Expression Characteristic Of Esterase Genes Suffering The Stress Of Pesticide In Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), one of the most important pests on cotton and most vegetables, is widely distributed in China. Because of high fecundity and short generation time, the mite can rapid formation of a certain number of stocks in a short period of time, develop resistance easily and thus it is difficult to prevent and control this mite.As a large class of enzymes that metabolize a variety of pesticides, esterases play important roles in priority hydrolysis of water-soluble short-chain esters of the ester. The research of esterase is very active in foreign and relative behindhand in domestic.This study was based on the esterase genes TCE1 and TCE2 that had been cloned from carmine spider mite, and a real-time PCR method based on SYBR Green I dye was developed. The mRNA expression differences of the two genes in different developmental period (egg, protonymph, nymphae and adults), different strains (abamectin-resistant, AbR; fenpropathrin-resistant, FeR; omethoate-resistant, OmR and susceptive strains, S) and these three pesticides induced from carmine spider mite were also detected using real-time PCR technology, respectively. TCE1 and TCE2 genes were inserted into pET-43.1a(+) of prokaryotic expression vector, and recombinant expression vector was obtained, named pET43a-TCE1 and pET43a-TCE2. The recombinant expression vector could express fusion proteins in E. coli. The key results were as follows.1. Seven candidate genes has been cloned from carmine spider mite using RT-PCR technology, and submitted them to the GenBank, GenBank number and length were RPS18 (FJ608659):262bp, 5.8SrRNA (FJ526334):470bp, GAPDH (FJ526335):293bp, RPL13a (FJ608662):242bp, TBP (FJ608661):365bp, SDHA (FJ608660):594bp, a-TUB (FJ526336):962bp, respectively. The qrtPCR primers were designed and the stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. In this study, the ranking of gene expression stability value (M) of 5.8s rRNA,α-TUB, RPS18, GAPDH, RPL13a and P-actin were 0.381,0.449,0.352, 0.460,0.445 and 0.643, and the ranking of gene expression stability were RPS18> 5.8srRNA> RPL13a> 0.449>GAPDH>P-actin in different strains, the ranking of gene expression stability value (M) of this six reference genes were 0.762,0.555,0.537,0.855,0.574 and 0.768, the ranking of gene expression stability were RPS18>a-TUB> RPL13a> 5.8srRNA>P-actin> GAPDH in different life stages. The geNorm program was subsequently used to calculate the optimal number of reference genes was two. To further validate our findings regarding the most optimal reference gene to normalize transcript expression data, we assessed our data set with NormFinder, Based on these data, the NormFinder program validated the findings with the geNorm algorithm, in which the most stable single gene was RPS18, and the best combination of the reference genes was RPS18 and 5.8SrRNA in different strains, RPS18 and a-TUB in different life stages of carmine spider mite, respectively.2. The mRNA expression levels of TCE1 and TCE2 genes were detected from egg, protonymph, nymph and adults of T. cinnabarinus through the method of qrtPCR technology with RPS18 and a-TUB as reference genes. The results showed that the mRNA expression level of TCE2 gene was higher than that of TCE1 gene, only by contrary in protonymph. The mRNA expression levels of two esterase genes in adults of T. cinnabarinus were significantly higher than those from other instars.3. The mRNA relative expression level of the TCE1 and TCE2 genes were also detected from T. cinnabarinus AbR, FeR, OmR and S. Compared with S, TCE1 gene mRNA expression of the three resistant strains were 1.06～1.07 fold, and 1.386～2.473, respectively for mRNA expression of the TCE2 gene. The results showed that the mRNA expression levels of TCE2 genes of the three resistant strains compared with that of S, and the difference was significant, but there was no obvious difference of the mRNA expression levels of TCE1 genes among the four strains.4. After induced by abamectin, fenpropathrin and omethoate, compared with susceptive strain that not induced with pesticide, the mRNA expression level of TCE1 gene from T. cinnabarinus decreased at 4h and then increased, but not preponderate over the control. The expression levels of TCE2 gene increased gradually at the beginning,12 to 16h reached the maximum after being treated with insecticides, then decreased. The highest expression level of TCE2 was 1.64-,2.92-and 2.24-fold of the control when treated with omethoate, abamectin and fenpropathrin, respectively, and the difference was significant, illustrate that TCE2 related to pesticide resistance closely.5. TCE1 and TCE2 genes were inserted into pET-43.1a(+) of prokaryotic expression vector, and recombinant expression vector was obtained, named pET43a-TCE1 and pET43a-TCE2. The recombinant expression vectors could express fusion proteins of recombinant TCE1 and TCE2 in E. coli Transetta. Specific strip appeared on gel in SDS-PAGE and on PVDF-Membran in Western blotting. It supported important theoretical and practical significance for further study.