Validation of housekeeping genes as internal controls for studying the gene expression in <i>Pyropia haitanensis</i> (Bangiales, Rhodophyta) by quantitative real-time PCR

LI Bing; CHEN Changsheng; XU Yan; JI Dehua; XIE Chaotian

doi:10.1007/s13131-014-0526-2

坛紫菜基因表达实时荧光定量PCR分析中内参基因的筛选

doi: 10.1007/s13131-014-0526-2

集美大学水产学院, 厦门, 361021

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.

计量
- 文章访问数: 2004
- HTML全文浏览量: 36
- PDF下载量: 1180
- 被引次数: 0
出版历程
- 收稿日期: 2013-04-02
- 修回日期: 2013-12-18

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR

Fisheries College, Jimei University, Xiamen 361021, China

摘要

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C_t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。
- 坛紫菜 /
- 实施荧光定量PCR /
- 内参基因 /
- 基因表达
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 (C_t) 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.
- Pyropia haitanensis /
- quantitative real-time PCR /
- internal control genes /
- gene expression

HTML全文

参考文献(45)

Andersen C L, Jensen J L, Ørntoft 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): 5245-5250

Asamizu E, Nakajima M, Kitade Y, et al. 2003. Comparison of RNA expression profiles between the two generations of Porphyra yezoensis (Rhodophyta), based on expressed sequence tag frequency analysis. Journal of Phycology, 39(5): 923-930

Blouin N A, Brodie J A, Grossma A C, et al. 2011. Porphyra: a marine crop shaped by stress. Trends Plant Sci, 16(1): 29-37

Brunner A M, Yakovlev I A, Strauss S H. 2004. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biology, 4: 14

Bustin S A, Benes V, Garson J A, et al. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4): 611-622

Chang Ermei, Shi Shengqing, Liu Jianfeng, et al. 2012. Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR. PLoS ONE, 7(3): e33278

Choi S, Hwang M S, Im S, et al. 2013. Transcriptome sequencing and comparative analysis of the gametophyte thalli of Pyropia tenera under normal and high temperature conditions. Journal of Applied Phycology, 25(4): 1237-1246

Cole K, Conway E. 1975. Phenetic implications of structural features of the perennating phase in the life history of Porphyra and Bangia (Bangiophyceae, Rhodophyta). Phycologia, 14(4): 239-245

Czechowski T, Stitt M, Altmann T, et al. 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology, 139(1): 5-17

Dundas J, Ling M. 2012. Reference genes for measuring mRNA expression. Theory Biosci, 131(4): 215-223

Faccioli P, Ciceri G P, Provero P, et al. 2007. A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies. Plant Molecular Biology, 63(5): 679-688

Fan Xiaolei, Fang Yongjun, Hu Songning, et al. 2007. Generation and analysis of 5318 expressed sequence tags from the filamentous sporophyte of Porphyra haitanensis (Rhodophyta). Journal of Phycology, 43(6): 1287-1294

Flintoft L. 2011. Transcriptomics: measuring gene expression in nonmodel organisms. Nature Reviews Genetics, 12(11): 742

Guénin S, Mauriat M, Pelloux J, et al. 2009. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. Journal of Experimental Botany, 60(2): 487-493

Guo R Y, Ki J S. 2012. Evaluation and validation of internal control genes for studying gene expression in the dinoflagellate Prorocentrum minimum using real-time PCR. European Journal of Protistology, 48(3): 199-206

Hong S Y, Seo P J, Yang M S, et al. 2008. Exploring valid reference genes for gene expression studies in Brachypodium distachyon by realtime PCR. BMC Plant Biology, 8: 112

Hu Ruibo, Fan Chengming, Li Hongyu, et al. 2009. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Molecular Biology, 10: 93

Iskandar H M, Simpson R S, Casu R E, et al. 2004. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Molecular Biology Reporter, 22(4): 325-337 Jain M, Nijhawan A, Tyagi A K, et al. 2006. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochemical and Biophysical Research Communications, 345(2): 646-651

Jian Bo, Liu Bin, Bi Yurong, et al. 2008. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Molecular Biology, 9: 59

Kakinuma M, Coury D A, Nakamoto C, et al. 2008. Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta). Cell Biology and Toxicology, 24(6): 629-639

Kim B R, Nam H Y, Kim S U, et al. 2003. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnology Letters, 25(21): 1869-1872

