JIA-2018-09

2063 ZHANG Bai-zhong et al. Journal of Integrative Agriculture 2018, 17(9): 2054–2065 other studies (Shen et al. 2010; Li et al. 2013; Chandra et al. 2014; Liang et al. 2014; Yuan et al. 2014; Ma et al. 2016). ACT and GAPDH were the most frequently used reference genes for normalizing RT-qPCR (Ruan and Lai 2007; Lord et al. 2010; Pan et al. 2010; Gu et al. 2013). However, we found that ACT and GAPDH are also not ideal reference genes for all the studies of S. graminum , they are only suitable under certain experimental conditions and needed to cooperate with other reference genes to obtain a reliable result. These results indicate that the commonly-used reference genes are often not well-suited for normalization of all RT-qPCR data, and that researchers should pay more attention than they typically do when selecting genes to use as internal controls for normalization. EF1 ( EF1α and EF1β ) had been proven to be the optimal candidate reference gene for normalization in Agrilus planipennis (Rajarapu et al. 2012), Rhodnius prolixus (Majerowicz et al. 2011), and cotton aphid Aphis gossypii (Ma et al. 2016). However, EF1β was not a preferable gene in S. graminum . RPL18 was the least suitable for normalization as a reference gene among different developmental stage, tissues, and insecticide treatments in S. graminum , though it was well used in Schistocerca gregaria (Hiel et al. 2009), Delphacodes kuscheli (Maroniche et al. 2011), and A . gossypii (Ma et al. 2016). In addition, α-TUB was also one of the most suitable for normalization as a reference gene in all conditions in our study while it was least stable in all conditions in A . gossypii (Ma et al. 2016). These results suggested that the commonly-used reference genes are not well suited for normalization for all circumstances. Thus, these traditionally-used reference genes are not persistently and stably expressed in many species or experimental treatments (Chen et al. 2011; Chandna et al. 2012; Cheng et al. 2013), emphasizing the need to evaluate reference genes in S. graminum . To validate these selected reference genes, the expression levels of HSP70 , an important stress-inducible heat shock protein gene (Bettencourt et al. 2007), and two detoxifying genes, SgraCYP18A1 and GST , were analyzed in different developmental stages, tissues, and insecticide treatments. We found that using unsuitable reference gene for normalization might lead to deviated results. We advise to choose α-TUB and 28S in the developmental stage, 18S and ACT in different tissues, and 28S and α-TUB in insecticide treatments. Therefore, choosing appropriate reference genes for normalization is a key precondition for the accurate estimation of target gene expression. 5. Conclusion We studied eight candidate reference genes by four popular programs and ∆Ct method, and confirmed that α-TUB and 18S was the most suitable reference genes for exploring gene expression profiles among different developmental stages, different tissues and insecticide treatments. This not only provided useful reference to Northern blot and RT-PCR techniques that require a reference gene for normalization, but also identified several potential reference genes to accurately evaluate target gene expression profiles in S. graminum . Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm Acknowledgements The authors are highly obliged to 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. References Al-Mousawi A H, Richardson P E, Burton R L. 1983. Ultrastructural studies of greenbug (Hemiptera: Aphididae) feeding damage to susceptible and resistant wheat cultivars. Annals of the Entomological Society of America , 76 , 964–971. Andersen C L, Jensen J L, Ørntoft T F. 2004. 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