| LI Bing,CHEN Changsheng,XU Yan,JI Dehua,XIE Chaotian. 2014. Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR. Acta Oceanologica Sinica, 33(9):152－159 |
| Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR |
| 坛紫菜基因表达实时荧光定量PCR分析中内参基因的筛选 |
| Received:April 02, 2013 Revised:December 18, 2013 |
| DOI：10.1007/s13131-014-0526-2 |
| Key words:Pyropia haitanensis quantitative real-time PCR internal control genes gene expression |
| 中文关键词: 坛紫菜 实施荧光定量PCR 内参基因 基因表达 |
| 基金项目:The National High Technology Research & Development Program of China under contract No. 2012AA10A411; the National Natural Science Foundation of China under contract Nos 41176151 and 41276177. |
| Author Name | Affiliation | E-mail | | LI Bing | Fisheries College, Jimei University, Xiamen 361021, China | | | CHEN Changsheng | Fisheries College, Jimei University, Xiamen 361021, China | | | XU Yan | Fisheries College, Jimei University, Xiamen 361021, China | | | JI Dehua | Fisheries College, Jimei University, Xiamen 361021, China | | | XIE Chaotian | Fisheries College, Jimei University, Xiamen 361021, China | ctxie@jmu.edu.cn |
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| Abstract: |
| Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes (18S rRNA (18S), ubiquitin-conjugating enzyme (UBC), actin (ACT), β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by two different software packages: geNorm and Norm-Finder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses. |
| 中文摘要: |
| 坛紫菜是我国南方沿海广泛栽培的一种大型经济海藻，其许多独特的生命现象都具有很高的研究价值，如抗逆性。为了解这些特有的生命现象，国内多个课题组已经在转录组水平开展了相关基因表达水平的研究。采用实时荧光定量PCR技术进行基因表达水平研究的首要问题是要有一个合适的看家基因作为内参。为此，本研究从坛紫菜中选择了6个看家基因[18S rRNA （18S），泛素连接酶（UBC）， 肌动蛋白（ACT）， β-微管蛋白（TUB）， 延伸因子2 （EF2）和3-磷酸甘油醛脱氢酶（GAPDH）]，采用实时荧光定量PCR技术测定了它们在坛紫菜不同品系、不同生活史阶段和不同胁迫条件下的绝对表达水平，并通过比较Ct值、geNorm和NormFinder软件分析3种方法评价了这6个看家基因在不同条件下的表达稳定性，结果表明：各样本之间表达水平最稳定的看家基因是UBC基因，而GAPDH基因的表达水平最不稳定。由此认为，UBC基因可以作为后续坛紫菜各生命现象相关基因表达研究的最佳内参基因。 |
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