JIA-2018-09

2055 ZHANG Bai-zhong et al. Journal of Integrative Agriculture 2018, 17(9): 2054–2065 expression under all experimental conditions, the studies of validating reference genes have been driven by several algorithms and freely available softwares, e.g., geNorm (Vandesompele et al. 2002), BestKeeper (Pfaffl et al. 2004), NormFinder (Andersen et al. 2004). RT-qPCR is generally characterized as one of effective, sensitive, and economical methods, which has been already widely applied to analyze gene expression in biological research (Lu et al. 2013; Liang et al. 2014). However, there remains a number of challenges. One of the biggest challenges in RT-qPCR analysis is normalization of the data to correct the variations created due to tissue amount, RNA extraction and purification, reverse transcription, efficiency of PCR amplification, etc. (Bustin et al. 2009). Several strategies have been proposed to normalize these variations in RT-qPCR analysis, including normalization of sample size, ensuring the quality and quantity of RNA, and removing DNA contamination (Huggett et al. 2005). Of such strategies, the most widely used one is the selection of appropriate reference gene to normalize nonspecific variation or errors (Liang et al. 2014). Actin ( ACT ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) have been demonstrated to change broadly under particular experimental conditions or in response to external stimuli (Glare et al. 2002; Ma et al. 2016). Clearly, for a given set of experimental biological samples, selecting suitable reference genes for use in the normalization of RT-qPCR data is quite urgent. To date, several studies have systematically assessed reference genes in various insects across different experimental conditions. The greenbug, Schizaphis graminum is one of the most serious pests of cereal crops, especially for wheat and is extensively distributed in relative temperate areas (Al- Mousawi et al. 1983; Lage et al. 2003; Newman 2005). This pest is the most important factor affecting wheat yield (Burton et al. 1985; Kieckhefer and Kantack 1988; Riedell et al. 1999; Kindler et al. 2002; Ranjan 2006; Hussain et al. 2015). In addition, it also injects toxins into host plants and causes serious plant damage (Reavy and Mayo 2002). However, the mechanisms including evolutionary history, resistance to insecticides, transmission of viruses and so on, were seldom studied. In recent years, RT-qPCR has been widely used to quantify gene expression level in pests, such as studies of insecticide resistance and ecological adaption (Shang et al. 2016; Zhang et al. 2016). Some studies have shown that at least two or three reference genes should be used to achieve accurate normalization (Thellin et al. 1999; Vandesompele et al. 2002). However, in the aforementioned reports, only one reference gene ( 18S , GAPDH , or ACT ) was used to normalize the variation in mRNA levels of genes of interest for all of the diverse experimental conditions. No validated reference gene for S. graminum was used in gene expression of developmental stages, different tissues and insecticide treatments. Thus, a systematic validation of reference gene is required to ensure suitable normalization in S. graminum . So, we conducted the present study to ameliorate this situation and to enable the empirically informed selection of suitable reference genes for future study with S. graminum . Eight commonly used normalization 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 selected for analyzing their performance under several different experimental conditions in S. graminum . Whereafter, three target genes, heat shock protein gene ( HSP70 ), cytocrome P450 gene ( SgraCYP18A1 ), and glutathione S -transferase ( GST ) were selected and used as the validation on the performance of the reference genes. To our knowledge, our study is critical systematic study to validate a set of candidate reference genes for RT-qPCR in S. graminum . This should be useful for the selection of suitable, reliable reference genes in modern molecular genetic analyses in S. graminum . 2. Materials and methods 2.1. Insect culture The strain of S. graminum used was collected from the Department of Entomology, China Agricultural University, which has been maintained in laboratory for more than 3 years. The aphids were reared on wheat seedlings under controlled conditions of 15–23°C, 60% relative humidity (RH), and a photoperiod of 16 h L:8 h D, as described previously (Lu and Gao 2007). 2.2. Biotic factors Developmental stages Ten of first-instar nymphs, third-instar nymphs, alate adults, and apterous adults of S. graminum were collected in RNase-free tubes for each replication, respectively. Then snap frozen in the liquid nitrogen before stored at –80°C for RNA extraction. The experiments were replicated at least three times. Different tissues Heads, thoraces, and abdomens were collected from 30 third-instar nymphs in RNase-free tubes for each replication. Then snap frozen in the liquid nitrogen before stored at –80°C for RNAextraction. The experiments were conducted at least three times. 2.3. Abiotic factors (Insecticide treatments) Leaf-dipping with aphids was performed according to