Department of Biological Sciences, North Dakota State University, Fargo, ND, 58108-6050, USA; Punjab Agricultural University, Regional Research Station, Faridkot, Punjab, India.
Punjab Agricultural University, Regional Research Station, Faridkot, Punjab, India.
Gene Expr Patterns. 2022 Mar;43:119233. doi: 10.1016/j.gep.2022.119233. Epub 2022 Feb 4.
In light of a number of recent studies highlighting the increasing research interest in bruchids, it is crucial to validate suitable reference genes that could be used in quantitative gene expression studies. Callosobruchus maculatus is a serious pest of stored grains and field legumes in which reference genes have not been assessed and validated to date. The present study aimed to identify and validate reference genes in different developmental stages of C. maculatus shortlisted from commonly used reference genes such as VATPase, TRIP12, TBP, TF11D, ACTIN, GST, ANNEXIN, PTCD3, RPL32, and β -Tub in various insects. Dedicated algorithms like GeNorm, NormFinder, and BestKeeper were used to analyze the stability of these candidate genes, which revealed GST for third instar, ANNEXIN and PTCD3 for the fourth instar, TF11D and VATPase for male pupa, RPL32 and β-tub for female pupa, β-tub and TBP for adult male and VATPase and GST for adult females as suitable reference genes for expression studies in C. maculatus. The final comprehensive ranking using RefFinder identified GST and TBP as the best reference genes for all the developmental stages of C. maculatus. To the best of our knowledge, this is the first report which evaluates and validates stable reference genes in C. maculatus. The information of stage-specific gene expression, generated in this study will be useful for future molecular, physiological, and biochemical studies on C. maculatus and other closely related bruchids.
鉴于最近有许多研究强调了人们对象鼻虫的研究兴趣日益增加，因此，验证可用于定量基因表达研究的合适参考基因至关重要。斑皮蠹是一种严重危害储粮和田间豆类的害虫，迄今为止，尚未对其参考基因进行评估和验证。本研究旨在从 VATPase、TRIP12、TBP、TF11D、ACTIN、GST、ANNEXIN、PTCD3、RPL32 和 β -Tub 等常用参考基因中鉴定和验证不同发育阶段的斑皮蠹参考基因。专门的算法，如 GeNorm、NormFinder 和 BestKeeper，用于分析这些候选基因的稳定性，结果表明 GST 适用于三龄幼虫，ANNEXIN 和 PTCD3 适用于四龄幼虫，TF11D 和 VATPase 适用于雄性蛹，RPL32 和 β -Tub 适用于雌性蛹，β -Tub 和 TBP 适用于雄性成虫，VATPase 和 GST 适用于雌性成虫，可用于 C. maculatus 的表达研究。使用 RefFinder 进行的最终综合排名确定 GST 和 TBP 是 C. maculatus 所有发育阶段的最佳参考基因。据我们所知，这是首次评估和验证 C. maculatus 稳定参考基因的报告。本研究中生成的特定阶段基因表达信息将有助于未来对 C. maculatus 和其他密切相关的象鼻虫进行分子、生理和生化研究。
