高光胁迫下坛紫菜定量PCR内参基因的筛选

为了筛选不同高光光强胁迫下坛紫菜中表达水平最为稳定的内参基因,本研究采用qRT-PCR技术测定了植物中常用的6种内参基因UBC、TUB、18S、EF2、ACT和GAPDH在不同光照强度下培养的坛紫菜藻体中的绝对表达水平,并采用比较Ct值法、Ge Norm、Bestkeeper和NormFinder软件分析4种方法对6种候选内参基因的表达稳定性进行评估。结果显示,UBC的Ct值变化范围最小,18S的Ct值变化范围最大;ge Norm软件分析结果显示最稳定的内参基因为UBC和EF2;Bestkeeper和NormFinder软件分析结果类似,UBC基因的稳定值M均为最低值(分别为0.044和0.80),说明其表达水平最稳定。研究表明,UBC基因可以作为不同光照条件下坛紫菜基因表达qRT-PCR分析的最适内参基因。

Selection of the internal control gene for expression analyses of Pyropia haitanensis under high light stress by quantitative real-time PCR

Pyropia haitanensis is a sessile marine macroalgae that inhabits the intertidal zone. High light was the main environmental stressor of P. haitanensis during low tides and the key factor affecting yield. To explore the molecular mechanisms of high light tolerance, gene expression analysis was the first step. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression levels of six housekeeping genes 18S rRNA (18S), ubiquitin-conjugating enzyme (UBC), actin (ACT), β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] were examined by the quantitative real-time PCR in samples corresponding to different high light stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by three different software packages: ge Norm, Bestkeeper and Norm Finder. The results showed that the Ct value of UBC had the least variation, while that of 18S obviously changed under different light intensities treatments. The analysis of ge Norm also exhibited that the most stable housekeeping genes were UBC and EF2. The analysis of both Bestkeeper and Norm Finder exhibited that stability measure M of UBC was the lowest (0.044 and 0.80, respectively) among six candidate genes. Thus the most stable housekeeping gene is UBC. Based on the above results, it is proposed that the most appropriate internal control gene for expression analyses under high light stress of P. haitanensis is UBC.