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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