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

作　　者：YAN Lulu SU Jiaqi WANG Zhaoping YAN Xiwu YU Ruihai 

机构地区：[1]Fisheries College,Ocean University of China [2]South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences [3]Engineering Research Center of Shellfish Culture and Breeding of Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University

出　　处：《Journal of Ocean University of China》2017年第6期1139-1147,共9页中国海洋大学学报（英文版）

基　　金：supported by the National Natural Science Foundation of China (No.31172403)

摘　　要：Quantitative real-time polymerase chain reaction(q RT-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 q RT-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, 28 S ribosomal protein S5(RPS5), ribosomal protein L35(RPL35), and 60 S ribosomal protein L29(RPL29); three structural genes, tubulin gamma(TUBγ), 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(EF1α and EF1β); and one protein synthesis gene(ubiquitin(UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, ge Norm, Norm Finder and Best Keeper, were used to evaluate the expression stability of these candidate genes. Best Keeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α 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, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. FurtheQuantitative 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 (TUBγ), 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 (EF1α and EF1β); 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 EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α 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, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyste

关 键 词：CRASSOSTREA sikamea CRASSOSTREA angulata HYBRID OYSTER reference gene quantitative real-time PCR 

分 类 号：S917.4[农业科学—水产科学]