海藻糖酶基因TRE2-like和TRE2在异色瓢虫羽化过程中的表达与功能

【目的】异色瓢虫Harmonia axyridis是一种重要的捕食性天敌昆虫,海藻糖在异色瓢虫的变态发育、羽化等整个生命过程都起着重要的作用。本研究以前期获得的类似膜结合型海藻糖酶(TRE2-like)与膜结合型海藻糖酶(TRE2)基因为基础,探讨在异色瓢虫羽化阶段这两个海藻糖酶的潜在功能,为阐明异色瓢虫从蛹发育到成虫时海藻糖代谢机制提供参考。【方法】根据TRE2-like和TRE2基因序列设计双链RNA(dsRNA)区域片段并合成对应的dsRNA,通过RNAi将其注射到异色瓢虫2日龄蛹中。采用实时荧光定量PCR(RT-qPCR)检测RNAi处理后羽化第1天的异色瓢虫成虫糖代谢相关基因的表达;同时采用蒽酮比色法、酶标法等分别测定RNAi处理后羽化第1天的异色瓢虫成虫主要糖类物质含量及TRE活性变化,并观察异色瓢虫羽化后的表型变化。【结果】结果表明,与对照组(dsGFP注射组)相比,异色瓢虫2日龄蛹被注射TRE2-like或TRE2 dsRNA后,其新羽化成虫体内TRE2-like和TRE2表达量均极显著下调,且少数个体出现了蜕皮与翅形成困难等畸形表型。可溶性海藻糖酶活性在注射dsTRE2-like后显著降低,膜结合型海藻糖酶活性在注射dsTRE2后显著降低;注射dsTRE2后糖原含量显著下降,注射dsTRE2-like后糖原和海藻糖含量显著下降,注射dsTRE2-like+dsTRE2后糖原和葡萄糖含量显著下降,且海藻糖含量极显著下降。注射dsTRE2-like, dsTRE2和dsTRE2-like+dsTRE2后可溶性海藻糖酶基因TRE1-1和TRE1-2表达下降或显著下降,而TRE1-5表达上升或显著上升,海藻糖合成酶(trealose-6-phosphate synthase, TPS)、糖原磷酸化酶(glycogen phosphorylase, GP)、糖原合成酶(glycogen synthase, GS)基因的表达均显著下调。【结论】TRE2-like和TRE2基因表达被抑制后,异色瓢虫海藻糖等代谢受到影响。研究结果为探究异色瓢虫体内膜结合型海藻糖酶的潜在功能和调控机制奠定了基础。

Expression and function of trehalase genes TRE2-like and TRE2 during adult eclosion in Harmonia axyridis (Coleoptera: Coccinellidae)

【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.