Cite this paper:
WANG Xia, FENG Jianhua, HUANG Aiyou, HE Linwen, NIU Jianfeng, WANG Guangce. Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis[J]. HaiyangYuHuZhao, 2017, 35(6): 1432-1441

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

WANG Xia1,2,4, FENG Jianhua1, HUANG Aiyou1,2,3, HE Linwen1,2,3, NIU Jianfeng1,2,3, WANG Guangce1,2,3
1 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
2 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
3 Nantong Branch, Institute of Oceanology, Chinese Academy of Sciences, Nantong 226006, China;
4 University of Chinese Academy of Sciences, Beijing 100049, China
Abstract:
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 internal 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.
Key words:    real-time quantitative PCR|housekeeping genes|internal control genes|stress responding|Pyropia haitanensis   
Received: 2016-03-28   Revised: 2016-06-22
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