Glutathione S-transferases (GSTs) are a large family of multifunctional enzymes, known for their role in cellular detoxification. Here we report a cytosolic GST with optimal activity at alkaline pH (8.3) from the visceral fat body of late-last instar (LLI) larvae of a lepidopteran insect rice moth Corcyra cephalonica. All previously known GSTs are active between pH 6.0 to 6.5. Purification and characterization revealed the Corcyra cephalonica GST (CcGST) as a 23-kDa protein. HPLC and 2D analysis showed a single isoform of the protein in the LLI visceral fat body. Degenerate primer based method identified a 701-nucleotide cDNA and the longest open reading frame contained 216 amino acids. Multiple sequence and structural alignment showed close similarity with delta-class GSTs. CcGST is present mainly in the fat body with highest activity at the late-last instar larval stage. Juvenile hormone (JH) negatively inhibits the CcGST activity both ex vivo and in vivo. We speculate that high expression and activity of CcGST in the fat body of the late-last instar larvae, when endogenous JH titer is low may have role in the insect post-embryonic development unrelated to their previously known function.
"GSTs from Anopheles gambiae and cutworms Spodoptera litura are regulated variously in different developmental stages (Strode et al., 2006; Huang et al., 2011). A delta GST identified from Corcyra cephalonica has been proposed to be involved in development, according to its expression profile and hormone-regulated activity (Gullipalli et al., 2010). The expression of the sigma and delta classes of GST in Bombyx mori is regulated by a juvenile hormone analog (JHA) and 20-hydroxyecdysone (Zou et al., 2011). "
[Show abstract][Hide abstract] ABSTRACT: The juvenile hormone titer during the last instar is a crucial determining switch for metamorphosis in lepidopterans. We previously observed that the induction of glutathione S-transferase (GST) activity by a juvenile hormone analog (JHA) is reversely proportional to the occurrence of JHA-induced supernumerary instars following topical application of JHA to Spodoptera litura during the last instar period. In this paper, at least five JHA-induced GSTs were purified by glutathione-affinity chromatography, and one was further isolated for determination of the N-terminal sequence. The corresponding cDNA was cloned and named SIGST1 (GenBank accession no. AY506545). SIGST1 was classified into the epsilon class of GSTs by phylogenetic analysis. Northern blot analyses further showed that the SIGST1 in fat bodies can be induced by JHA, particularly in 0-clay-old sixth-instar larvae, in which induction was much higher than that in 1- and 2-day-old sixth-instar larvae. To our knowledge, this is the first JHA-inducible GST to have been identified, and we believe that it will provide new insight into the role of GST in insect development, particularly during metamorphosis.
"Although most eukaryotic GSTs are known to have optimal pH ranging between 6.0 and 6.5 , some reports suggest that insect GSTs also have considerable activities at higher pH. For example, Corcyra cephalonica GST has an optimal pH 8.3 . A recombinant GSTt1 expressed in E.coli from B. mori showed broadly optimal pH ranging between 4.0 and 9.0 . "
[Show abstract][Hide abstract] ABSTRACT: Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni(2+)-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu(2+) and Cd(2+)). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos.
PLoS ONE 03/2013; 8(3):e58410. DOI:10.1371/journal.pone.0058410 · 3.23 Impact Factor
"Although the Delta subclasse is absent in T. castaneum, one unnigene assigned to Delta subclasse was identified in T. yunnanensis. In addition, the majority of T. yunnanensis GSTs were assigned to Epsilon subclasse, which is in accordance with studied insect GSTs with extensive cases of gene expansions in Epsilon and Delta types . "
[Show abstract][Hide abstract] ABSTRACT: The pine shoot beetle Tomicus yunnanensis (Coleoptera: Scolytinae) is an economically important pest of Pinus yunnanensis in southwestern China. Developed resistance to insecticides due to chemical pesticides being used for a long time is a factor involved in its serious damage, which poses a challenge for management. In addition, highly efficient adaptation to divergent environmental ecologies results in this pest posing great potential threat to pine forests. However, the molecular mechanisms remain unknown as only limited nucleotide sequence data for this species is available.
In this study, we applied next generation sequencing (Illumina sequencing) to sequence the adult transcriptome of T. yunnanensis. A total of 51,822,230 reads were obtained. They were assembled into 140,702 scaffolds, and 60,031 unigenes. The unigenes were further functionally annotated with gene descriptions, Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genome (KEGG). In total, 80,932 unigenes were classified into GO, 13,599 unigenes were assigned to COG, and 33,875 unigenes were found in KO categories. A biochemical pathway database containing 219 predicted pathways was also created based on the annotations. In depth analysis of the data revealed a large number of genes related to insecticides resistance and heat shock protein genes associated with environmental stress.
The results facilitate the investigations of molecular resistance mechanisms to insecticides and environmental stress. This study lays the foundation for future functional genomics studies of important biological questions of this pest.
PLoS ONE 02/2012; 7(2):e32291. DOI:10.1371/journal.pone.0032291 · 3.23 Impact Factor
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