Cloning, expression analysis, and molecular modeling of the gamma-aminobutyric acid receptor alpha2 subunit gene from the common cutworm,Spodoptera litura

摘要

Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor α2 (SlGABARα2) subunit gene inSpodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of re-verse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence ofSlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence ofSlGABARα2 was 1965 bp, with an open read-ing frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, O-glycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities thatSlGABARα2 shared with the GABA receptor α2 subunit ofSpodoptera exigua, Heliothis vires-cens,Chilo suppressalis,Plutella xylostella,Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated thatSlGABARα2 was expressed in all developmental stages ofS. litura. The relative expression level ofSlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions ofSlGABARα2 in larvae varied among dif-ferent tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body.