Kim E, Park H S, Jung Y, et al. 2011. Identification of the high-temperature response genes from Porphyra seriata (Rhodophyta) expression sequence tags and enhancement of heat tolerance of Chlamydomonas (Chlorophyta) by expression of the Porphyra htr2 gene. Journal of Phycology, 47(4): 821-828 Le Bail A, Dittami S M, de Franco P O, et al. 2008. Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus. BMC Molecular Biology, 9: 75

Lee C, Kim J, Shin S G, et al. 2006. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. Journal of Biotechnology, 123(3): 273-280

Libault M, Thibivilliers S, Bilgin D D, et al. 2008. Identification of four soybean reference genes for gene expression normalization. Plant Genome, 1(1): 44-54

Mukai L S, Craigie J S, Brown R G. 1981. Chemical composition and structure of the cell walls of the conchocelis and thallus phases of Porphyra tenera (Rhodophyceae). Journal of Phycology, 17(2): 192-198

Muers M. 2011. Gene expression: transcriptome to proteome and back to genome. Nat Rev Genet, 12(8): 518

Nakamura Y, Sasaki N, Kobayashi M, et al. 2013. The first symbiontfree genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis). PLoS ONE, 8(3): e57122

Nicot N, Hausman J F, Hoffmann L, et al. 2005. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany, 56(421): 2907-2914

Nikaido I, Asamizu E, Nakajima M, et al. 2000. Generation of 10, 154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis. DNA Research, 7(3): 223-227

Niu Jianfeng, Gao Shenghan, Luo Yingfeng, et al. 2011. The analysis of the low coverage Porphyra yezoensis draft genome. Marine Sciences (in Chinese), 35(6): 76-81

Paolacci A R, Tanzarella O A, Porceddu E, et al. 2009. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Molecular Biology, 10: 11

Pfaffl M W, Tichopad A, Prgomet C, et al. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnology Letters, 26(6): 509-515

Radonić A, Thulke S, Mackay I M, et al. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 313(4): 856-862

Remans T, Smeets K, Opdenakker K, et al. 2008. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta, 227(6): 1343-1349

Rosic N N, Pernice M, Rodriguez-Lanetty M, et al. 2011. Validation of housekeeping genes for gene expression studies in Symbiodinium exposed to thermal and light stress. Marine Biotechnology, 13(3): 355-365

Sahoo D, Tang X R, Yarish C. 2002. Porphyra-the economic seaweed as a new experimental system. Current Science, 83(11): 1313-1316 Silver N, Best S, Jiang J, et al. 2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Molecular Biology, 7: 33

Sutherland J E, Lindstrom S C, Nelson W A, et al. 2011. A new look at an ancient order: generic revision of the Bangiales (Rhodophyta). Journal of Phycology, 47(5): 1131-1151

Tong Zhaoguo, Gao Zhihong, Wang Fei, et al. 2009. Selection of reliable reference genes for gene expression studies in peach using realtime PCR. BMC Molecular Biology, 10: 71

Udvardi M K, Czechowski T, Scheible W R. 2008. Eleven golden rules of quantitative RT-PCR. Plant Cell, 20(7): 1736-1737

Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3: RESEARCH0034

Whelan J A, Russell N B, Whelan M A. 2003. A method for the absolute quantification of cDNA using real-time PCR. Journal of Immunological Methods, 278(1-2): 261-269

Wu Xiaojie, Niu Jianfeng, Huang Aiyou, et al. 2012. Selection of internal control gene for expression studies in Porphyra haitanensis (Rhodophyta) at different life-history stages. Journal of Phycology, 48(4): 1040-1044

Xie Chaotian, Chen Changsheng, Xu Yan, et al. 2010. Construction of a genetic linkage map for Porphyra haitanensis (Bangiales, Rhodophyta) based on sequence-related amplified polymorphism and simple sequence repeat markers. Journal of Phycology, 46(4): 780-787

Yang Hui, Mao Yunxiang, Kong Fanna, et al. 2011. Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology. Chinese Science Bulletin, 56(20): 2119-2130

Zhang Xuecheng, Qin Song, Ma Jiahai, et al. 2005. The Genetics of Marine Algae (in Chinese). Beijing: China Agriculture Press, 184-186

施引文献

资源附件(0)

访问统计

点击查看大图

计量

文章访问数: 2004
HTML全文浏览量: 36
PDF下载量: 1180
被引次数: 0

坛紫菜基因表达实时荧光定量PCR分析中内参基因的筛选

doi: 10.1007/s13131-014-0526-2

计量

出版历程

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR

计量

出版历程

目录