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: geNorm, Bestkeeper and NormFinder. The results showed that the Ct value ofUBC had the least variation, while that of 18S obviously changed under different light intensities treatments. The analysis of geNorm also exhibited that the most stable housekeeping genes were UBC and EF2. The analysis of both Bestkeeper and NormFinder 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.