Selection of Reference Genes for Expression Analysis of Kumamoto and Portuguese Oysters and Their Hybrid

文献类型： 外文期刊

第一作者： Yan Lulu

作者： Yan Lulu;Wang Zhaoping;Yu Ruihai;Yan Lulu;Su Jiaqi;Yan Xiwu

作者机构：

关键词： Crassostrea sikamea;Crassostrea angulata;hybrid oyster;reference gene;quantitative real-time PCR

期刊名称：JOURNAL OF OCEAN UNIVERSITY OF CHINA （ 影响因子：0.913； 五年影响因子：1.012 ）

ISSN： 1672-5182

年卷期： 2017 年 16 卷 6 期

页码：

收录情况： SCI

摘要： Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts (expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of qRT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea (SS), Crassostrea angulata (AA) and their hybrid (SA), which included three ribosomal genes, 28S ribosomal protein S5 (RPS5), ribosomal protein L35 (RPL35), and 60S ribosomal protein L29 (RPL29); three structural genes, tubulin gamma (TUB gamma), annexin A6 and A7 (AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase (OD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSP); two transcription factors, elongation factor 1 alpha and beta (EF1 alpha and EF1 beta); and one protein synthesis gene (ubiquitin (UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, geNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of these candidate genes. BestKeeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1 alpha was stable under low salinity stress, and the expression of OD, GAPDH and EF1 alpha was stable under low temperature stress in hybrid (SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1 beta and AA7 was stable under low salinity stress, and the expression of RPL35, EF1 alpha, GAPDH and EF1 beta was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.

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