Discovery And Activity Evaluation Of Candidate Chemical And Nucleic Acid Insecticides Targeting Chitin Metabolic Enzymes

Irrational and absurd use of traditional pesticides leads to serious environmental and resistance problems.The development of highly selective pesticides is an effective approach to solve these problems..The chitinase Chi-h is exclusively present in lepidopteran insects,which is indispensable for their growth and development.Therefore,Chi-h has been confirmed to be an ideal target for the development of highly selective pesticides.However,few specific inhibitors of this enzyme have been identified.On the other hand,RNA interference(RNAi)is a suitable technique for developing highly selective pesticides due to its unique molecular mechanism.However,poor oral effects of RNA pesticides impedes its application.Aiming to solve the above two problems,a specific inhibitor targeting Of Chi-h with insecticidal activity were obtained via inhibitor screening and bioactivity evaluation.On the other hand,a candidate oral RNA nanopesticide suitable for locust control were prepared by targeting the chitin synthase.The main results of this thesis are as follows:1.Screening for highly selective inhibitor of Of Chi-h.Lynamicin B was identified as a selective inhibitor of Of Chi-h,which had almost no inhibitory activity against other GH18family chitinases.The K_i value of lynamicin B against Of Chi-h was 8.76μM.The co-crystal structure of lynamicin B and Of Chi-h demonstrated that the monochloroindolyl group of lynamicin B is located at the+1 subsite of the substrate-binding cleft and forms aπ-πstacked with Trp268,the dichloroindolyl group of lynamicin B occupied an unexplored pocket below subsites+1 and+2 of the substrate-binding cleft and formed multiple hydrogen bonds with surrounding residues,which was vital for its selectivity.2.Bioactivity of lynamicin B.Lynamicin B severely inhibited the growth and development of lepidopteran pests(Ostriniae.furnacalis,Mythimna separata,and Spodoptera frugiperda).The insects treated with lynamicin B were dead with severely shriveled bodies.Moreover,lynamicin B showed non-toxicity to Trichogramma ostriniae,a natural enemy of O.furnacalis.3.Preparation and characterization of RNA pesticides.The weight ratio of PEG-PLys(SH)and ds RNA was 10:1.The particle size of the ds RNA@PEG-PLys(SH)was 55 nm,the zeta potential was 21 m V,and the PDI was 0.295.The disulfide bond formed by crosslinking thesulfhydryl groups in PEG-PLys(SH)could significantly improve the structural stability of ds RNA@PEG-PLys(SH).The PEG-PLys(SH)imparted substantial protection of the ds RNA in the physiological environments of the digestive tract.In addition,the ds RNA@PEG-PLys(SH)was capable of efficiently extravasating the lining of midgut epithelial cells through the narrow fenestrations into the hemolymph enabling extensive biodistribution.Moreover,PEG-Plys(SH)improved the adhesion ability of ds RNA to leaves.4.Evaluation of biological activity of RNA pesticide targarting chitin synthase.PEG-Plys(SH)can significantly increase the efficiency of oral RNAi in locusts,causing distinct phenotypes.The expression level of midgut specific gene Lm CHS2 was down-regulated by approximately 60%in the Locusta migratoria nymphs fed with ds Lm CHS2@PEG-PLys(SH).Approximately 20%of the nymphs was dead with abnormal phenotype,including thin abdomens,amorphous and discontinuous peritrophic matrix.The expression level of non-midgut specific gene Lm CHS1 was down-regulated by approximately 50%in the L.migratoria nymphs fed with ds Lm CHS1@PEG-PLys(SH).Approximately 10%of the nymphs appeared distinctive lethal phenotype with molting failure,15%of the nymphs molted successfully,but the tibiae of the newly molted nymphs were curved and unable to locomote,the chitin content in the cuticle was significantly reduced.