Cloning And Characterization Analysis Of Glutathione S-Transferase BoGSTd1 And BoGSTd2 In Bradysia Odoriphaga

Glutathione S-transferases(GSTs, EC 2.5.1.18), a superfamily of multifunctional enzymes, have been found widely exsisting in plants, animals and microorganisms. GSTs play a crucial role in metabolizing xenobiotics and endogenous compounds to protect organisms from damage of the toxins and are also closely related to insecticide resistance. Bradysia odoriphaga is the major pest of Chinese chives, which mainly feeds on the roots and stems of Chinese chives. The primary management practice for controlling B. odoriphaga is the application of synthetic insecticides. However, excessive use of chemical insecticides causes pollution in the environment and leaves high residues on marketed Chinese chives which affects the human health. Furthermore, it is difficult to control because of increasing resistance. In this study, the glutathione S-transferases Bo GSTd1 and Bo GSTd2 in B. odoriphaga were cloned, and the expression levels of these genes in different developmental stages and tissues were analyzed by qPCR. Their functions were preliminarily analyzed to understand the mechanisms of resistance. The results are summarized as follows: 1. Cloning and sequence analysis of glutathione S-transferase genes in B. odoriphagaBy RT-PCR and RACE techniques,two glutathione S-transferase genes,BoGSTd1 and BoGSTd2 were cloned in B. odoriphaga. BoGSTd1 contained an open reading frame of 636 bp that encodes a protein of 211 amino acids with a predicted molecular weight of 23.72 kDa and with a theoretical isoelectric point of 5.94. BoGSTd2 has an open reading frame of 642 bp that encodes a protein of 213 amino acids with a predicted molecular weight of 23.78 kDa and with a theoretical isoelectric point of 5.12. The absence of a signal peptide and transmembrane domain in BoGSTd1 and BoGSTd2 secondary structures suggests that both of them are cytosolic GST proteins. By sequence alignment and phylogenetic analysis, the results indicate that BoGSTd1 and BoGSTd2 belong to Delta class. There was no intron in the BoGSTd1 and BoGSTd2 genes. Amino acids sequences of the two GSTs share 55.61% identity. 2. The spatiotemporal expression pattern of BoGSTd1 and BoGSTd2The expression patterns in different developmental stages and tissues of BoGSTd1 and BoGSTd2 were measured by quantitative real-time PCR. The results showed that both BoGSTd1 and BoGSTd2 were expressed in all developmental stages. BoGSTd1 was mainly expressed in the egg, followed by the 3rd- and 4th-instar larvae stage, but was weakly expressed in the 1st- and 2nd-instar larvae stage and the pupal stage. BoGSTd2 was mainly expressed in the egg, but expression of BoGSTd2 was weakly with no significant differences in other stages. Both BoGSTd1 and BoGSTd2 were mainly expressed in the abdomen, followed by the head and thorax, and were weakly expressed in legs and wings, and there is almost no expression of BoGSTd1 and BoGSTd2 in the antenna. 3. Prokaryotic expression and catalytic activity analysis of BoGSTd1 and BoGSTd2The BoGSTd1 and BoGSTd2 were expressed in E. coli BL21(DE3). The target recombinant proteins were purified through Ni2+-NTA agarose and two ~ 25 kDa soluble recombinant proteins were obtained. The concentrations of the purified proteins were 37.29mg/ml and 21.25mg/ml, respectively. Kinetic parameters of recombinant BoGSTd1 and BoGSTd2 were measured using CDNB as substrate. The results show that the Vmax and Km of BoGSTd1 were 1062μmol/min/mg and 0.35 mM, respectively. The Vmax and Km of BoGSTd2 were 290.4 μmol/min/mg and 0.24 mM, respectively. The activities of both BoGSTd1 and BoGSTd1 first increased and then declined with increasing pH. The optimal reaction pH range of the BoGSTd1 is 8.0- 8.5; the optimal reaction pH of BoGSTd2 is about 7.0. When the temperature is below 40℃, both BoGSTd1 and BoGSTd2 show high activities. 4. Effect of insecticides and inhibitor on BoGSTs activitiesIn vitro the effects of insecticides on inhibition of GSTs activities were measured. The results indicated that the activities of the enzymes were obviously inhibited in the precence of 0.25 mM chlorpyrifos-methyl, lambda-cyhalothrin and carbaryl. After treatment chlorpyrifos-methyl, the remaining enzyme activities of BoGSTd1 and BoGSTd2 were 11.7% and 5.44% as compared with the control, respectively. The activity of BoGSTd1 reduced with the increasing concentration of insecticides. The IC50 values were 0.19 mM, 0.21 mM and 0.22 mM for chlorpyrifos-methyl, lambda-cyhalothrin and carbaryl, respectively. The activity of the BoGSTd2 protein was increased at low concentrations of insecticides. With the increase of insecticide concentration, the activity of BoGSTd2 reached a peak and eventually started to decline. The activity of BoGSTd2 was inhibited totally in the presence of 0.25 mM chlorpyrifos-methyl, 0.3125 mM lambda-cyhalothrin or 0.3125 mM carbaryl. The values of IC50 of the Diethyl maleate(an inhibitor of GSTs) for BoGSTd1 and BoGSTd2 were 0.041 mM and 0.038 mM.