Selection of Suitable Internal Control Genes in Microalgae Under Radiation Condition
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摘要: 作为空间辐射的一种主要成分,紫外辐射可广泛引起陆地植物与水生生物细胞及其组份的破坏.荧光定量PCR技术广泛应用于各类胁迫环境下,研究目的基因的转录水平.在荧光定量PCR中选用合适的内参基因,能够更加准确地校正和标准化目的基因转录水平.本实验研究了辐射条件下水生生物莱茵衣藻6个传统内参基因18SrRNA,GAPDH,β-actin,β-tubulin,EF1-α和UBC基因表达的稳定性.经GeNorm软件研究分析,在辐射条件下,莱茵衣藻18SrRNA基因表达最不稳定,而选用β-actin和GAPDH作为双内参,可以得到更精确的实验结果.Abstract: As a major component of space radiation, ultraviolet radiation can extensively destruct terrestrial plants and aquatic. Real-time PCR technique has been widely used to detect the level of target mRNA expression when cell or tissues exposed to various types environmental stress. Choosing an appropriate internal control gene is important to accurately analyze the level of target gene transcription with real-time PCR technique. Therefore, our experiment tries to choose the most appropriate and stability reference gene from six traditional internal control genes, i.e., 18S rRNA, GAPDH, β-actin, β-tubulin, EF1-α and UBC in the Chlamydomonas reinhardtii under radiation conditions. The results showed that C. reinhardtii 18S rRNA gene expression is the most unstable through GeNorm analysis, but β-actin and GAPDH gene were finally selected as a pair of suitable internal control genes in expression analysis, with which more accurate experimental results can be obtained.
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Key words:
- Radiation /
- Chlamydomonas reinhardtii /
- Real-time PCR /
- Internal control gene
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[1] Li Taosheng, Chen Jun, Wang Zhiqiang. Brief introduction to space radiation environment[J]. Radiat. Prot. Bull., 2008, 4(2):1-10. In Chinese (李桃生, 陈军, 王志强. 空间辐射环境概 述[J]. 辐射防护通讯, 2008, 4(2):1-10) [2] Hanelt D, Roleda M Y. UVB radiation may ameliorate photoinhibition in specific shallow-water tropical marine macrophytes[J]. Aquat. Bot., 2009, 91(1):6-12 [3] Rao M V, Paliyath G, Ormrod D P. Differential response of photosynthetic pigments, rubisco activity and rubisco protein of Arabidopsis thaliana exposed to UVB and ozone[J]. Photochem. Photobiol., 2008, 62(4):727-735 [4] Schmidt É C, dos Santos R, Horta P A, et al. Effects of UVB radiation on the agarophyte Gracilaria domingensis (Rhodophyta, Gracilariales): Changes in cell organization, growth and photosynthetic performance[J]. Micron, 2010, 41(8):919-930 [5] Hu Ruibo, Fan Chengming, Fu Yongfu. Reference gene selection in plant real-time quantitative reverse transcription (qRT-PCR)[J]. J. Agr. Sci. Tech., 2009, 11(6):30-36. In Chinese (胡瑞波, 范成明, 傅永福. 植物实时荧光定量PCR内参基因的选 择[J]. 中国农业科技导, 2009, 11(6):30-36) [6] Goossens K, Poucke Van M, Soom Van A, et al. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos[J]. BMC Develop. Biol., 2005, 5(1):27-29 [7] Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol., 2002, 3(7):34-45 [8] Brattelid T, Winer H L, Levy Olav F, et al. Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies[J]. BMC Mol. Biol., 2010, 11(1):22-24 [9] Brugé F, Venditti E, Tiano L, et al. Reference gene validation for qPCR on normoxia-and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies[J]. J. Biotech., 2011, 156(3):153-162 [10] Hema R, Senthil-Kumar M, Shivakumar S, et al. Chlamydomonas reinhardtii, a model system for functional validation of abiotic stress responsive genes[J]. Planta, 2007, 226(3):655-670 [11] D'haene B, Vandesompele J, Hellemans J. Accurate and objective copy number profiling using real-time quantitative PCR[J]. Methods, 2010, 50(4):262-270 [12] Infante C, Matsuoka P M, Asensio E, et al. Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR[J]. BMC Mol. Biol., 2008, 9(1):28 [13] Etschmann B, Wilcken B, Stoevesand K, et al. Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm[J]. Vet Pathol., 2006, 43(6):934-942 [14] Schmid H, Cohen C D, Henger A, et al. Validation of endogenous controls for gene expression analysis in micro-dissected human renal biopsies[J]. Kidney Intern., 2003, 64(1):356-360 [15] Hou Weihai, Sun Peng, Chen Quanjia, et al. Selection of the reference genes for gene expression studies in rehmannia glutinosa by real-time quantitative PCR[J]. Chin. Agr. Sci. Bull., 2011, 27(17):76-82. In Chinese (侯维海, 孙鹏, 陈全家, 等. 地黄实时定量PCR内参基因的筛选[J]. 中国农学通报, 2011, 27(17):76-82) [16] Kadegowda A K, Bionaz M, Thering B, et al. Identification of internal control genes for quantitative polymerase chain reaction in mammary tissue of lactating cows receiving lipid supplements[J]. J. Dairy Sci., 2009, 92(5):2007-2019 [17] Bionaz M, Loor J J. Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle[J]. Physiol. Genom., 2007, 29(3):312-319 [18] Tang R, Dodd A, Lai D, et al. Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization[J]. Acta Biochim. Biophys. Sin., 2007, 39(5):384-390 [19] Kim B R, Nam H Y, Kim S U, et al. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice[J]. Biotech. Lett., 2003, 25(21):1869-1872 [20] Tricarico C, Pinzani P, Bianchi S, et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies[J]. Anal. Biochem., 2002, 309(2):293-300 [21] Nicot N, Hausman J F, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. J. Exp. Bot., 2005, 56(421):2907-2914
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