Acta Entomologica Sinica ›› 2016, Vol. 59 ›› Issue (4): 427-437.doi: 10.16380/j.kcxb.2016.04.008

• RESEARCH PAPERS • Previous Articles     Next Articles

Identification and selection of valid reference genes for assaying gene expression in the chemosensory tissues of Monochamus alternatus (Coleoptera: Cerambycidae) by RT-qPCR

FENG Bo, GUO Qian-Shuang, MAO Bi-Peng, DU Yong-Jun*   

  1. (Institute of Health and Environmental Ecology, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China)
  • Online:2016-04-20 Published:2016-04-20

Abstract: 【Aim】 This study aims to select suitable internal control genes for gene expression analysis in chemosensory tissues of Monochamus alternatus. 【Methods】 Candidate reference genes were identified from our transcriptome results, and their expression patterns in chemosensory tissues were investigated by RT-qPCR in different developmental stages and both genders. Their expression stabilities were also compared with each other by using softwares geNorm, NormFinder and BestKeeper. 【Results】 Nine candidate reference genes were identified in M. alternatus, including Actin, TUB, 18S rRNA, RPS27A, RPS3, RPL10, AK and GAPDH, of which the last seven were firstly identified in this insect species. Candidate reference genes in M. alternatus have high nucleotide sequence identity with those from other insects. Designed primers of nine candidate reference genes worked well with high amplification efficiencyand specificity to M. alternatus. It was found that 18S rRNA had the highest expression level while EF1A had the lowest expression level in M. alternatus. 18S rRNA and Actin had the most variable expression levels between studied chemosensory tissues, but GAPDH and TUB had the least variable expression levels. GAPDH was the most stable reference gene, TUB was a moderately stable reference gene, and 18S rRNA and Actin were the least stable reference genes in different chemosensory tissues, which was referred from analysis with softwares geNorm and NormFinder. Analysis using BestKeeper software showed that GAPDH and TUB but not 18S rRNA and Actin were the suitable reference genes in chemosensory tissues of M. alternatus. The combination of two reference genes, GAPDH and TUB, was referred as the best internal control combination genes for normalization of RT-qPCR data conducted with chemosensory tissues of different developmental stages (larva, pupa and adult) and different genders of M. alternatus. 【Conclusion】 The results of this study provide important data for selecting internal reference genes for gene expression analysis of M. alternatus and other longhorn beetles, and also for gene expression analysis of chemosensory tissues of other insects by RT-qPCR.

Key words: Monochamus alternatus, reference gene, chemosensation, development, RT-qPCR