Bai Yu, Lv Ya-Nan, Zeng Mei, Jia Pei-Yao, Lu Hu-Na, Zhu Yi-Bo, Li Sheng, Cui Ying-Ying, Luan Yun-Xia
Guangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Genes (Basel). 2020 Dec 25;12(1):21. doi: 10.3390/genes12010021.
Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from , a representative species of Zygentoma, including (), (), (), (), and (), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of and was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of (), a crucial gene-regulating insect metamorphosis, in ametabolous , and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.
石蛃目昆虫在理解昆虫变态发育的进化过程中占据着关键的进化地位，但在遗传分析方面却很少受到关注。为了开展对这种昆虫的功能基因组学研究，我们从石蛃目的一个代表性物种中评估了五个用于定量逆转录聚合酶链反应（qPCR）研究的候选内参基因，包括肌动蛋白（Actin）、微管蛋白（Tubulin）、核糖体蛋白L32（RPL32）、甘油醛-3-磷酸脱氢酶（GAPDH）和18S核糖体RNA（18S rRNA），在不同发育阶段、不同身体部位以及在双链RNA显微注射和饥饿胁迫条件下，使用四种算法（delta Ct、geNorm、NormFinder和BestKeeper）以及一种比较算法（RefFinder）进行评估。针对特定实验条件推荐了特定合适的内参基因，并且肌动蛋白和微管蛋白的组合适用于所有测试样本。利用我们选定的内参基因组合，我们研究了调控昆虫变态发育的关键基因Broad-complex（BR-C）在无变态发育的石蛃目中的基因表达模式，并证明了RNA干扰（RNAi）在石蛃若虫中的有效性。本研究为基因的可靠定量研究提供了基础，并极大地有益于石蛃目的进化和功能基因组学研究。
