Identification And Characterzation Of Glutathione S-transferase Genes From Locusta Migratoria

Glutathione S-transferases (GSTs) are enzymes widespread in most organisms and play a major role in detoxification of chemical insecticides. Locusta migratoria is one of the most important agriculture pests and the Locust plagues are still poses a serious threat to agriculture in China. The destructive outbreaks of locust will be increasing in the near future due to the climate changes and the deteriorated ecological environment. Frequently applications of chemical insecticides have inevitably resulted in reducing sensitiveness of the insecticides in some natural populations of the locust. In order to carrying out effective management strategies of locust, glutathione S-transferases of L. migratoria were characterized in molecular and proteinic level. the content of this paper are as follows:1. Cloning of glutathione S-transferase genes from L. migratoriaTen different GST transcripts were identified from L. migratoria EST database and the identities were revealed by BLASTX search against the non-redundant database at NCBI. The complete cDNA sequence was obtained and verified with the coding region of each gene by RACE-PCR. The phylogenetic analysis of the ten GSTs deduced from their cDNAs revealed nine GSTs that belonged to only three different cytosolic classes, including one in delta (LmGSTd1), seven in sigma (LmGSTs1, LmGSTs2, LmGSTs3, LmGSTs4, LmGSTs5, LmGSTs6 and LmGSTs7) and one in theta (LmGSTt1), based on their sequence similarities to other insect GSTs. The remaining one GST (LmGSTu1) was unclassified due to its low relatedness to the currently known classes of insect GSTs.2. Characterization of glutathione S-transferase genes from L. migratoriaStage-specific expression patterns of ten L. migratoria GST genes were determined in eggs, five different nymphs (first-, second-, third-, fourth-, and fifth-instar), and adults by using RT-PCR. Among the ten genes, four genes (LmGSTs2, LmGSTs4, LmGSTs6 and LmGSTs7) showed significantly fewer expressions in eggs than other developmental stages. LmGSTu1 appeared to be the only gene, of which expressions could not be detected in eggs. In contrast, five GST genes (LmGSTd1, LmGSTs1, LmGSTs3, LmGSTs5 and LmGSTt1) showed similar levels of expressions in all the tested stages. Tissue-specific expression patterns of ten L. migratoria GST genes were analyzed in nine different tissues, including foregut, midgut, gastric caecum, hindgut, Malpighian tubules, fatbodies, muscles, spermaries and ovaries by using RT-PCR. Our results indicated that six of the ten GST genes, including LmGSTd1, LmGSTs1, LmGSTs3, LmGSTs5, LmGSTs6 and LmGSTt1, were expressed in all tissues examined. LmGSTs2, LmGSTs4, LmGSTs7 and LmGSTu1 were expressed in several tissues. Real-time quantitative PCR showed that deltamethrin at 0.08μg/mL and/or 0.12μg/mL increased almost all the ten GSTs gene expressions in third-instar nymph locusts. However, deltamethrin at 0.16μg/mL and/or 0.2μg/mL decreased the expressions of LmGSTd1, LmGSTs1, LmGSTs5 and LmGSTs6. The increases of GSTs gene expressions after deltamethrin exposure in L. migratoria might result to its elevating tolerance to other insecticides and xenobiotics.3. Prokaryotic expression and characterization of glutathione S-transferases from L. migratoriaTwo recombinant proteins (LmGSTtl and LmGSTs1) were functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTt1 and LmGSTs1 were able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. LmGSTt1 and LmGSTs1 were relatively stable during incubations at temperatures below 40℃. In the presence of 0.05mM ECA and RB, LmGSTt1 showed 37±1% and 6±0.1% of the original activities, respectively. The I50 of ECA was 12.6μM. The I50 of RB was 4.41μM; LmGSTs1 showed 81±3% and 41±3% of the original activities, respectively. The I50 of RB was 18.5μM. In the presence of 0.05 mM chlorpyrifos, deltamethrin, malathion and carbaryl, LmGSTt1 showed 71±3%,88±2%,94±2% and 83±1% of the original activities, respectively. But no significant effects were observed in LmGSTs1 after insecticids treatment.