Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress
[Objective]To clone the glutathione S-transferases(GST)genes of Monochamus alternatus(Coleoptera:Cerambycidae)and to determine the expression levels of these genes under heat stress in adult males and females,as well as at different developmental stages of M.alternatus,we used molecular biology and bioinformatics methods.[Method]Three GST cDNA sequences were cloned,and their structural characteristics were analyzed using DNAMAN 9.0 and I-TASSER software.qRT-PCR was employed to measure the relative expression levels of GST genes in adult and fourth instar larvae of M.alternatus under varying temperatures and treatment durations.The role of these GST genes in protecting the organism from oxidative stress was assessed through disc diffusion assays.[Result]Three GST cDNA sequences were cloned and named MaltGSTe1,MaltGSTe2,and MaltGSTt1.MaltGSTe1 and MaltGSTe2 belong to the Epsilon family,while MaltGSTt1 belongs to the Theta family.Analysis of three-dimensional protein structures indicated that these GST are cytoplasmic.The relative expression levels of MaltGSTe1,MaltGSTe2 and MaltGSTt1 in fourth instar larvae significantly changed under heat stress,with MaltGSTe2 showing the greatest change.In males,the expression level of MaltGSTt1 was significantly down-regulated.Escherichia coli expressing the GST genes demonstrated enhanced antioxidant capacity,with MaltGSTe2 exhibiting the strongest activity.[Conclusion]We successfully cloned three GST genes and investigated their expression characteristics under high temperature stress in M.alternatus.High temperature stress was found to induce up-regulation of GST gene expression.Disc diffusion assays confirmed that heterogeneously expressed GST proteins have antioxidant capabilities.These findings suggest that GST genes play a role in the response mechanism to high temperature stress by protecting against oxidative stress,providing a theoretical foundation for exploring the heat resistance mechanisms of M.alternatus in subtropical regions.
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