Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR

作     者：DENG Yunyan HU Zhangxi MA Zhaopeng TANG Ying Zhong 

作者机构：Key Laboratory of Marine Ecology and Environmental SciencesInstitute of OceanologyChinese Academy of SciencesQingdao 266071China Laboratory for Marine Ecology and Environmental ScienceQingdao National Laboratory for Marine Science and TechnologyQingdao 266071China University of the Chinese Academy of SciencesBeijing 100049China 

出 版 物：《Acta Oceanologica Sinica》 (海洋学报（英文版）)

年 卷 期：2016年第35卷第8期

页      面：106-112页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 090102[农学-作物遗传育种] 

基　　金：The National Natural Science Foundation of China-Shandong Joint Fund for Marine Science Research Centers under contract No.U1406403 the State Key Program of National Natural Science of China under contract No.61533011 China Postdoctoral Science Foundation under contract Nos 2014M551969 and 2015T80754 

主　　题：Akashiwo sanguinea dinoflagellate internal control qRT PCR resting cyst reference gene 

摘      要：The accurate measurement of gene expression via quantitative real-time reverse transcription PCR（q RT-PCR）heavily relies on the choice of valid reference gene（s） for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA（18S）, glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, α-tubulin（TUA）, β-tubulin（TUB）, actin（ACT） and cytochrome oxidase subunit 1（COX1）, were evaluated for their expression stability with q RT-PCR and three statistical algorithms（Ge Norm, Norm Finder, and Best Keeper） for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages（resting cysts）, different growth stages, in darkness, exposed to abscisic acid（ABA） and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates