Collaborative Innovation Center for Modern Grain Circulation and Safety, College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu, 210023, China.
State Key Laboratory of Grassland Agro-Ecosystems, College of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, Gansu, 730020, China.
Sci Rep. 2017 Aug 1;7(1):7047. doi: 10.1038/s41598-017-07430-2.
Cryptolestes ferrugineus is a serious pest of stored grain and has developed high levels of resistance to phosphine fumigants in many countries. Measuring differences in expression levels of certain 'resistant' genes by quantitative real-time PCR (qRT-PCR) may provide insights into molecular mechanisms underlying resistance to phosphine in C. ferrugineus, but reliable qRT-PCR results depend on suitable reference genes (RGs). We evaluated the stability of nine candidate RGs across different developmental stages and phosphine strains of C. ferrugineus, using four softwares. The results showed that RPS13 and EF1α were the most stable RGs, whereas α-TUB was the least under developmental stages. Across the different strains, RPS13 and γ-TUB were the most stable RGs, whereas CycA and GAPDH were the least. We confirmed the reliability of the selected RGs by qRT-PCR analyses of the mitochondrial cox1 gene. Expression of cox1 was not significantly different in the phosphine-resistant strain compared with the phosphine-susceptible strain, but three mitochondrial genes (nad3, atp6 and cob) were significantly down-regulated. These results suggest that alterations in the expressions of these three genes may be associated with phosphine resistance in C. ferrugineus. The findings will facilitate future functional genomics studies on the development and phosphine resistance in C. ferrugineus.
窃蠹属的一种严重的仓储害虫，在许多国家已对磷化氢熏蒸剂产生了高水平的抗性。通过实时荧光定量 PCR(qRT-PCR)测量某些“抗性”基因的表达水平差异，可能有助于了解窃蠹属对磷化氢抗性的分子机制，但可靠的 qRT-PCR 结果取决于合适的参考基因(RGs)。我们使用四种软件评估了 9 个候选 RG 在不同发育阶段和磷化氢菌株中的稳定性。结果表明，RPS13 和 EF1α 是最稳定的 RG，而在发育阶段，α-TUB 是最不稳定的。在不同菌株中，RPS13 和γ-TUB 是最稳定的 RG，而 CycA 和 GAPDH 是最不稳定的。我们通过 cox1 基因的 qRT-PCR 分析证实了所选 RG 的可靠性。与磷化氢敏感菌株相比，磷化氢抗性菌株中 cox1 的表达没有显著差异，但 3 个线粒体基因(nad3、atp6 和 cob)的表达显著下调。这些结果表明，这些三个基因的表达变化可能与窃蠹属对磷化氢的抗性有关。这些发现将有助于未来对窃蠹属的发育和磷化氢抗性进行功能基因组学研究。
