Identification Of Target And Resistant Genes Related To Abamectin In Tetranychus Cinnabarinus

The avermectins are extensively used in agriculture and animal husbandry and used to control the plant pest, spider mite, nematode as well as parasitic diseases. And Japanese scientist Satoshi ōmura and Irish scientists William C. Campbell won the 2015 Nobel Prize for medicine for discovering avermectin. However, research on the mechanism of avamectin as well as mite resistance to abamectin are still rare and in progress. In present, there are mainly two problems in the toxicological mechanism study of mite resistance to abamectin. On the one hand, traditional research on abamecin resistance in mites mainly focuses on the three major metabolism enzyme systerms(cytochrome P450, glutathione S-transferase, carboxylesterase). If other new genes be involved in the abamectin metabolism and the mite resistance formation to abamectin? On the other hand, the target of mite to abamectin and target resistance mechanism are still controversial. Therefore, this dissertation set the above problems as ultimate aim and achieved the following results. 1. Microarray detected the differentially expressed genes between the susceptible and abamectin resistant strains of T. cinnabarinusIn previous study, RNA sequencing(RNA-seq) and digital gene expression profile were executed to establish a repository of differentially expressed genes(DEGs) in T. cinnabarinus, and in this dissertation, a custom-made microarray was utilized to confirm the DEGs between susceptible(SS) and abamectin resistant strains(Ab R). The results showed that a total of 52 DEGs including 21 up-regulated and 31down-regulated genes were detected in the abamectin resistant strain compared to the susceptible strain. The GO anotation discovered that 14 DEGs could be annotated into 12 GO clusters. When considering the metabolic enzyme genes potentially involved in resistance, the strong response of the resistant strains against acaricide selection through transcription level modifications was confirmed. Detection of gene expression revealed that 2 GST genes, 1 P450 gene and 1 esterase gene were upregulated in the Ab R strain, while with 6 cuticle protein genes being down-regulated. We also detected several other biotransformation enzymes or proteins maybe participate in the insecticide resistance, including intradiolring-cleavage dioxygenase(ID-RCDs), serine protease and Cathepsin L, etc. 2. Cloning and expresson patten in four life stages of GABA and glutamate gated chloride channels genes in T. cinnabarinusFirstly, three full lengths of γ-aminobutyric acid(GABA) gated chloride channel(Tc GABACl) and five Glutamate gated chloride channel(Tc Glu Cl) subunits were obtained by T. cinnabarinus transcriptome analysis. Cloning of eight full length c DNA was performed using specific primers and submitted to the Gen Bank under accession number KF284894- KT284901. An alignment of their protein sequences shows that Tc Glu Cls have high similarity with homologous genes of other species especially with sibling species T. urticae. A maximum likelihood phylogenetic analysis of Glu Cls and GABACls subunits of T. cinnabarinus, Glu Cls and GABACls subunits of other insects, and 28 cys-loop LGIC subfamily gens of T. urticae indicated orthologous relationships between the mite and other insects and these genes could be classified into one subbranch. Furthermore, all of the three GABACls were classified as RDL homologous subunits(Tc RDL1-3) and no orthologues of GRD and LCCH3 was found in T. cinnabarinus. We next investigated the expression of the transcripts of eight genes in different developmental stages by quantitative PCP(q PCR). The expression of all 8 genes where lowest in eggs slightly different in other stages. 3. Functional expressions of Tc RDLs and Tc Glu Cls in Xenopus laevis oocytesUsing the Xenopus laevis oocytes and two-electrode voltage clamp, three GABACls and five Glu Cls subunits were successfully expressed. The phenomena of inward currents in the oocyte substantiated the function of channel proteins. Along with the increasing concentrations of GABA or L-glutamate, the current of oocytes also increased until up to the equilibrium concentration. Then the value of EC50 and electrophysiological parameters of channel proteins were calculated based on the current response with the Tc Glu Cls EC50 of Tc Glu Cl2(3.019±0.0354 M)> Tc Glu Cl3(2.983±0.0181 M)> Tc Glu Cl1(2.884±0.0472 M)> Tc Glu Cl4(2.851±0.0340 M)> Tc Glu Cl5(2.701±0.0380 M); and Tc GABACls of Tc RDL3(1.086±0.032 M)> Tc RDL2(0.677±0.042 M)> Tc RDL1(0.445±0.047 M). 4. Identification of abamectin target in T. cinnabarinusThe sensitivity of T. cinnabarinus to abamectin also declined after successful inhibition of 5 Tc Glu Cls and 3 Tc GABACls genes respectively by using RNAi technique, which illustrated that 5 Tc Glu Cls and 3 Tc GABACls are abamectin targets in T. cinnabarinus. On the other hand, the two-electrode voltage clamp test certificated that 5 Tc Glu Cls subunits were sensitive to abamectin and ivermectin, while 3 Tc GABACls are diametrically opposed. Finally, the voltage clamp and RNAi both demonstrated that Glu Cls are the molecular targets for abamectin and ivermectin in T. cinnabarinus. But GABACls as molecular targets for abamectin also cannot be excluded. 5. Target resistance mechanism analysis in T. cinnabarinusThere is no significant difference of expression levels of 8 chloride channels genes in the susceptible and abamectin resistant strains, which indicated a fact that mite resistance to abamectin has no relationship with the expression levels of these genes. And no mutation was discovered in the alignment of 8 chloride channels genes in the susceptible and abamectin resistant strains. It also indicates that mite resistance to abamectin has nothing to do with mutation in 8 chloride channels genes.