Gene Clone And Functional Study Of Insecticides Targets In Central Neuronal System In Bombyx Mori

With the widely application of chemical insecticides, pesticdes resistance has been the major problem in pest control. Many studies had indicated that the point mutation of target genes can induce the sencitivity decline is the most important mechanism in pesticdes resistance development. But there is no exact the action mode and researchers haven't to table a proposal about this problem, and most studies focus on the epidemical pests such as Culex pipens, Drosophila melanogaster, Musca domestica, and agriculture pest such as Heliothis virescens, Aphis gossypii, Myzus persicae. Until now there is no relevant study in Bombyx mori which has abundant genetic resources, good research basic and accumulated genome informations, so we take advantage of the silkworm genome database to do some research on the insecticides target genes, hope we can find some molecular clues which will help us to understand the mechanism of these phenomena deeply.The Central Neural System (CNS) has important role in regulating the development of insect, especially in the metamorphism development course. So, most insecticides target are focus on the insect central neural system, including Acetylcholinesterase(AChE), Acetylcholine receptor(AChR), para Sodium channel, GABA-gated chlorine channel, and Ecdysone hormone network. Through the gene levels study of insecticides targets, we can find out the mechanism of insecticides and the pesticides resistance. So, learning about the structure and function of insecticides neural targets is the basic of analysis insect's behavior, toxicological mechanism and new pesticide design.Combining with the silkworm genome database information, we use the electro-clone and RACE method to obtain the full length cDNAs of AChE (GenBank No. EF52189), AChR (GenBank No.EF521820) and GABA-gated chlorine channelαsubunit (GenBank No. EF521821), and partial cDNAs of para Sodium channel (GenBank No. EF521818) in Bombyx mori. Combining with the reported genes and we cloned genes, did the homology analysis and constructed phylogenetic tree of AChE1 and AChE2 from different species to ananlysis the evolution relations. We constructed the prokaryotic expression plasmid of AChE, named pET41b-AChE and transformed into E.coli to express the target gene, successfully expressed the AChE of silkworm, it is about 100kDa fusion protein with His and GST tags. Dissolubility analysis of the AChE fusion protein indicated that the expression products exist in inclusion bodies.In order to avoid the activity of expression protein affected by inclusion body, we select the insect Bac-to-Bac expression system to construct the eukaryotic expression plasmids of AChR and GABA-gated chlorine channelαsubunit. The constructed donor plasmids of pFASTBAC-HTa-AChR and pFASTBAC-HTa-GABA were transfected into the DH10BAC, and then select the positive clone and extract the Bacmid, using Bacmid to transfect the Tn-5 cells obtain the recombine virus, then amplificate the first generation virus and infect the Tn-5 cells to express the target protein. They were all efficiecently expressed in different expression systems.In the Ecdysone Hormone Network regulate system, we get a start research about the mechanism of Azadirachtin (Az), and this part work is done in Spodoptera litura. With the degenerate primer strategy we clone a 600bp cDNA fragment of Ecdysone Receptor (EcR) gene, using this sequence information design a pair of pimers to analysis the effect of Az treatment on the SL cells. We found that the Az treatments affect the normal adherent growth of cells and suspend of most cells, the higher concentration of Az will induce the cell cytosis and cell death. We also use the RNAi method to study the EcR function related with the Az toxicology. Injected the third instar larvae of Spodoptera litura with the EcR-dsRNA, we found that the EcR RNAi will influence the development course from larva to pupa. In our experiment, we found two abnormal phenotypes that one only the last three segments develop into pupa, but the other segments are still keep the larva characters; another sample has differently develop speed between abdomen and backside, which resulted in the back develop into pupa but the abdomen were still the larva characters. When we add Az in the feed of injected EcR-dsRNA samples, in the 13 live larvae we found that 6 have the abnormal proleg phenotypes (details in paper). It is very interesting and based on these results we deduced that the EcR gene may be involved in the construcation of tectology during insect metamorphosis and the Az maybe intervention this regulation process.