Abstract: E75D is one of the important early transcription factors in the molting process of insects. The cDNA of E75D was cloned from Spodoptera litura by RT-PCR and RACE technology for the first time in this experiment and named Sli-E75D (GenBank accession no. JQ266225). Sli-E75D consists of a 1 836 bp open reading frame encoding 611 amino acids, with a 341 bp 5′ untranslated regions (UTR) and a 358 bp 3′UTR. E75, especially its 3′UTR, is highly conserved among the Lepidoptera insects; however, it shows significant difference in 5′UTR among four isoforms because of the difference of their promoter. The deduced amino acid sequence of E75D in S. litura share 98.4%, 79.3% and 76.5% identity with the homologues in Spodoptera littoralis, Manduca sexta and Bombyx mori, respectively. The miRNAs miR-14, miR-33 and miR-87, which are the most possible regulator of E75, were predicted by PITA and RNAhybird programs based on the highly conserved 3′UTR of E75. The recombinant vector pET28a-Sli-E75D was constructed and transformed into Escherichia coli BL21(DE3) and Transetta(DE3), respectively, to study the effects of rare codons on E75D expression. SDS-PAGE analysis of prokaryotic protein showed that the Transetta(DE3) which transformed pET28a-Sli-E75D recombinant vector expressed much more prokaryotic recombinant E75D protein (consisting of 67.19 kD Sli-E75D, and 7.6 kD T7·Tag and His·Tag) than BL21(DE3). The result of SDS-PAGE analysis demonstrated that Transetta(DE3) is better for the prokaryotic expression of E75D because it supplies tRNAs corresponding to six rare codons in E. coli. The expression levels of Sli-E75 in developmental stages from the last instar larva to adult were detected by qRT-PCR. The results of qRT-PCR revealed that Sli-E75 was expressed at a low level in 6th instar larva, and its expression increased rapidly from prepupa and reached the peak in the middle pupal stage. Then, the expression level of Sli-E75 decreased quickly in the last stage of pupa and rebounded again in the adult. These results can contribute to the in-depth study of E75 in molting process in insects.

Key words: Spodoptera litura, E75D, cloning, prokaryotic expression, microRNA