Biochemical And Toxicological Properties And CDNA Cloning Of Glutathione S-transferases In Liposcelis Bostrychophila Badonnel

Glutathione S-transferases (GSTs) are a supergene family of detoxification enzymes found ubiquitously in organisms. They could catalyse the conjugation of the thiol group of reduced glutathione (GSH) with electrophilic compounds, generally making the resultant products more water soluble and excretable than the non-GSH conjugated substrates. Interest in insects GSTs has primarily focused on their imporatant roles in insecticide resistance. To elucidate the role of GSTs and the mechanism of GSTs-mediated resistance, the biochemical and toxicological characterizations and gene cloning of GSTs were investigated in the resistant (DDVP-R and PH₃-R) and susceptible strains of Liposcelis bostrychophila Badonnel and the study was supported by the program for New Century Excellent Talents in University (NCET-04-0854), Ministry of Education, China.The systematic investigation on biochemical and toxicological characterizations of GSTs in L. bostrychophila were conducted by the microplate reader. The biochemical results showed that compared to their susceptible counterparts, the activities per insect and specific activities of GSTs in DDVP-R and PH₃-R were significantly higher (P < 0.05). Apparent K_m values for CDNB were significantly lower for DDVP-R and PH₃-R compared with the susceptible strain (P < 0.05), indicating GSTs might have changed in quality. In contrast, the catalytic activity of GSTs toward GSH in the susceptible strain was significantly higher than those in resistant strains. Meanwhile, V_max values were approximately four times as high for DDVP-R compared with the susceptible strain. These results suggested compared to their susceptible counterparts, GSTs were overexpressed to various extents in DDVP-R and PH₃-R. And the continuous selection pressure of insecticides and fumigants contributed to the overexpression of GSTs. The inhibition kinetics of insecticides to GSTs in vitro revealed that dichlorvos and paraoxon possessed excellent inhibition effects on GSTs. These suggested that an elevated detoxification ability of GSTs developed in the resistant strains. In contrast, the inhibition effect of cypermethrin on GSTs was quite poor. It was noticeable that for chlorpyrifos, there was some facilitated effect of chlorpyrifos with a lower concentration (0.0002-0.0228 μM) on GSTs activities, while higher concentration (2.2825-22.8245 μM) possessed some inhibitory impact.A fragment was amplified with the degenerate primers from the conseverd peptide sequences of Delta class GSTs in insect species by reverse transcriptase-polymerase chain reaction (RT-PCR) method and the corresponding cDNA was as follows: KTPGQAACLQVDDL SSMLNRRSLQGLV GTDIQASTDPRTICLRTWISRNNDVQLAPT. The homologous analysis with BLAST software...