Selection and Validation of Reference Genes for Real-time Florescence Quantitative PCR Analysis on Gene Expression of White Tea
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摘要: 利用GeNorm，NormFinder，BestKeeper软件对茶树不同叶位及白茶萎凋过程中7个候选内参基因的表达稳定性进行分析。获得3个稳定性较好的内参基因β-Actin、GAPDH和RUBP，RUBP适合作为白茶萎凋过程叶片荧光定量PCR的内参基因，β-Actin适合作为白茶不同叶位叶片荧光定量PCR的最佳内参基因。因此，在白茶萎凋过程中，使用GAPDH+RUBP组合作为内参基因进行荧光定量PCR检测。Abstract: To select appropriate reference genes to be used in the real-time florescence quantitative PCR analysis for studying the gene expression in of white tea leaves during withering, the software of GeNorm, NormFinder, and BestKeeper were applied. Three stable reference genes, β-Actin, GAPDH and RUBP, were identified. Among them, RUBP was found most adequate for comparing the expressions at various stages of withering, while β-Actin for differentiating them in leaves from different parts of a tea plant. The combination of GAPDH+RUBP was determined as choice reference genes for the analysis.
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Key words:
- white tea /
- withering /
- reference genes /
- stability /
- real-time fluorescence quantitative PCR
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图 3 运用GeNorm评价候选基因在萎凋历时的表达稳定性系数M值
Figure 3. M values of expression stability during withering on potential reference genes as calculated by GeNorm
图 4 运用GeNorm评价候选基因在不同叶位的表达稳定性系数M值
Figure 4. M values of expression stability of potential reference genes in tea leaves from different parts of plant as calculated by GeNorm
图 5 GeNorm软件评价候选内参基因最适数目
Figure 5. Optimal number of reference genes as determined by GeNorm evaluation
图 6 NormFinder评价候选内参基因在不同叶位的表达稳定系数M值
Figure 6. M values of expression stability of potential reference genes in tea leaves from different parts of plant as calculated by NormFinder
图 7 NormFinder评价候选内参基因在萎凋历时的表达稳定系数M值
Figure 7. M values of expression stability during withering on potential reference genes as calculated by NormFinder
表 1 7个候选内参基因qRT-PCR分析的引物序列
Table 1. Primer sequences of 7 candidates in selection for reference genes used in qRT-PCR analysis
基因名称 上游(5′-3′) 下游(5′-3′) β-Actin GCCATCTTTGATTGATTGGAATGG GGTGCCACAACCTTGATCTT GAPDH TTGGCATCGTTGAGGGTCT CAGTGGGAACACGGAAAGC 18s rRNA CGGCTACCACATCCAAGGAA GCTGGAATTACCGCGGCT cyc CTCACTCAGGCGAAGAAATC GACCCATGACATACGACCAG RUBP CGATGGGCGATACTGGACAA GCCAGGAGGCTTGCACAAT EF1a TTCCAAGGATGGGCAGAC TGGGACGAAGGGGATTTT α-tubulin GGGTGTCAATGCTGGACAA GCCAGGAGGCTTGTAGCAAA 
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表 2 NormFinder得出的各候选内参基因的表达稳定性
Table 2. Expression stability of potential reference genes calculated by NormFinder
基因 表达稳定值 不同叶位 萎凋历时 β-Actin 0.019 0.112 GAPDH 0.198 0.040 cyc 0.072 0.158 18s rRNA 0.183 0.197 RUBP 0.073 0.107 EF1a 0.076 0.113 α-tubulin 0.049 0.093
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表 3 Bestkeeper软件分析内参基因在不同叶位的表达稳定性值
Table 3. M values of expression stability of potential reference genes in tea leaves from different parts of plant as calculated by Bestkeeper
内参基因 β-Actin GAPDH cyc 18s rRNA RUBP EF1a α-tubulin n 5 5 5 5 5 5 5 GM[Ct] 14.50 15.12 10.14 16.07 8.44 14.79 18.77 AM[Ct] 14.51 15.56 10.57 16.08 9.37 14.81 18.77 Min[Ct] 13.73 13.40 4.41 15.16 3.08 13.58 17.70 Max[Ct] 15.54 33.02 13.26 16.90 16.21 16.03 19.54 SD[±Ct] 0.41 2.33 1.89 0.46 3.39 0.43 0.36 CV[%Ct] 2.81 14.97 17.89 2.85 36.16 2.93 1.92 r 0.997 0.865 0.320 0.174 0.565 0.141 -0.079 注：n为样品数目；GM[Ct]为Ct值几何平均数；AM[Ct]为Ct值算数平均数；Min[Ct]and Max[Ct]为Ct的最大/小值；SD[±Ct]为Ct的标准误差；CV[%Ct]为方差；r为变异系数。表 4同。
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表 4 Bestkeeper分析内参基因在萎凋历时的表达稳定性值
Table 4. M values of expression stability during withering on potential reference genes as calculated by Bestkeeper
