Gene Clone And Characterzation Analysis Of Glutathione S-transferase From Locusta Migratoria

Glutathione S-transferases(GSTs) are encoded by multiple genes with variety biological functions, existing in animals, plants and microorganisms. They are important detoxification enzymes in insects involved in insecticide resistance. This study focus on molecular characterization and biochemical testing indicated the expression patterns and activity characteristics of glutathione S-transferases from locusts.1. Cloning and sequense analysis of glutathione S-transferase genes from LocustaThirty-two different GST genes were identified from the locust transcriptome dates. All of their full-length cDNA sequences were obtained by RACE-PCR. twenty-eight of them belong to the cytosolic GSTs by the phylogenetic relationship analysis, respectively dispersed in six super-families, including ten in sigma (LmGSTsl, LmGSTs2, LmGSTs3, LmGSTs4, LmGSTs5, LmGSTs6, LmGSTs7, LmGSTs8, LmGSTs9, LmGSTs10), three in omega (LmGSTo1, LmGSTo2, LmGSTo3), five in epsilon (LmGSTel, LmGSTe2, LmGSTe3, LmGSTe4, LmGSTe5), seven in delta (LmGSTdl, LmGSTd2, LmGSTd3, LmGSTd4, LmGSTd5, LmGSTd6, LmGSTd7), two in theta(LmGSTtl, LmGSTt2), and only one in zeta (LmGSTzl). The other four belong to MAPEG family (LmGSTMl, LmGSTM2, LmGSTM3and LmGSTM4).2. The expression patterns of GSTs genes in locustsThe expression of the GST genes in different tissues(oregut, midgut, hindgut, gastric cecum, malpighian tubule, cuticle, spermary, ovary, fat body, trachea, antenna, muscle) in adults and different stages(eggs, five different instar nymphs and adults) of locusts was analysed by real-time quantitative PCR. Quantitative tissue expression analysis showed that mRNA levels of GSTs widely expressed in the different tissues of adults. LmGSTs2, LmGSTs4, LmGSTs6, LmGSTs10, LmGSTd4, LmGSTd6, LmGSTe2, LmGSTe5and LmGSTM3mainly expressed in the digestive and excretory systems; LmGSTsl, LmGSTo2, LmGSTo3, LmGSTd3, LmGSTd5, LmGSTd7, LmGSTe3, LmGSTtl and LmGSTt2showed relatively higher expressions in muscle; LmGSTs3, LmGSTs7, LmGSTo1, LmGSTd1, LmGSTe4and LmGSTM2showed relatively higher expressions in antenna; LmGSTM1significantly expressed in spermary; LmGSTs8and LmGSTs9mainly expressed in spermary, antenna and muscle; LmGSTs5and LmGSTel were predominantly expressed in fat body; The others have no significantly expression in all of the tissues. Stage-specific expression patterns showed the GSTs were respectively expressed in egg period and post-egg development period. Twelve genes, including LmGSTs3, LmGSTs8, LmGSTdl, LmGSTd3, LmGSTd3, LmGSTd7, LmGSTe2, LmGSTe3, LmGSTe4, LmGSTe5, LmGSTol and LmGSTz1were mainly expressed in eggs. Although the two groups of LmGSTs2, LmGSTs7, LmGSTsd6and LmGSTs4, LmGSTs5, LmGSTs6, LmGSTs10, LmGSTM3starting expression in different stages, the expression level of them as higher as the nymphs growth, and seven genes reached the highest spot but the LmGSTs10is exceptionally reduced in the adult stage. LmGSTd4was expressed starting in1st instar nymph and reached peak in4th instar nymph. LmGSTo2, LmGSTo3, LmGSTM2showed low expression level in the late of egg stage. The others have no obvious high expression in all of the stages.The twelve genes expressed in egg stage were selected for further study. The expression pattern of different development days in the egg stage showed eight genes, including LmGSTdl, LmGSTd3, LmGSTd5, LmGSTe2, LmGSTe3, LmGSTe4, LmGSTs3, LmGSTs8were significantly expressed in the early days. The expression of LmGSTol enhanced as the development of the egg. LmGSTd7and LmGSTe5showed significantly fewer expressions in the interim stage, and displayed a "V-shaped expression pattern. Oppositely, LmGSTz1were expressed higher in the interim stage of egg development. The diverse expression patterns of GST genes indicated they may play different biological functions in different tissues and different developmental phases.3. Prokaryotic expression and catalytic activity analysis of glutathione S-transferases from locustSeventeen recombinant cytoplasmic GSTs were obtained by E. coli expression systems. Kinetic parameters of recombinant LmGSTs were measured while CDNB as substrate, and the value of Vmax, Kcat, KmCDNB and KmGSH indicated all of them have different catalytic ability to CDNB and considerably variable kinetic parameters, and the KmCDNB valves were relatively difference, however the KmGSH valves were relatively consistent in the same family. Although The specific activity of six different compounds (CNDB, DCNB, FNDB, NDB-C1, pNBC and pNPB) as substrates were significant variation between different recombinant LmGSTs, it’s relatively consistent in the same families. Epsilon family have higher catalytic activity to all of the six substrates, and sigma family showed highest catalytic activity to FNDB but the catalytic activity were undetected when pNBC as substrate. Delta family indicated significant catalytic activity both to FNDB and NDB-C1, what is more, the catalytic activity to FNDB of LmGSTd1and LmGSTd6respectively reached125μmol/min/mg and76μmmol/min/mg. The activities of LmGSTtl were optimal when pNBC and pNPB as substrates. LmGSTo2and LmGSTzl showed powerless activity to the six substrates, particularly when DCNB, pNPB and pNBC were used as substrates, the activities were undetected. Glutathione peroxidase (GPX) activity was measured of the recombinant LmGSTs. LmGSTt2and LmGSTe1indicated significantly GPX activity reached306U/mg and92U/mg respectively. LmGSTs4, LmGSTs9, LmGSTe4, LmGSTd7, LmGSTo2and LmGSTz1have not detected GPX avtivty. The others showed low GPX avtivty which were between4-25U/mg. The diverse activity patterns of recombinant LmGSTs may suggested they play different biological functions in locusts, and the functions are correlation in the same family. What’s more, LmGSTt2and LmGSTe1may participate in the process of peroxide elimination.