Abstract: 【Aim】 Cuticular proteins (CPs) are abundant in insects, playing important roles in insect growth and development. This study aims to enrich different types of CPs in Antheraea pernyi and to determine the expression patterns of CPs during different developmental stages. 【Methods】 Two CP genes were cloned from cuticle of A. pernyi larvae using PCR and 3′ RACE technology, and the phylogenetic relationship was analyzed by bioinformatics methods. The expression patterns of the two genes in different tissues of larvae and during embryonic development of A. pernyi were analyzed by semi-quantitative RT-PCR, and the relative expression levels of the two genes in A. pernyi at different developmental stages and in the 3rd instar larvae after RNAi treatment were measured using real-time quantitative PCR (qPCR) technology. 【Results】 Two CP genes were cloned from A. pernyi and named ApCP12 (GenBank accession number: MF318874) and ApCP23 (GenBank accession number: MF318875), respectively. Bioinformatic analysis indicated that ApCP12 is 690 bp in length with an open reading frame (ORF) of 336 bp, encoding a protein of 111 amino acid residues with the calculated molecular weight of 12 kD, while ApCP23 is 1 243 bp in length with an ORF of 594 bp, encoding a protein of 197 amino acid residues with the calculated molecular weight of 23 kD. Both proteins contain RR-1 consensus and are classified in different clusters with RR-2 cuticular proteins by phylogenetic analysis. Tissue-specific mRNA expression profiling showed  that ApCP12 was more widely distributed than ApCP23. In the progress of embryonic development, the expression level of ApCP12 increased gradually. During larval ecdysis stage, the expression level of ApCP12 increased by 3-fold, and that of ApCP23 increased by 13-fold in 3 d after ecdysis than in molting stage. During pupal melanization, the expression level of ApCP12 was higher than that of ApCP23. The expression levels of the two genes had no significant changes during the earlier stage of pupal eclosion, while on the last day before eclosion, the expression levels of ApCP12 and ApCP23 increased by 3.5- and 3-fold, respectively, as compared with that on the first day of 20E injection. After RNAi treatment, the expression levels of ApCP12 and ApCP23 in the 3rd instar larvae were downregulated 5- and 3-fold, respectively. 【Conclusion】 The results suggest that these two CPs of subtribe RR-1 participate in the cuticle building of larva, pupa and adult and have a close relationship with the whole life cycle of A. pernyi development.

Key words: Antheraea pernyi; cuticular protein, developmental stage, gene expression, RNAi