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基于实时荧光定量PCR对镉处理下黄花蒿内参基因稳定性的分析

Keywords: 实时荧光定量PCR,黄花蒿,,内参基因,稳定性分析

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Abstract:

研究选取黄花蒿的Actin,18SrRNA,PAL,GAPDH,CPR作为候选内参基因,设计特异性引物,通过实时荧光定量PCR,利用geNorm,NormFinder,BestKeeper,DeltaCT以及RefFinder软件和方法对5种内参基因在不同浓度镉处理下黄花蒿叶片中的表达稳定性进行分析,为黄花蒿基因差异表达研究提供可靠的内参基因,确保黄花蒿基因表达分析结果的可靠性。结果表明在不同浓度镉处理下,黄花蒿叶片中候选内参基因的表达稳定性存在差异,综合分析得出候选内参基因表达稳定性顺序依次为Actin>18SrRNA>PAL>GAPDH>CPR。因此,在黄花蒿基因差异表达分析中,可以考虑选取Actin,18SrRNA,PAL作为内参基因,采用多内参校正结果。此外,研究还发现同浓度镉处理下黄花蒿叶片中的候选内参基因表达稳定性也存有差异,这说明即使在同一处理条件下也有必要进行内参基因的筛选。总之,该研究首次提供了在不同浓度镉处理下黄花蒿叶片中比较理想的内参基因,也为其他条件下黄花蒿的基因表达分析提供了参考。

References

[1]  Huggett J, Dheda K, Bustin S, et al. Real-time RT-PCR normalization; strategies and considerations[J]. Genes and Immun, 2005, 6:279.
[2]  Radonic\'A, Thulke S, Mackay I M, et al. Guideline to reference gene selection for quantitative real-time PCR[J]. Biochem Biophys Res Commun, 2004, 313(4):856.
[3]  Suzuki T, Higgins P J, Crawford D R. Control selection for RNA quantitation[J]. Biotechniques, 2000, 29:332.
[4]  Bustin S A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR):trends and problems[J]. J Mol Endocrinol, 2002, 29:23.
[5]  Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards:use and limits[J]. J Biotechnol, 1999, 75:291.
[6]  Lee P D, Sladek R, Greenwood C M T, et al. Control genes and variability:absence of ubiquitous reference transcripts in diverse mammalian expression studies[J]. Genome Res, 2002, 12:292.
[7]  Gutierrez L, Mauriat M, Gunin S, et al. The lack of a systematic validation of reference genes:a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants[J]. Plant Biotechnol J, 2008, 6(6):609.
[8]  Vandesompele J, De P 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):research00341.1.
[9]  Andersen C L, Jensen J L, Orntoft T F. 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[J]. Cancer Res, 2004, 64:5245.
[10]  Pfaffl M W, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:best Keeper-Excel-based tool using pair-wise correlations[J]. Biotechnol Lett, 2004, 26:509.
[11]  Silver N, Best S, Jiang J, et al. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR[J]. BMC Mol Biol, 2006, 7:33.
[12]  Xie F, Sun G, Stiller J W, et al. Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database[J]. PLoS ONE, 2011, 6(11):e26980.
[13]  韩晓丽,黄璐琦,郭兰萍,等.土壤-黄花蒿系统中镉(Cd)迁移规律及Cd对黄花蒿生长和黄花蒿素含量的影响[J].中国中药杂志,2010,35(13):1655.
[14]  Li X, Zhao M X, Guo L P, et al. Effect of cadmium on photosynthetic pigments, lipid peroxidation, antioxidants, and artemisinin in hydroponically grown Artemisia annua [J]. J Environ Sci, 2012, 24(8):1511.
[15]  Zhu X Y, Li X P, Chen W X, et al. Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions[J]. PLoS ONE, 2012, 7(8):e44405.
[16]  Kim B R, Nam H Y, Kim S U, et al. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice[J]. Biotechnol Lett, 2003, 25:1869.
[17]  Olofsson L, Engstr m A, Lundgren A, et al. Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L.[J]. BMC Plant Biol, 2011, 11:45.
[18]  Nolan T. Hands R E. Bustin S A. Quantification of mRNA using real-time RT-PCR[J]. Nat Protoc, 2006, 1:1559.
[19]  Dheda K, Huggett J F, Chang J S, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization[J]. Anal Biochem, 2005, 344:141.
[20]  Zhu J, He F, Song S, et al. How many human genes can be defined as housekeeping with current expression data?[J]. BMC Genomics, 2008, 9:172.
[21]  Logan J, Edwards K, Saunders N. Real-time PCR:current technology and applications[M]. Poole:Caister Academic Press, 2009:47.
[22]  Jain M, Nijhawan A, Tyagi A K, et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR[J]. Biochem Bioph Res Co, 2006, 345(2):646.
[23]  Yan J W, Yuan F R, Long G Y. Selection of reference genes for quantitative real-time RT-PCR analysis in citrus[J]. Mol Biol Rep, 2011, 39(2):1831.
[24]  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.
[25]  Hu R, Fan C M, Li H Y, et al. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR[J]. BMC Mol Biol, 2009, 10:93.

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