showed that the dsRNA/Chl@MON-PEI nanocarriers were successfully prepared,with uniform particle size,chlorantraniliprole loading efficiency of 14.40%,and dsRNA loading efficiency was about 10%.(3)The stability test showed that MON-PEI can improve the stability of dsRNA,protect dsRNA from RNAase degradation,and prolong the storage time of dsRNA.Cell experiments showed that MON can effectively reduce the cytotoxicity of PEI,and MONPEI can effectively increase the uptake of dsRNA by cells.Compared with naked dsRNA treatment,the expression of target genes in cells treated with dsRNA@MON-PEI was significantly reduced by 31.92%.The results of bioassay showed that the co-delivery nanocarrier could effectively overcome the resistance of chlorantraniliprole to C.suppressalis and enhance the toxicity of chlorantraniliprole against C.suppressalis.Compared with Chl@MON-PEI treatment,the median letheal concentration of ds CsCYP321F3/Chl@MON-PEI and ds CsUGT40AL1/Chl@MON-PEI for C.suppressalis significantly decreased from 48.64 mg/L to 32.37 mg/L and 30.14 mg/L.qRT-PCR was used to measure the changes in the expression of target genes in C.suppressalis after treatment with dsRNA and dsRNA@MON-PEI.The results showed that the application of nanocarriers could effectively deliver dsRNA into C.suppressalis and plays an important role of RNAi to silence the target genes.