Chinese Journal of Oceanology and Limnology

, Volume 35, Issue 6, pp 1432–1441 | Cite as

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

  • Xia Wang (王霞)
  • Jianhua Feng (冯建华)
  • Aiyou Huang (黄爱优)
  • Linwen He (何林文)
  • Jianfeng Niu (牛建峰)
  • Guangce Wang (王广策)
Biology
  • 67 Downloads

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.

Keywords

real-time quantitative PCR housekeeping genes internal control genes stress responding Pyropia haitanensis 

Abbreviations

18S

18S ribosomal RNA

EF3

elongation factor 3

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

RPS

30S ribosomal protein

TubA

Alpha-tubulin

ACT

actin

TubB

beta-tubulin

PAM

pulse-amplitude-modulation fluorometry

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Notes

Acknowledgment

We would like to thank SUN Qinghai of Haihu Seaweed Farming Company for kindly provision of experiment material.

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Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Xia Wang (王霞)
    • 1
    • 2
    • 4
  • Jianhua Feng (冯建华)
    • 1
  • Aiyou Huang (黄爱优)
    • 1
    • 2
    • 3
  • Linwen He (何林文)
    • 1
    • 2
    • 3
  • Jianfeng Niu (牛建峰)
    • 1
    • 2
    • 3
  • Guangce Wang (王广策)
    • 1
    • 2
    • 3
  1. 1.Key Laboratory of Experimental Marine Biology, Institute of OceanologyChinese Academy of SciencesQingdaoChina
  2. 2.Laboratory for Marine Biology and BiotechnologyQingdao National Laboratory for Marine Science and TechnologyQingdaoChina
  3. 3.Nantong Branch, Institute of OceanologyChinese Academy of SciencesNantongChina
  4. 4.University of Chinese Academy of SciencesBeijingChina

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