Cloning, Sequence Analysis, And Prokaryotic Expression Of Carboxylesterase Gene In The Silkworm, Bombyx Mori

Carboxylesterases (COEs) are a multi-function enzymes that occur in animals, plants, and microbes. Based on sequence similarity and substrate specificity, insect COE genes can be subdivided into eight subfamilies:α-esterase,β-esterase, juvenile hormone esterase, acetylcholinesterase, neurotactin, neuroligin, gliotactin and glutactin class. Carboxylesterases have a broad range of functions, some of carboxylesterases belonging toα/β-esterase were expressed in insect antennae, these COEs are important odorant-degrading enzymes that eliminate pheromones and allelochemiacls.Recently, the studies on antennae esterases as a crucial role in inactivating pheromones and plant allelochemicals provided us with useful information for understanding the new functions of these enzymes. Antennae esterases are localized in insect olfaction sensilla and able to degrade odorants compounds. This suggests that they participate in the sense of smell reaction, and play important roles in maintaining the normal physiology reaction. Previous studies mainly focused on the function of insect antanne esterase, which can degrade odorants compounds. However, if the insect larval COEs are also expressed in olfaction tissue, and if they can hydrolyze endogenous, all those questions are still unclear. Bombyx mori is the model organism of Lepidopteran insects and play important role in studying genetics. Silkworm larva is different from Drosophila melanogaster and Anopheles gambiae. Silkworm is phytophagous insect and grows well on mulberry leaves, which will encounter a low chemical signals from its host plant and toxic allelochemicals, such as insecticide. Thus, study on silkworm COEs involved in detoxifying metabolism and olfactory detection will provide useful information for preventing Lepidopteran pests.Previous research in our laboratory suggested that more than 11 COEs were expressed in silkworm larval antenna and maxilla, and some of them are the putative orthologs of insect odorant-degrading esterases and pheromone-degrading esterases. In present study, we cloned three carboxylesterases gene Bmae32, Bmae33 and Bmae35, which were expressed in larval olfactory tissue in the silkworm. According to EST assembling results, we designed primers and cloned those sequence from silkworm head. Then using bioinformatics methods we presumed and analyzed these protein sequences. Based on the semi-quantitative RT-PCR, we determined tissue expression patterns of these genes. At last, The Bmae32 and Bmae35 genes were sub-cloned into the prokaryotic expression vectors. The Bmae35 recombinant protein was expressed in Escherichia coli, purified by immobilized Ni2+ absorption chromatograph column, and proved by Western blotting. All the results are as follows:1. The CDS length of Bmae32, Bmae33, Bmae35 is 1623bp, 1656bp and 1581bp, respectively. The three genes are all composed of 3 exons and 2 introns, and all the boundaries between exons and introns follow the GT-AG rule. Bmae32 encodes 540 amino acids, its molecular mass and isoelectric point are 61.7 kD and 8.72, respectively. It potentially comprises 1 N-glycosylation site and 19 phosphorylation sites. Bmae33 encodes 551 amino acids, its molecular mass and isoelectric point are 62.15 kD and 5.87, respectively. It potentially comprises 7 N-glycosylation site and 27 phosphorylation sites. Bmae35 encodes 526 amino acids, its molecular mass and isoelectric point are 59.19 kD and 6.39, respectively. It potentially comprises 5 N-glycosylation site and 18 phosphorylation sites.2. Signal IP 3.0 analysis revealed that Bmae32, Bmae33 and Bmae35 probably comprise signal peptide with 24, 24 and 15 amino acids long, respectively. Alignment of the Bmae32, Bmae33 and Bmae35 protein sequences with other esterases revealed that all of them have the structure characteristics of insect esterases, such as the catalytic triad (Ser, Glu and His) and the conserved pentapeptide Gly-x-Ser-x-Gly in theα-esterase family. Phylogenetic tree and sequence similarity analysis results indicated that the three COEs belong toα-esterase family, Bmae32 is an putative ortholog of Mbra-EST in Mamestra brassicae with 58.8% identity, Bmae33 is an putative ortholog of Apol-EST in Antheraea polyphemus and Slit-EST in Spodoptera littoralis, with 73.1% and 64.6% identities, respectively, Bmae35 is the putative ortholog of antennal esterase Snon-EST in Sesamia nonagrioide with 48.6% identity.3. The expression pattern showed that Bmae32 was highly expressed in head, hemocyte, and silk gland in day 3 of the fifth instar. Bmae33 was highly expressed in malpighian tubule, epidermis and head. Bmae35 gene was highly expressed in head, fat body, malpighian tubule, epidermis, and silk gland. In addition, the expression of Bmae35 showed positive correlation with sex pheromone content. It suggested that Bmae35 might play important role in pheromone synthesizing.4. We cloned Bmae32 and Bmae35 genes again after removed the signal peptide and analyzed the restriction enzyme site, then constructed the recombined plasmid with pET28(a)vector. The correct recombinant plasmid Bmae35/pET28(a) was transferred into Escherichia coli BL21 (DE3). The recombinant protein was obtained by isopropyl β-D-1-thiogalactopyranoside (IPTG) inducement. Electrophoresis analysis showed that Bmae35 was expressed in Escherichia coli as inclusion body. The recombinant protein Bmae35 was purified by immobilized Ni2+ absorption chromatograph column. Western blotting analysis suggested that Bmae35 was correctly expressed in E. coli and purified.