Cloning And Heterogenous Expression Of Carboxylesterase Gene Bmae33in The Silkworm, Bombyx Mori

Carboxylesterases (COEs) are a kind of multifunctional enzymes which catalyzehydrolysis of many kinds of esters. COEs are widely distributed in the organic sphere,and play important roles in organisms. COEs were subdivided into8classes based onsequence similarity and substrate specificity: α-esterases, β-esterases,acetylcholinesterases, juvenile hormone esterases, gliotactins, neuroligins, neurotactinsand glutactins. The functions of carboxylesterases are highly diversity in organisms,they paly important roles in detoxification of exogenous substance, degradation of sexpheromone and regulation of nerve/development. The functions of carboxylesterases inthe degradation of odorants and resistance to insecticides were investigated in previousstudies mainly using fruit flies or mosquitoes as model organisms.It was found in previous studies that antennal carboxylesterases participate in thedegradation of ester pheromone in order to maintain the sensitivity of the olfactorysystem in the adult insects. Recent studies have shown that antennal carboxylesterasescould also degrade volatile odorants of host plants, which expanded our understandingof the function of the antennal esterase. This suggested that carboxylesterases in insectolfactory system not only could terminate the ester signal, but also may play veryimportant roles in the maintenance of normal physiological function of the olfactorysystem.Studies of antennal esterases of Lepidoptera were mostly focused on the functionsof adult antennal esterases in degradation of volatile odorants before, and there werefew reports about larval antennal esterases. In this study we analyzed a carboxylesterasegene Bmae33, which expressed in olfactory system of silkworm larval. The main resultsare as follows:1. We removed the signal peptide of Bmae33gene, and designed primers withtwo restriction enzyme sites, then cloned the gene and obtained a1605bp DNA product.2. Aligment of protein sequences of Bmae33and other insect carboxylesterasesrevealed that all of these proteins have the characteristic structure of α-/β-esterase:Gly-x-Ser-x-Gly, and three requisite amino acids which constructed catalytic triad. Theobservation of the3-D structure of Bmae33protein by Raswin verified that the threeamino acids Ser196, Glu327and His114were aggregated together and formed catalytictriad, which suggested that Bmae33could be an activited hydrolase. In addition, phylogenetic tree analysis indicated that Bmae33is a putative orthologous gene ofApol-ODE and Slit-EST.3. RT-PCR analysis showed that Bmae33gene was highly expressed in head,fatbody and integument of the3rd day of the5th instar silkworm, suggesting that itmight play important roles in xenobiotic detoxification and odorant degradation.Moreover, after fumigated with OP insecticide phoxim, the expression level of Bmae33in larval head and male adult antennae were both up regulated, suggested that Bmae33might play important roles in the degradation of phoxim.4. The gene we obtained was ligated with prokaryotic expression vector pET-28a(+) and eukaryotic expression vector pPIC9K, respectively, to construct recombinantexpression vectors Bmae33/pET-28a(+) and Bmae33/pPIC9K. After sequencing, therecombinant vectors were transformed separately into E. coli BL21(DE3) strain andPichia pastoris X-33strain, and induced with IPTG and methanol, respectively.SDS-PAGE analysis showed that Bmae33was expressed as inclusion body in E. coli.The recombinant Bmae33protein was purified by His-tagged Ni2+affinitychromatograph column, and the purified protein was identified with Western blotting.Moreover, the purified protein was used to prepare polyclonal antiboby of Bmae33.5. In order to obtain activated protein of Bmae33, we optimized some of thecodons of Bmae33gene based on the Pichia pastoris’ codon bias. After that we clonedthe optimized gene using overlap extension PCR, and constructed the recombinantvector for the expression of Bmae33in Pichia pastoris.In this paper, a series of studies on the silkworm olfactory system carboxylesteraseBmae33were conducted, and polyclonal antibodis were prepared. In order to obtainactivated protein, the gene sequence was optimized and eukaryotic expression vectorwere constructed. All results presented in this study provided the foundation for futureresearch of immunohistochemistry, activity and functions of Bmae33, consequently,provided a basis for in-depth studies of functions of insect carboxylesterases.