Cloning And Expression Of Genes Correlated With Target Resisitance Against Avermectin In Tetranychus Cinnabarinus

The carmine spider mite(CSM), Tetranychus cinnabarinus (Boisduval), is one of the most important pests on cotton, vegetables and other crops. It is widely distributed in China. Chemical control is still the main way to control the mtie at present. Resistance against acaricides in CSM develops more quickly than other crops pests because of short generation cycle, a high fecundity, more opportunities to contact pesticides and a high inbreeding rate in CSM.Avermectin is one of macrocyclic lactone compounds which is effective in killing insects, mites, and nematodes. As a broad-spectrum and highly effective pesticide, abamectin has been widely used to prevent and control pests in agriculture, forestry and hygiene. With increased frequency of application, there are a lot of pests having developed resistance against it. So, target resistance against avermectin arises as the focus of research in recent years.In this sduty, the possible γ-aminobutyric acid receptors (GABARs) and Glutamate-gated chloride channels (Glucls), which may be correlated with target resisitance Against avermectin in Tetranychus cinnabarinus, were cloned via molecular biology techniques; Amino acid sequences of cloned genes in susceptive strains (SS) and in abamectin-resistant strains(AbR) were compared; The mRNA expression differences of cloned genes in different developmental periods (egg, protonymph, nymphae and adults) and different strains (SS, AbR and FeR) were analysed. Interaction bewteen resisitance against avermectin and GABARs or Glucls were investigated preliminarily at molecular level, which is expected to pave the way to locate the target of Avermectin, and inspire ideas for development of novel acaricides. The main results obtained from this research are as follows:Complete cDNA of two Glucl genes in CSM, TcGlucl1and TcGlucl2, were cloned, with the accession numbers JX501237, JX548319respectively in GenBank. Open reading frame (ORF) of TcGlucl1is1365bp that encoded a protein subnit containing454amino acids; The open reading frame (ORF) of TcGlucl2is1650bp that encoded a protein subnit containing550amino acids. Tansmembrane domain projections speculated that TcGlucl1has four transmembrane domains, and TcGlucl2has five transmembrane domains. Molecular phylogenetic tree analysis showed in the18species chosen for analysis, two Glucls in CSM are the closest with those in Tetranychus urticae, the second is Glucl in Ixodes scapularis, the last is the Glucl in Nematoda. According to the root edge relations of molecular phylogenetic tree, The Glucls in CSM and Tetranychus urticae of Arachnoidea and in Cooperia oncophora of Nematoda are relatively primitive ones among Glucls in the18species. The sequence homology between TcGlucl1and TcGlucl2is48.56%, and the major differences are the number of structure domains and the location of loop, according to the space structure simulation.A full length and four fragment cDNA of GABAR genes named TcGABAA2, TcGABAA1, TcGABAA3, TcGABAB1and TcGABAB2respectively were cloned, with the accession numbers EU362111, KC708069, KC708070, KC708071and KC708072respectively in GenBank. Sequence alignment conducted in NCBI indicated they are GABAR.Amino acid sequences of GABARs and Glucls between in SS and AbR were compared. The result showed that no differences at amino acid levels were found among TcGABAA1, TcGABAA2, TcGABAB2, TcGlucl1and TcGlucl2, and that TcGABAA3and TcGABAB1in AbR showed1and2amino acid mutations respectively, compared with those in SS. Detailedly, amino acids of TcGABAA3showed N280D mutation, and amino acids of TcGABAB1showed N219S and S396P mutations. These amino acid differences might be associated with avermectin resistance.The mRNA expression levels of GABARs and Glucls in different developmental period (eggs, protonymphae, nymphae and adults) were detected by Real-Time PCR technique. The results showed that the GABARs and Glucls were expressed at all developmental period of CSM. Gene expression levels in protonymphae and nymphae were generally highest. Those in eggs were generally lowest. While those in adults were generally between in protonymphae, nymphae and in eggs.The mRNA expression levels of GABARs and Glucls in in different strains (SS, AbR and FeR) were detected by Real-Time PCR technique. The results showed there are not significant differences in mRNA expression of TcGlucl1, TcGlucl2, TcGABAA1and TcGABAA2between in AbR and FeR, but those in AbR and FeR are significantly lower than in SS. No significant differences in mRNA expression of TcGABAB1and TcGABAB2were found among SS, AbR and FeR. The mRNA expression of TcGABAA3in AbR is significantly lower than those in SS and FeR, while no significant differences in latter two strains were found.