昆虫学报 ›› 2016, Vol. 59 ›› Issue (4): 427-437.doi: 10.16380/j.kcxb.2016.04.008

• 研究论文 • 上一篇    下一篇

松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选

冯波, 郭前爽, 毛必鹏, 杜永均*   

  1. (温州医科大学健康与环境生态研究所, 浙江温州 325035)
  • 出版日期:2016-04-20 发布日期:2016-04-20

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

摘要: 【目的】本研究拟选择适合用于分析松墨天牛Monochamus alternatus 化学感受组织中基因表达的内参基因。【方法】依据转录组测序结果进行内参基因鉴定,利用RT-qPCR技术分析内参基因在松墨天牛不同发育阶段和不同性别化学感受组织间的表达差异,并利用软件 geNorm, NormFinder和 BestKeeper比较其表达的稳定性。【结果】松墨天牛转录组中鉴定出9个候选内参基因(Actin, TUB, 18S rRNA, RPS27A, RPS3, RPL10, AK, GAPDHEF1A),其中后7个候选内参基因在松墨天牛中被首次鉴定,松墨天牛候选内参基因和其他昆虫相应基因的同源性很高。9个候选内参基因引物均具有良好的扩增效率,18S rRNA的表达水平最高,EF1A的表达水平最低;18S rRNA和Actin在不同样品间的表达水平差异最大,GAPDHTUB表达水平在不同样品间差异最小。geNorm和NormFinder软件分析认为,GAPDH是最稳定的内参基因,TUB是较为稳定的内参基因,18S rRNA和Actin是最不稳定的内参基因;BestKeeper软件分析认为,GAPDHTUB是合适的内参基因,18S rRNA和Actin是不适合的内参基因。最适合校正松墨天牛化学感受组织中基因表达数据的内参基因数量为2个,即GAPDHTUB,并且这样的内参基因组合可以用于不同发育阶段和不同性别的不同化学感受组织。【结论】本研究结果为利用RT-qPCR技术准确分析松墨天牛和其他天牛基因包括化学感受组织基因相对表达量的内参基因选择提供了重要参考。

关键词: 松墨天牛, 内参基因, 化学感受, 发育, 荧光定量PCR

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