Molecular cloning, characterization and positively selected sites of the glutathione S-transferase family from Locusta migratoria

Abstract

Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes--sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses--insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily.

Figure 1. Phylogenetic analysis of cytosolic GSTs from L. migtatoria and representative insect species.

The bootstrap neighbor-joining tree was generated using MEGA from ClustalW alignments. Branch numbers represent bootstrap values (1000 replicates). The 28 L. migratoria GSTs are marked with filled circles. The sequences used to reconstruct the NJ tree are available as S3 Data.

Figure 2. Phylogenetic analysis of microsomal GSTs from L. migratoria and representative insect species.

The bootstrap neighbor-joining tree was generated using MEGA from ClustalW alignments. Branch numbers represent bootstrap values (1000 replicates). The four L. migratoria GSTs are marked with red circle and green square, respectively. The species names and amino acid sequences used to reconstruct the NJ tree are available as S4 Data.

Figure 3. Expression patterns of GST genes from L. migratoria.

(A) Tissue-specific expression patterns of GST genes as evaluated using RT-qPCR: foregut (FG), midgut (MG), hindgut (HG), gastric caecum (GC), Malpighian tubules (MT), cuticle (CU), spermary (SP), ovary (OV), trachea (TR), fat bodies (FB), anterior (AN) and muscles (MU). (B) Developmental stage-specific expression patterns of GST genes as evaluated using RT-qPCR: 5-day and 10-day eggs (E1 and E2), first-(1st), second-(2nd), third-(3rd), fourth-(4th) and fifth-(5th) instar nymphs and adults (AD). The β-actin gene was used as a reference gene. For each gene, relative expression levels are shown with the highest as red and the lowest as green. (C) Relative expression levels of GST genes in egg stage as determined by RT-qPCR. β-actin was used as a reference gene. Data are expressed as means±SE of three biological replications. Small letters and capital letters indicate significant differences at p<0.05 and p<0.01 level, according to ANOVA and LSD test, respectively.

Figure 4. Homology structure of delta and sigma class GSTs from L. migratoria.

a, Ribbon representation of the 3D-structures of LmGSTD1. b, c Surface representation of LmGSTD1 (b front, c back). N-domain, C-domain and link region are colored yellow, red and green, respectively. The glutathione binding site (G-site), the hydrophobic ligand binding site (H-site) and the positive selective sites are labeled with cyan, orange and blue, respectively. The residues located at the interface of the N- and C-domain are shown in gray. In order to conspicuously display structure details, the homology structure of LmGSTD1 were selected as representative structure of each class in ribbon and surface representation.