Cloning And Functional Characterization Of A Glutathione S-transferase Genen BmGSTe2 In The Silkworm

Glutathione S-transferases(GSTs, EC2.5.1.18) are a superfamily of multifunctional enzymes, which have been found in both prokaryotic and eukaryotic cells. Except for their noteworthy catalytic role in detoxification by conjugation reduced glutathione(GSH) to xenobiotics, these multifunctional enzymes are also involved in the homeostasis of endogenous compounds, which can act as selenium-independent glutathione peroxidases(non-Se GPx) in cellular antioxidant defense against oxidative stress. GSTs also have non-catalytic functions, mainly binding hydrophobic compounds such as drugs, hormones, and other metabolites. Based on amino acid sequence similarities and immunological relationships, the ubiquitous omega, sigma, theta, and zeta classes, insect specific delta and epsilon classes, as well as unclassified GSTs, have been identified in insects.The silkworm Bombyx mori was domesticated from Bombyx mandarina. B. mori is an important economic insect. The silk of the silkworm has been used in many field. Furthermore, the silkworm is one of the model organisms in Lepidoptera, which is widely used for insect research in genetics and molecular biology. With the release of whole genome sequence and functional genomics data of the silkworm, silkworm GSTs have been identified in the silkworm genome. In our study, we cloned Bm GSTe2, which can be induced by insecticids, and performed functional study. The main results are as follows: 1. Cloning and sequences analysis of Bm GSTe2 geneBm GSTe2 was cloned by RT-PCR from the midgut of the silkworm and deposited in Gen Bank(Accession No. DQ355376). Bm GSTe2 contained an open reading frame of 648 bp encoding 215 amino acid residues. The theoretical molecular mass and p I were estimated to be 24.72 k Da and 5.98, respectively. Based on sequence similarities, we selected some other insect GSTs used for reconstructing phylogenetic tree with Bm GSTE2 protein. The result indicated that Bm GSTE2 was clustered with Epsilon members. Three putative conserved domains of Bm GSTE2 were detected, including dimer interface polypeptide binding site, C-terminal domain substrate binding site(H-site), N-terminal domain glutathione binding site(G-site). 2. Heterologous expression and enzyme assay of silkworm Bm GSTe2 geneWe construed the expression vector p ET28a-Bm GSTe2, which was heterologously expressed in Escherichia coli. SDS-PAGE analysis revealed that Bm GSTE2 was in a soluble form. The soluble recombinant protein was purified by anion-exchange chromatography and ultrafiltration. The purified recombinant Bm GSTE2 showed 5.24 μmol/min/mg activity toward the model substrate CDNB. The non-Se GPx activity of recombinant Bm GSTE2 with cumene hydroperoxide was 0.10 μmol/min/mg. As for the thermostability, the recombinant Bm GSTE2 was examined at various temperatures and measurement of residual activity. Bm GSTE2 was relatively stable during incubations at temperatures below 50°C.The results from inhibition experiments of recombinant Bm GSTE2 with two organophosphate insecticides(phoxim and chlorpyrifos) and one pyrethroid insecticide(fenpropathrin) shown that the inhibitory patterns of three insecticides on Bm GSTE2 were similar to one another. The residual activities gradually decreased with increasing amounts of each insecticide. The IC50 values of two organophosphate phoxim and chlorpyrifos were close to each other, 0.15 m M and 0.18 m M respectively. Relatively, fenpropathrin was the most effective inhibitor on Bm GSTE2, and its IC50 value was only 0.09 m M. 3. Protein expression of silkworm Bm GSTE2Western blotting was used to detect expression profiles of Bm GSTE2 at translational level. We examined four important tissues from the day 3 of the 5th instar larvae(19-440 strain). Bm GSTE2 was only expressed in the midgut tissue. In laboratory, we sequentially selected the silkworms(19-440 strain) with phoxim(organophosphate) and fenpropathrin(pyrethroid), respectively. Using Western blotting, we detected the expression of Bm GSTE2 in the midgut of the insecticide-selected strains and the parental strain. The band intensities of Bm GSTE2 in phoxim and fenpropathrin-selected strains were obviously stronger than that in the parental strain. Thus, the expression of Bm GSTE2 increased in the insecticide-selected strains.4. Localization of silkworm Bm GSTE2 proteinImmunohistochemical analysis was performed to investigate the location of Bm GSTE2. Because Bm GSTE2 was specifically expressed in the midgut, only this tissue was used to show the distribution of the protein in immunohistochemical analysis. Our results shown that Bm GSTE2 was localized in the epithelium, columnar cells, goblet cells and regenerative cells of the midgut.In summary, we cloned Bm GSTe2 from the B. mori larvae after long-term exposure to phoxim and fenpropathrin caused its insecticide tolerance. Bm GSTe2 was specifically expressed in the midgut at transcription and protein levels. The results of heterologous expression, insecticide inhibition analysis, and immunoblotting suggested that Bm GSTe2 might play roles in enhancing insecticide resistance of the silkworm larvae. Our results can be used for silkworm resistance breeding. At the same time, silkworm is the model organism in Lepidoptera, Bm GSTe2 might be used for the target of pest management.