关键词: 异色瓢虫, 海藻糖酶, TRE2-like, TRE2, 基因表达, 酶活性, RNA干扰

Abstract: 【Aim】 Harmonia axyridis is an important predatory natural enemy, and trehalose plays an important role in the whole life of H. axyridis, such as metamorphosis and adult eclosion. This study aims to explore the potential functions of two trehalase genes TRE2-like and TRE2, which were obtained earlier, on trehalose utilization during adult eclosion of H. axyridis, and to provide reference for clarifying the potential mechanism of trehalose metabolism in the development from pupa to adult in H. axyridis. 【Methods】 The dsRNAs of TRE2-like and TRE2 were synthesized by designing double-stranded RNA (dsRNA) region fragments based on the cloned gene sequences of TRE2-like and TRE2, and injected into the 2-day-old pupae of H. axyridis by RNAi technology. The expression levels of carbohydrate metabolism-related genes in the 1-day-old adults of H. axyridis after RNAi were detected by real-time quantitative PCR (RT-qPCR). At the same time, the changes in the contents of main saccharides and the trehalase activity after RNAi in its 1-day-old adults were determined by fluorenone colorimetric method and enzyme labeling method, respectively, and the phenotypic changes in eclosed adults after RNAi were observed. 【Results】 The results showed that after the dsRNA of TRE2-like or TRE2 was injected into the 2-day-old pupae of H. axyridis, the expression levels of TRE2-like and TRE2 in its newly emerged adults were significantly decreased as compared with the control group (dsGFP injection group), and a few adults showed abnormal phenotypes such as difficulty in molting and wing formation. The soluble trehalase activity decreased significantly after injection of dsTRE2-like, while the membrane-bound trehalase activity decreased significantly after injection of dsTRE2. As compared with the control group, the dsTRE2 injection group had significantly decreased glycogen content, the dsTRE2-like injection group had significantly decreased glycogen and trehalose contents, and the dsTRE2-like plus dsTRE2 injection group had significantly decreased glycogen and glucose contents and extremely significantly decreased trehalose content. Meanwhile, the expression levels of soluble trehalase genes TRE1-1 and TRE1-2 in the dsTRE2-like, dsTRE2 and dsTRE2-like plus dsTRE2 injection groups decreased or significantly decreased, that of TRE1-5 increased or significantly increased, and those of trealose-6-phosphate synthase (TPS), glycogen phosphorylase (GP) and glycogen synthase (GS) genes were significantly decreased as compared to the control group. 【Conclusion】 The metabolism of trehalose and the like in H. axyridis is affected after the expression of TRE2-like and TRE2 is inhibited. The results of this study lay a foundation for exploring the potential function and regulation mechanism of membrane-bound trehalases in H. axyridis.

Key words: Harmonia axyridis, trehalase, TRE2-like, TRE2, gene expression, enzyme activity, RNA interference