Zhang Songdou, An Shiheng, Li Zhen, Wu Fengming, Yang Qingpo, Liu Yichen, Cao Jinjun, Zhang Huaijiang, Zhang Qingwen, Liu Xiaoxia
Department of Entomology, China Agricultural University, Beijing 100193, China.
State Key Laboratory of Wheat and Maize Crop Science (College of Plant Protection), Henan Agricultural University, Zhengzhou 450002, China.
Gene. 2015 Jan 25;555(2):393-402. doi: 10.1016/j.gene.2014.11.038. Epub 2014 Nov 18.
Recent studies have focused on determining functional genes and microRNAs in the pest Helicoverpa armigera (Lepidoptera: Noctuidae). Most of these studies used quantitative real-time PCR (qRT-PCR). Suitable reference genes are necessary to normalize gene expression data of qRT-PCR. However, a comprehensive study on the reference genes in H. armigera remains lacking.
Twelve candidate reference genes of H. armigera were selected and evaluated for their expression stability under different biotic and abiotic conditions. The comprehensive stability ranking of candidate reference genes was recommended by RefFinder and the optimal number of reference genes was calculated by geNorm. Two target genes, thioredoxin (TRX) and Cu/Zn superoxide dismutase (SOD), were used to validate the selection of reference genes. Results showed that the most suitable candidate combinations of reference genes were as follows: 28S and RPS15 for developmental stages; RPS15 and RPL13 for larvae tissues; EF and RPL27 for adult tissues; GAPDH, RPL27, and β-TUB for nuclear polyhedrosis virus infection; RPS15 and RPL32 for insecticide treatment; RPS15 and RPL27 for temperature treatment; and RPL32, RPS15, and RPL27 for all samples.
This study not only establishes an accurate method for normalizing qRT-PCR data in H. armigera but also serve as a reference for further study on gene transcription in H. armigera and other insects.
最近的研究集中于确定棉铃虫（鳞翅目：夜蛾科）中的功能基因和微小RNA。这些研究大多使用定量实时PCR（qRT-PCR）。合适的内参基因对于标准化qRT-PCR的基因表达数据是必要的。然而，关于棉铃虫内参基因的全面研究仍然缺乏。
选择了棉铃虫的12个候选内参基因，并评估了它们在不同生物和非生物条件下的表达稳定性。RefFinder推荐了候选内参基因的综合稳定性排名，并通过geNorm计算了最佳内参基因数量。使用两个靶基因，硫氧还蛋白（TRX）和铜锌超氧化物歧化酶（SOD）来验证内参基因的选择。结果表明，最合适的候选内参基因组合如下：发育阶段为28S和RPS15；幼虫组织为RPS15和RPL13；成虫组织为EF和RPL27；核型多角体病毒感染为GAPDH、RPL27和β-TUB；杀虫剂处理为RPS15和RPL32；温度处理为RPS15和RPL27；所有样本为RPL32、RPS15和RPL27。
本研究不仅建立了一种准确标准化棉铃虫qRT-PCR数据的方法，也为棉铃虫和其他昆虫基因转录的进一步研究提供了参考。
