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

DENG Yunyan; HU Zhangxi; MA Zhaopeng; TANG Ying Zhong

doi:10.1007/s13131-016-0887-9

藻华甲藻红色赤潮藻(Akashiwo sanguinea)实时荧光定量PCR内参基因的筛选

doi: 10.1007/s13131-016-0887-9

1.
中国科学院海洋研究所, 海洋生态与环境科学重点实验室, 青岛, 266071
2.
中国科学院海洋研究所, 海洋生态与环境科学重点实验室, 青岛, 266071;中国科学院大学, 北京, 100049
3.
中国科学院海洋研究所, 海洋生态与环境科学重点实验室, 青岛, 266071;青岛海洋科学与技术国家实验室海洋生态与环境科学功能实验室, 青岛, 266071

基金项目: 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.

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- 收稿日期: 2015-07-23
- 修回日期: 2016-02-18

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

1.
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2.
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;University of the Chinese Academy of Sciences, Beijing 100049, China
3.
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China

摘要

摘要: 实时荧光定量PCR（qRT-PCR）技术是研究功能基因表达的常用方法之一。准确的进行基因表达量检测需要表达稳定的管家基因作为内参对实验过程校正。形成休眠孢囊是许多甲藻类藻华原因种一项重要的生活史适应策略。休眠孢囊在孢囊类甲藻种源延续、藻华生消、年际复发和地理扩散等方面发挥着非常关键的作用，但目前在该生物学过程中的表达稳定的内参基因尚未见报道。本文以世界广布性有毒有害藻华甲藻—红色赤潮藻（Akashiwo sanguinea）为研究对象，选取编码18S核糖体RNA（18S ribosomal RNA,18S），甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase,GAPDH）,微管蛋白α亚基（α-tubulin,TUA）,微管蛋白β亚基（β-tubulin,TUB),肌动蛋白（actin,ACT）和细胞色素氧化酶亚基1（cytochrome oxidase subunit 1,COX1)的6个常用管家基因作为候选基因。利用3种内参筛选软件，GeNorm,NormFinder和BestKeeper，对红色赤潮藻休眠孢囊、生长不同阶段、不同温度变化、不同黑暗时间和植物激素脱落酸处理条件下候选基因的表达稳定性进行检测。结果表明，对于所有检测条件下的细胞，TUA，18S和GAPDH表达量相对稳定；在不同生长阶段和黑暗条件下，ACT和TUA,18S和GAPDH分别是表达最稳定的内参基因组合；在休眠孢囊和脱落酸处理条件下，TUA和TUB分别是表达稳定性最高的内参基因；在不同温度条件下，ACT和COX1是最稳定的内参组合。本文首次对适用于甲藻休眠孢囊qRT-PCR检测的内参基因进行筛选，相关研究结果为后续开展藻华甲藻功能基因表达规律、在分子水平上探索红色赤潮藻休眠孢囊生物学机制的研究奠定基础。
- 红色赤潮藻 /
- 藻华甲藻 /
- 休眠孢囊 /
- 内参基因 /
- 实时荧光定量PCR
Abstract: The accurate measurement of gene expression via quantitative real-time reverse transcription PCR (qRT-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 qRT-PCR to the HAB-forming group. In this study, six candidate reference genes, 18S 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 qRT-PCR and three statistical algorithms (GeNorm, NormFinder, and BestKeeper) 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, 18S 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, 18S 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 regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of qRT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.
- Akashiwo sanguinea /
- dinoflagellate /
- internal control /
- qRT-PCR /
- resting cyst

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藻华甲藻红色赤潮藻(Akashiwo sanguinea)实时荧光定量PCR内参基因的筛选

doi: 10.1007/s13131-016-0887-9

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

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