内参基因 β-Actin GAPDH cyc 18s rRNA RUBP EF1a α-tubulin n 9 9 9 9 9 9 9 GM[Ct] 18.30 18.81 13.69 18.81 16.18 20.44 22.08 AM[Ct] 18.33 18.86 13.77 18.83 16.43 20.48 22.11 Min[Ct] 16.98 16.08 11.33 17.32 12.26 18.11 20.07 Max[Ct] 20.59 20.99 16.50 20.55 21.28 22.76 23.94 SD[±Ct] 1.00 1.21 1.27 0.75 2.59 1.15 0.93 CV[%Ct] 5.47 6.39 9.21 3.99 15.79 5.62 4.22 r 0.804 0.928 0.445 0.715 0.815 0.778 0.454
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[1] 马春雷. 茶树查尔酮异构酶、黄酮醇合成酶和无色花色素还原酶等基因的克隆与表达分析[D]. 北京: 中国农业科学院, 2007. http://d.wanfangdata.com.cn/Thesis/Y1056911 [2] 史成颖, 李正国, 徐乾.茶愈伤组织实时定量PCR分析中内参基因的选取[J].安徽农业大学学报, 2014, 41(6):905-910. http://www.cnki.com.cn/Article/CJFDTotal-REST201405035.htm [3] 曾威, 赵昕梅, 薛佩, 等.茶树α-tubulin基因实时荧光定量RT-PCR方法的建立[J].生物技术通讯, 2012, 23(2):245-247. http://d.wanfangdata.com.cn/Periodical_swjstx201202023.aspx [4] 刘圆, 王丽鸳, 韦康, 等.不同氮处理茶树实时定量PCR内参基因筛选和验证[J].茶叶科学, 2016, 36(1):92-96. http://www.jabiotech.org/CN/abstract/abstract10710.shtml [5] 叶乃兴.白茶科学·技术与市场[M].北京:中国农业出版社, 2010:33-42. [6] 陈静, 俞滢, 张丹丹, 等.白茶萎凋过程中儿茶素合成关键酶基因表达分析[J].南方农业学报, 2016, 47(8):1364-1369. http://www.cnki.com.cn/Article/CJFDTotal-FJNX201509006.htm [7] VANDESOMPELE J D P K P. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 7(3):H34. [8] ANDERSEN C L J J L. 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 Research, 2004, 15(64):5245-5250. http://cancerres.aacrjournals.org/content/64/15/5245/T1.expansion.html [9] PFAFFL M W T A P C. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations[J]. Biotechnol Lett, 2004, 6(26):509-515. http://www.gene-quantification.com/pfaffl-dechema-2003.pdf [10] NICOT N H J L E. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 1973, 2(3):295-309. https://academic.oup.com/jxb/article-abstract/56/421/2907/593469/Housekeeping-gene-selection-for-real-time-RT-PCR [11] DIE J V R B N S. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions[J]. Planta, 2010, 232(1):145-153. doi: 10.1007/s00425-010-1158-1 [12] YI S, QIAN Y, HAN L, et al. Selection of reliable reference genes for gene expression studies in Rhododendron micranthum Turc[J]. Scientia Horticulturae, 2012, 138:128-133. doi: 10.1016/j.scienta.2012.02.013 [13] MIGOCKA M, PAPIERNIAK A. Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators[J]. Molecular Breeding, 2011, 28(3):343-357. doi: 10.1007/s11032-010-9487-0 [14] WANG H, CHEN S, JIANG J, et al. Reference gene selection for cross-species and cross-ploidy level comparisons in Chrysanthemum spp[J]. Scientific Reports, 2015, 15(4):112-116. http://www.ncbi.nlm.nih.gov/pubmed/25627791 [15] ZHANG D H, SUN Y L, ZHAO L, et al. Reference gene selection for quantitative real-time PCR normalization in Medicago Lupulina under zinc stres[J]. Zhongguo Huanjing Kexue/china Environmental Science, 2015, 35:833-838. https://www.researchgate.net/publication/288366698_Cloning_and_expression_analysis_of_a_LEAFY_gene_from_lotus_nelumbo_nucifera_gaertn [16] LACERDA A L M, FONSECA L N, BLAWID R, et al. Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems[J]. Plos One, 2015, 10(8):1-17. http://ainfo.cnptia.embrapa.br/digital/bitstream/item/135008/1/document-1.pdf [17] 孙美莲. 茶儿茶素生物合成相关基因表达的实时荧光定量PCR分析[D]. 合肥: 安徽农业大学, 2010. http://www.wanfangdata.com.cn/details/detail.do?_type=degree&id=Y1735208 [18] 郝姗. 茶树不同逆境条件下qRT-PCR适宜内参基因的筛选[D]. 南京: 南京农业大学, 2012. http://www.wanfangdata.com.cn/details/detail.do?_type=degree&id=Y2361490 [19] PAUL A, LAI L, AHUJA P S, et al. Alpha-tubulin (CsTUA) up-regulated during winter dormancy is a low temperature inducible gene in tea[Camellia sinensis (L.) O. Kuntze]. Mol Biol Rep, 2002, 39(4):3485-3490. doi: 10.1007/s11033-011-1121-7 -
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