Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

摘要

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China.Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta.Here,the effects of salinity and light intensity on the quantum yield of photosystem Ⅱ in Py.haitanensis were investigated using pulse-amplitude-modulation fluorometry.Total RNA and genomic DNA of the samples under different stress conditions were isolated.By normalizing to the genomic DNA quantity,the RNA content in each sample was evaluated.The cDNA was synthesized and the expression levels of seven potential intemal control genes were evaluated using qRT-PCR method.Then,we used geNorm,a common statistical algorithm,to analyze the qRT-PCR data of seven reference genes.Potential genes that may constantly be expressed under different conditions were selected,and these genes showed stable expression levels in samples under a salinity treatment,while tubulin,glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light.Based on the results of the pulse amplitude-modulation fluorometry,an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples.The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening.Based on the results of the software analysis and absolute quantification,we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py.haitanensis blades were subjected to salinity stress,and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light.In general,our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py.haitanensis.