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 po...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.展开更多
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)+2 种基金Liaoning Provincial Natural Science Foundation (2012010263)Knowledge Innovation Programs of Dalian Institute of Chemical Physics,CAS (K2010A13)China Postdoctoral Science Foundation Funded Project (2014M551139)
文摘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.
