Glutathione-S-transferase plays a regulatory role in the oxidative stress response of Megalurothrips usitatus under environmental stress conditions
Description
The escalating environmental pollution, coupled with the degradation of the ozone layer, has led to an increase in ultraviolet radiation (UV) at the Earth’s surface. There is also a growing accumulation of pesticide residues in the environment. These stressors are exerting a profound impact on insect populations. When insects are subjected to adverse environmental stressors, their antioxidant enzymes can quickly respond with appropriate feedback adjustments, facilitating their adaptation to environmental changes. Glutathione S-transferases (GSTs), integral members of a multifunctional supergene family in insects, are pivotal in countering environmental stress and detoxifying chemical agents. Through transcriptomic screening and RT-qPCR, this investigation identified MuGSTS1 as a gene whose expression is significantly altered under UV stress. The application of RNAi confirmed the gene’s function in managing oxidative stress induced by UV and lambda-cyhalothrin. The research demonstrated that Megalurothrips usitatus, the M. usitatus caterpillar, adapts to these stressors by modulating the activity of antioxidant enzymes, thereby exhibiting a robust adaptability to UV light and lambda-cyhalothrin exposure. Experimental silencing of MuGSTS1 has been shown to impair the M. usitatus’s oxidative stress management, resulting in accelerated cellular apoptosis and an increased susceptibility to lambda-cyhalothrin, with sensitivity being augmented by a factor of 2.89. These findings provide a theoretical framework for understanding the adaptive mechanisms of insects to environmental stress.
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1.Materials and methods 1.1Transcriptome analysis M. usitatus was subjected to a 12-h dark treatment and subsequently transferred to various light environments for rearing, with wavelengths of 365 (UV), 460 (blue light), and 700 nm (white light). The rearing conditions of temperature and humidity were maintained at constant levels, and samples were collected at 3, 6, and 9 h post-transfer. Transcriptome sequencing was conducted based on the M. usitatus genome (Genebank: GCA_026979955.1). The transcriptome data of M. usitatus under UV stress (Genebank: SRP453564) were analyzed to identify the GST genes. Phylogenetic trees and conserved domain analysis were employed to determine the GST gene subfamilies, and the genes were subsequently named. Accession numbers were obtained upon uploading the results to the National Center for Biotechnology Information (NCBI) website, as detailed in Table 1. Enrichment analysis of the GST genes was carried out, utilizing topGO (v2.50.0) for Gene Ontology (GO) enrichment analysis and clusterProfiler (v4.6.0) for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. 1.2Analysis of the expression patterns of GST genes 1.3RNA extraction, cDNA synthesis, and RT-qPCR Following sample collection, the samples were rapidly frozen in liquid nitrogen and stored at −80 ℃ for subsequent analysis, with three replicates per treatment. Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Shanghai). The concentration of RNA was determined using a Micro Drop device, and its integrity was assessed via 1% agarose gel electrophoresis. cDNA was synthesized from total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) according to the manufacturer’s guidelines. RT-qPCR was performed using the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) kit. The reference genes were actin (ACT) and ribosomal proteins (RPL), which are known to be stably expressed under UV light and pesticide treatments, as reported in Hou (2023)[31]. The primer sequences for GSTs are provided in Table 2. 1.4Data analysis The bioassay data were processed utilizing PoloPlus software to determine the slope of the toxicity regression equation, the Lc50 value, and the associated 95% confidence interval. Graphpad Prism 9.5 was employed for data visualization and subsequent analysis. Statistical comparisons between the two groups were conducted using a T-test, while differences among multiple groups were assessed through two-way ANOVA.