Molecular Cloning And Oxidative Stress Response Of A Sigma Class Glutathione S-transferase In Bombus Ignitus

Bombus ignitus, one of the bumblebees, is an important pollinator of various greenhouse crops. Because of their various merits, such as lower development, weak phototaxis, resistance to low temperature and adaptability in green house, they are widely used in pollination for greenhouse crops. Research of functional genomics in Bombus ignitus provides significant information for molecular regulation mechanism and also physiological characterization.Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainly involved in the xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Our research mainly focus on the exogenous oxidative response of GST in Bombus ignitusWe identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTS gene spans 1490bp and consists of 3 introns and 4 exons that encode 201 amino acids. The N-terminal domain of BiGSTS has a conserved Tyr residue, as well as conserved Phe, Trp, Lys, Gln, Pro, Ser, Glu and Asp residues that involved in the GSH-binding site of GST. Comparative analysis indicates that the deduced amino acid sequence of BiGSTS showed high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). The GenBank accession number of BiGSTS gene is HM101135. BiGSTS was close to the insect-specific Sigma class of GSTs in a phylogenetic tree.ESTs screened from cDNA library of Bombus ignitus was used for identification of the plasmid of pBluescript-BiGSTS. The cDNA fragment of BiGSTS was subcloned into pBacPAK9 vector and then was expressed in sf9 cells which was tranfected by the recombinant baculovirus. By SDS-PAGE, we analyzed the molecular weight of recombinant protein BiGSTS for approximately 28 KDa.Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H₂O₂ overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees.At last, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. BiGSTS and BiGSTD are highly induced at 9 h after H₂O₂ exposure in all tissues examined. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.