Selection of reference genes for RT-qPCR of ovarian gene expression in Calliptamus italicus (Orthopera: Acrididae)

Author of the article:XU Ya-Li, DONG Bin, DING Guo-Chan, SANG Di, JI Rong, WANG Han

Author's Workplace：International Research Center for the Collaborative Containment of Cross-Border Pests in Central Asia, Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology, College of Life Sciences, Xinjiang Normal University, Urumqi 830054, China

Key Words：Calliptamus italicus; reference gene; expression stability; ovarian development; RT-qPCR

Abstract：[Objectives]  To identify stable reference genes for RT-qPCR investigation of the expression and regulatory functions of genes related to ovarian development of Calliptamus italicus (L.), an important pest in Xinjiang’s grasslands. [Methods]  Nine genes including β-actin, EF1-a, β-tubulin, α-tubulin, GAPDH, SDHA, GST, RPL32 and TBP, were selected as candidate reference genes. The expression levels of these genes were detected with quantitative real-time PCR and their stability of expression evaluated with GeNorm, NormFinder, BestKeeper, and RefFinder. Their stability in different tissues and developmental stages was verified using Hex2, VgR, Sox9a and Cyp6bg10 as the target gene. [Results]  β-tubulin, RPL32, GAPDH and EF1-α were the most stable genes. Based on comprehensive analysis using GeNorm and RefFinder, the best combination of reference genes for the oocytes of different developmental stages was RPL32 and EF1-α. [Conclusion]  The most stable genes identified in this study could be suitable reference genes for analyzing gene expression in C. italicus.