Determination of Internal Controls for Quantitative Gene Expression of Isochrysis zhangjiangensis at Nitrogen Stress Condition  

作　　者：WU Shuang ZHOU Jiannan CAO Xupeng XUE Song 

机构地区：[1]Marine Bioproducts Engineering Group,Dalian Institute of Chemical Physics,Chinese Academy of Sciences

出　　处：《Journal of Ocean University of China》2016年第1期137-144,共8页中国海洋大学学报（英文版）

基　　金：supported by the Hundred Talent Program of the Chinese Academy of Sciences (No.A1097);National High Technology Research and Development Program ‘863’ (2012AA052101);Liaoning Provincial Natural Science Foundation (2012010263);Knowledge Innovation Programs of Dalian Institute of Chemical Physics,CAS (K2010A13);China Postdoctoral Science Foundation Funded Project (2014M551139)

摘　　要：lsochrysis zhangfiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under ni- trogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction （qPCR） provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin （ACT）, α-tubulin （TUA）, β-tubulin （TUB）, ubiquitin （UBI）, 18S rRNA （18S） and one target gene, diacylglycerol acyltransferase （DGAT）, were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed 1. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experi- mental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expres- sion under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of/. zhangfiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production,which accumulates lipid under nitrogen limitation conditions,but the mechanism on molecular level is veiled.Quantitative real-time polymerase chain reaction(q PCR) provides the possibility to investigate the gene expression levels,and a valid reference for data normalization is an essential prerequisite for firing up the analysis.In this study,five housekeeping genes,actin(ACT),α-tubulin(TUA),β-tubulin(TUB),ubiquitin(UBI),18 S r RNA(18S) and one target gene,diacylglycerol acyltransferase(DGAT),were used for determining the reference.By analyzing the stabilities based on calculation of the stability index and on operating the two types of software,ge Norm and bestkeeper,it showed that the reference genes widely used in higher plant and microalgae,such as UBI,TUA and 18 S,were not the most stable ones in nitrogen-stressed I.zhangjiangensis,and thus are not suitable for exploring the m RNA expression levels under these experimental conditions.Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions.Our findings will contribute not only to future q PCR studies of I.zhangjiangensis,but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

关 键 词：CHRYSOPHYTA Isochrysis zhang/iangensis nitrogen deficiency lipid accumulation reference gene selection 

分 类 号：Q943.2[生物学—植物学]