JIA-2018-09

2062 ZHANG Bai-zhong et al. Journal of Integrative Agriculture 2018, 17(9): 2054–2065 tissues, significant difference of GST transcript levels was observed between heads, thoraces and abdomens no matter whether it was normalized by the most stable reference gene ( 18S ), the combination of the two most stable reference genes ( 18S and ACT ), or the least stable reference gene ( GAPDH ). The expression level of GST normalized by the most stable reference gene or the combination of the two best reference genes was significantly different from the expression level calculated using the least suitable reference gene in the abdomens ( P <0.05) (Fig. 6-B). Across insecticide treatments, GST transcript levels increased significantly in insecticide treatments compared with controls no matter whether it was normalized by the most stable reference gene ( 28S ), the combination of the two most stable reference genes ( 28S and α-TUB ), or the least stable reference gene ( ACT ). Furthermore, the expression level of GST normalized by the most stable reference gene or the combination of the two best reference genes was significantly different from the expression level calculated using the least suitable reference gene in the insecticide treatments ( P <0.05) (Fig. 6-C). 4. Discussion We found that 28S and α-TUB were the best reference genes according to the average expression stability or stability values acquired by ∆Ct method, geNorm, NormFinder and RefFinder while BestKeeper selected ACT and 28S . There were also some differences in developmental stages, tissues and insecticide treatments when the outcomes of the five methods were compared. Considering the developmental stages, ∆Ct method, BestKeeper, NormFinder, geNorm, and RefFinder all identified α-TUB and 28S as the most stable genes. Among different tissues, the most stable genes were RPL7 and EF1α (∆Ct method, NormFinder), 28S and α-TUB (BestKeeper), EF1β and TBP (geNorm), and 18S and ACT (RefFinder), respectively. For insecticide treatments, the most stable genes were α-TUB and 28S (∆Ct, NormFinder, geNorm, and RefFinder), and GAPDH and 28S (BestKeeper), respectively. Based on the rankings from RefFinder, which integrates outcomes of the four major statistic algorithms (∆Ct method, geNorm, Normfinder, and Bestkeeper), and it also assigns an appropriate weight to an individual gene and calculates the geometric mean of their weight, 28S and α-TUB had a good performance under specific conditions. Of these most commonly - used reference genes, GAPDH , EF1α , 18S , 28S and ACT tested in S. graminum varied considerably. rRNAs, including 18S rRNA and 28S rRNA, were commonly-used reference genes, as the levels of rRNA are thought to be less likely to vary due to under different conditions (Bustin 2000; Bagnall and Kotze 2010; Chandra et al. 2014). This is consistent with our results that 18S and 28S were the stable genes among the different developmental stages, tissues and insecticide treatments. However, 18S and 28S were the least suitable reference genes under the majority of the experimental conditions in Relative quantity First-instar nymphs Third-instar nymphs Apterous adults Alate adults 0 2 4 6 Relative quantity Heads and thoraxes Abdomens 0 0.5 1.0 1.5 2.0 2.5 Relative quantity Controls Imidacloprid treatments 0 1 2 3 4C B A NF1 NF1–2 NF8 Fig. 6 Relative expression levels of a target gene of interest ( GST ) calculated using different sets of reference genes. A, expression levels in different developmental stages. B, expression levels in different tissues. C, expression levels in insecticide treatments. NF1, the most stable reference gene; NF1–2, the top two stable reference genes; NF8, the least stable reference gene. Bars represent the mean±SE of three biological replicates.