光裸星虫全组织荧光定量PCR分析中内参基因的筛选  

Screening of Reference Genes for Real-time PCR in Whole Tissue from Sipunculus nudus

作　　者：张家炜[1] 郑哲[1] 王庆恒[1] 黄荣莲[1] 杜晓东[1] ZHANG Jia-wei;ZHENG Zhe;WANG Qing-heng;HUANG Rong-lian;DU Xiao-dong(Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China)

机构地区：[1]广东海洋大学水产学院,广东湛江524025

出　　处：《广东海洋大学学报》2018年第1期7-13,共7页Journal of Guangdong Ocean University

基　　金：广东省科技计划(2016A020209010);广东省自然科学基金(2017A030303075)

摘　　要：【目的】筛选光裸星虫体壁、肠道、肾管、吻、收吻肌以及体腔液细胞中稳定表达的内参基因。【方法】应用荧光定量PCR技术,对GAPDH、β-actin、TUB、TBP以及UBC等常用内参基因的表达情况进行分析,通过ge Norm、Norm Finder、Best Keeper以及在线内参筛选工具Ref Finder对8个内参基因表达稳定性进行评测。【结果】8个内参基因均可获得特异性扩增产物,但稳定性各异。通过累计积分法综合了4个软件给出的稳定性排序结果,候选内参基因表达稳定性综合排名为TBP(1)>TBP(2)>UBC>GAPDH>TUB(2)>TUB(1)>β-actin(2)>β-actin(1)。【结论】TBP(1)在光裸星虫全组织中的表达最稳定,可作为最适单内参基因。【Objective】To obtain the most stable reference gene in tissues of Sipunculus nudus(trunk muscle,intestinal,nephridium,introvert,retractor muscle,and coelomocytes).【Method】Quantitative Real-time PCR(qRT-PCR)was adopted to analyze eight traditional reference genes which including3-phosphate dehydrogenase(GAPDH),β-actin,tubulin(TUB),TATA box binding protein(TBP),and ubiquitin C(UBC).Combining geNorm,NormFinder,BestKeeper and RefFinder software,the expression stability of eight reference genes had been analyzed.【Result】The results showed that specific amplification products could be obtained by the eight reference genes.The results of the stability ranking given by the four software were integrated by the cumulative integration method.The reference genes stability was descended as follows:TBP(1)>TBP(2)>UBC>GAPDH>TUB(2)>TUB(1)>β-actin(2)>β-actin(1).【Conclusion】The TBP(1)could be used as a suitable reference gene in qRT-PCR analysis of S.nudus.The results identified a suitable reference gene and laid the foundation for the follow-up research in the molecular biology.

关 键 词：荧光定量PCR 内参基因 光裸星虫 

分 类 号：Q959.19[生物学—动物学]