Yang Chunxiao, Pan Huipeng, Noland Jeffrey Edward, Zhang Deyong, Zhang Zhanhong, Liu Yong, Zhou Xuguo
Hunan Academy of Agricultural Sciences, Institute of Plant Protection, Hunan, China.
Department of Entomology, University of Kentucky, Lexington, KY, USA.
Sci Rep. 2015 Dec 10;5:18201. doi: 10.1038/srep18201.
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.
逆转录定量聚合酶链反应（RT-qPCR）是一种用于量化各种生物过程中基因表达的可靠技术，该技术需要一组合适的内参基因来标准化表达数据。黄斑盘瓢虫（鞘翅目：瓢虫科）是田间用于防治节肢动物害虫的最广泛使用的生物防治剂之一。在本研究中，克隆并研究了从黄斑盘瓢虫中选出的16个管家基因的表达谱。通过geNorm、Normfinder、BestKeeper和ΔCt法等专用算法评估了这些候选基因在特定实验条件下作为内参的性能。此外，RefFinder这个整合了上述所有算法的综合平台，对这些候选基因的整体稳定性进行了排名。结果，针对涉及不同组织、发育阶段、性别以及用饮食双链RNA处理的黄斑盘瓢虫幼虫的实验，特别推荐了多组合适的内参基因。本研究是为瓢虫黄斑盘瓢虫的功能基因组学研究建立标准化RT-qPCR方案的关键第一步。此外，它为对非靶标生物（在这种情况下是一种关键的捕食性生物防治剂）进行基于RNAi的基因沉默生物技术的生态风险评估奠定了基础。
