关键词: 棉铃虫, ATP合酶亚基d, 20E, 幼虫变态, 可溶性海藻糖酶, 海藻糖

Abstract: 【Aim】 This study aims to analyze the function and molecular mechanism of the ATP synthase subunit d (ATPs-d) in trehalose metabolism regulating the development and metamorphosis of Helicoverpa armigera larva. 【Methods】 The open reading frame (ORF) of HaATPs-d of H. armigera was cloned by PCR, and its sequence and phylogeny were analyzed by bioinformatics methods. The qRT-PCR was employed to analyze the expression levels of HaATPs-d in the cuticle, midgut and fat body of the 5th instar larva at the molting stage and 6th instar larva, and in the fat body and cuticle of the 6th instar larva in response to 20-hydroxyecdysone (20E)(0.1 mg/mL). The subcellular localization of HaATPs-d in the Spodoptera frugiperda oocyte line Sf9 cells was studied by fluorescence photography. Yeast two-hybrid (Y2H) was emplyed to identify the protein interacting with HaATPs-d. The effects of the HaATPsd knockdown by RNAi on the larval development and metamorphosis, and soluble trehalase activity and trehalose content in the midgut after injecting dsHaATPs-d into the 6th instar larva of H. armigera were measured. 【Results】 The H. armigera HaATPs-d (GenBank accession number: LOC110375576) ORF is 525 bp in length and encodes 174 amino acids. HaATPs-d was highly conservative, and had close relationship to ATPs-d of S. frugiperda and S. litura. The HaATPs-d expression level peaked in the cuticle of the day-3 6th instar larva and in the midgut and fat body of the 5th instar larva at the molting stage. Compared with the control, 20E (0.1 mg/mL) significantly upregulated the expression levels of HaATPs-d in the fat body and cuticle of the 6th instar larva. HaATPs-d is a cytoplasmic protein. HaATPs-d directly bound with soluble trehalase of H. armigera. Knockdown of HaATPs-d in the 6th instar larva of H. armigera resulted in slower larval development, larval weight loss, increased larval mortality, decreased pupation rate and adult emergence rate, significantly decreased soluble trehalase activity and significantly increased trehalose content in the midgut compared with the control group (injected with dsGFP). 【Conclusion】 HaATPs-d controls the activity of soluble trehalase and trehalose content in larvae by binding with the soluble trehalase of H. armigera, thus affecting the sugar source and finally the larval metamorphosis.

Key words: Helicoverpa armigera, ATP synthase subunit d, 20E, larval metamorphosis, soluble trehalase, trehalose