Identification of Halloween genes involved in ecdysone synthesis and functional analysis of shade in melon fruit fly Zeugodacus cucurbitae
To clarify the role of Halloween genes in the ecdysone synthesis pathway of melon fruit fly Zeugodacus cucurbitae,a globally significant quarantine pest,reverse transcription-polymerase chain reaction(RT-PCR)was employed for cloning the Halloween genes.Real-time quantitative PCR(RT-qP-CR)was used to analyze their spatio-temporal expression patterns,and RNA interference(RNAi)was employed to investigate the effect of the ecdysone synthesis pathway gene Shade on the metamorphosic development of Z.cucurbitae.The results showed that five Halloween genes in the ecdysone synthesis pathway were identified:ZcSpook(abbr.ZcSpo),ZcPhantom(ZcPhm),ZcDisembodied(ZcDib),Zc-Shadow(ZcSad)and ZcShade(ZcShd).Amino acid sequence alignment exhibited a conserved cyto-chrome P450 domain in all five P450 enzymes.Phylogenetic analysis showed that these genes were closely related to homologs in Bactrocera dorsalis.These genes were highly expressed in the prothorac-ic gland complex in both larvae and adults before sexual maturity.Following RNAi-mediated silencing of ZcShd for 24 h and 48 h,gene expression was down-regulated by 80.54％and 60.59％,respectively.Statistical analysis of Z.cucurbitae pupation showed that 16.66％of larvae exhibited shrinkage and discoloration,33.33％died,and 33.33％displayed various malformations during pupation,indicating their involvement in regulating ecdysis,which could be used as a new target for controlling Tephritidae insects.
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