Screening and stability analysis of internal reference genes in Bombus terrestris

Author of the article:XIA Zhong-Yan LIU Fu-Gang YANG Fan ZHANG Zhi-Hao YANG Jun-Hao YANG Hui-Peng LI Ji-Lian

Author's Workplace：State Key Laboratory of Resource Insects Institute of Apicultural Research, Chinese Academy of Agricultural Science

Key Words：Bombus teresstris; internal-reference gene; gene selection; quantitative real-time PCR; stability analysis

Abstract：[Aim]  Selecting appropriate internal reference genes is crucial for the analysis of gene expression in insects. Using real-time PCR, this study assessed the expression stability of five candidate reference genes across four tissue types in bumble bees, aiming to identify the most suitable reference gene for quantitative gene expression analysis. [Methods]  Specific primers were designed for the five candidate internal reference genes of Bombus terrestris, including Actin5C, PLA2 (phospholipase A2), S18(ribosomal protein S18), S28(ribosomal protein S28), and EF-1 (elongation factor 1 alpha). Newly emerged workers, queens, and drones were labeled and reared in their original colonies. Total RNA was extracted from samples of the head, ovary, ventral nerve cord, and seminal vesicle taken at 1, 3, 5, 7, and 10 days of age. The fragments of candidate reference genes were cloned into a pMD19-T vector before being verified via sequencing. The amplification processes were then optimized and the standard curves established. The levels of candidate internal reference genes in queen, worker, and drone tissue samples were quantified using real-time PCR. The expression stability of the internal candidate internal reference genes was analyzed using NormFinder, BestKeeper, GeNorm, CT, and RefFinder. [Results]  The expression stability of the candidate reference genes depended on the method of analysis used. However, results from a comprehensive analysis using RefFinder showed that PLA2 had the most stable expression level in the head of queens and drones, ventral nerve cord of workers and drones, and ovaries of workers. Actin5C was most stably expressed in the ovaries of queens and workers, whereas EF-1 had the most stable expression level in the ventral nerve cord of queens. Finally, S18 was the stably expressed gene in the seminal vesicle of drones. [Conclusion]  These findings indicate that the quantitative stability of internal reference genes varies among castes and tissues of bumble bees. Therefore, it is crucial to select the most stable internal reference genes based on the sample tissue type to ensure the reliability of the results.