Abstract: 【Objective】Insect metamorphosis is initiated by ecdysone, which is synthesized in prothoracic gland through cytochrome P450 enzymes-catalyzed enzymatic reactions. Silkworm (Bombyx mori) is a silk-producing insect. Interrupting or delaying the pupa-adult transition in silkworm will help to reform the flow of silkworm cocoon-processing and to further improve the quality of silk. The objective of this study is to identify and characterize P450 genes involving in ecdysteroidogenesis in silkworm and to provide candidate genes for genetic controlling of silkworm metamorphosis. 【Method】 Ecdysteroidogenesis-related P450 genes were identified in silkworm and other insects through sequence homology search. Phylogenetic tree of insect ecdysteroidogenesis- related P450 genes was constructed by ClustalW program. The mRNA expression profiles of ecdysteroidogenesis-related P450 genes from silkworm were checked by microarray and RT-PCR analysis. RNAi experiment was used to examine the effects of down- regulation of expression level of ecdysteroidogenesis-related P450 genes on silkworm metamorphosis. 【Result】Four P450 genes involving in ecdysteroidogenesis, including Cyp306a1, Cyp302a1, Cyp315a1, and Cyp314a1, were identified in the silkworm genome. Comparative analysis revealed that each edysteroidogenesis-related P450 gene existed as a single copy in the surveyed insects and all its orthologs in insects were grouped together well in the phylogenetic tree, indicating edysteroidogenesis-related P450 genes and edysteroidogenesis pathway were very conserved during insect evolution. Spatio-temporal expression profiling found that Cyp302a1, Cyp315a1, and Cyp314a1 showed a high expression in the ovary, testis, and head of silkworm larvae and were continually expressed during the late stages of silkworm metamorphosis. Cyp314a1 expression could be highly detected before wandering, pupation, or eclosion, showing a consistency with the stage-specific changes of ecdysone pulses. RNAi of Cyp314a1 resulted in not only larval death before silkworm pupation but also developmental abnormality of adult ovary. Two key genes involving in ecdysone signaling, HR3 and Ftz-f1, were also down-regulated after Cyp314a1 RNAi.【Conclusion】Silkworm P450 genes involving in ecdysteroidogenesis are evolutionarily conserved and down-regulating the expression level of the selected P450 gene interrupts normal pupation, suggesting that ecdysteroidogenesis-related P450 genes might be used in altering genetically the progress of silkworm metamorphosis.

Key words: silkworm (Bombyx mori) , ecdysteroidogenesis , cytochrome P450 , metamorphosis , RNAi