期刊文献+

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

Selection of Reference Genes for Expression Analysis of Kumamoto and Portuguese Oysters and Their Hybrid
下载PDF
导出
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. 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. 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 (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 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.
出处 《Journal of Ocean University of China》 SCIE CAS CSCD 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)
关键词 CRASSOSTREA sikamea CRASSOSTREA angulata HYBRID OYSTER reference gene quantitative real-time PCR Crassostrea sikamea Crassostrea angulata hybrid oyster reference gene quantitative real-time PCR
  • 相关文献

参考文献1

二级参考文献39

  • 1Andersen C L, Jensen J L, Omtoft T F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 64(15): 5 245- 5 250.
  • 2Araya M T, Siah A, Mateo D, Markham F, McKenna P, Johnson G, Berthe F C J. 2008. Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus. Journal of Invertebrate Pathology, 99(3): 326-331.
  • 3Aursnes I A, Rishovd A L, Karlsen H E, Gjoen T. 2011. Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua L.) during temperature stress. BMC Research Notes, 4: 104.
  • 4Bustin S A. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology, 29(1): 23-29.
  • 5Castro L F C, Melo C, Guillot R, Mendes I, Queir6s S, Lima D, Reis-Henriques M A, Santos M M. 2007. The estrogen receptor of the gastropod (Nucella lapillus): modulation following exposure to an estrogenic effluent? Aquatic Toxicology, 84(4): 465-468.
  • 6Chen L, Xie L P, Xiong X H, Dai Y P, Fan W M, Zhang R Q. 2005. Cloning and characterization of a novel G protein [3-subunit of pearl oyster (Pinctada fucata), and its interaction sites with calmodulin. Comparative Biochemistry and Physiology Part B: Biochemistry & Molecular Biology, 142(2): 142-152.
  • 7Cho Y S, Lee S Y, Kim K H, Nam Y K. 2008. Differential modulations of two glyceraldehyde 3-phosphate dehydrogenase mRNAs in response to bacterial and viral challenges in a marine teleost Oplegnathus fasciatus (Perciformes). Fish & Shellfish Immunology, 25(5): 472- 476.
  • 8Cubero-Leon E, Ciocan C M, Minier C, Rotchell J M. 2011. Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure. Environmental Science and Pollution Research, 19(7): 2 728-2 733.
  • 9Dheilly N M, Lelong C, Huvet A, Favrel P. 2011. Development of a Pacific oyster (Crassostrea gigas) 31, 918-feature microarray: identification of reference genes and tissue- enriched expression patterns. BMC Genomics, 12(1): 468.
  • 10Dondero F, Dagnino A, Jonsson H, Capri F, Gastaldi L, Viarengo A. 2006. Assessing the occurrence of a stresssyndrome in mussels (Mytilus edulis) using a combined biomarker/gene expression approach. Aquatic Toxicology, 78(1): S13-$24.

共引文献1

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部