JIA-2018-09

Journal of Integrative Agriculture 2018, 17(9): 2054–2065 RESEARCH ARTICLE Available online at www.sciencedirect.com ScienceDirect Selection and evaluation of potential reference genes for gene expression analysis in greenbug ( Schizaphis graminum Rondani) ZHANG Bai-zhong 1, 2* , LIU Jun-jie 1* , YUAN Guo-hui 2 , CHEN Xi-ling 1 , GAO Xi-wu 3 1 Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, P.R.China 2 College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, P.R.China 3 Department of Entomology, China Agricultural University, Beijing 100193, P.R.China Abstract In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For greenbug, Schizaphis graminum , a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored. In our study, eight reference genes, elongation fator 1 beta ( Ef1β ), TATA box binding protein ( TBP ), alpha-tubulin ( α-TUB ), 18S ribosomal ( 18S ), 28S ribosomal ( 28S ), glyceraldehyde-3-phosphate ( GAPDH ), actin ( ACT ), and ribosomal protein L18 ( RPL18 ) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments. To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ∆Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene ( HSP70 ), cytocrome P450 gene ( SgraCYP18A1 ), and glutathione S -transferase ( GST ). We found that the most suitable reference genes varied considerably under different experimental conditions. For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data. In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments. This should be useful for the selection of the suitable reference genes to obtain reliable RT- qPCR data in the gene expression of S. graminum . Keywords: Schizaphis graminum , gene expression, normalization, RT-qPCR, reference gene 1. Introduction The internal control of target gene measurement refers to the use of reference gene expression and is the currently preferred method for normalizing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) data as reference genes could capture all nonbiological variations (Ciric 2010). Although no gene exhibits constant Received 21 September, 2017 Accepted 27 January, 2018 ZHANG Bai-zhong, E-mail: 281341651@qq.com ; LIU Jun- jie, E-mail: 15560148574@163.com; Correspondence YUAN Guo-hui, E-mail: hnndygh@126.com; CHEN Xi-ling, E-mail: chenxiling@hist.edu.cn ; GAO Xi-wu, E-mail: gaoxiwu@263.net. cn * These authors contributed equally to this study. © 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(18)61903-3