Selection and evaluation of potential reference genes for gene expression analysis in greenbug(Schizaphis graminum Rondani)  

作　　者：ZHANG Bai-zhong LIU Jun-jie YUAN Guo-hui CHEN Xi-ling GAO Xi-wu 

机构地区：[1]Postdoctoral Research Base, Henan Institute of Science and Technology [2]College of Plant Protection, Henan Agricultural University [3]Department of Entomology, China Agricultural University

出　　处：《Journal of Integrative Agriculture》2018年第9期2054-2065,共12页农业科学学报（英文版）

基　　金：the National Key Research and Development Program of China (2017YFD0201700);the Key Science and Technology Program (Agriculture) of Henan,China (182102110053);the Major Projects of Henan Institute of Science and Technology,China (14QN024);the Project of High-Level Talent Introduction of Henan Institute of Science and Technology,China (208010616003);the Scientific and Technological Innovation of Henan Institute of Science and Technology,China (208010616005) for the financial support given to the present research work

摘　　要：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 （a-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）, ACt 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, a-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACTwere suitable reference genes; for insecticide treatments, 28S and a-TUB were suitable for normalizations of expression data. In addition, 28S and a-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.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 （a-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）, ACt 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, a-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACTwere suitable reference genes; for insecticide treatments, 28S and a-TUB were suitable for normalizations of expression data. In addition, 28S and a-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.

关 键 词：Schizaphis graminum gene expression NORMALIZATION RT-QPCR reference gene 

分 类 号：Q78[生物学—分子生物学]