目的筛选铁皮石斛(Dendrobiumofficinale)实时定量PCR(realtimequantitativePCR，qPCR)最佳内参基因。方法采用同源克隆法、RT-PCR分离候选内参基因；qPCR分析基因的引物扩增效率及相对表达量；geNorm分析内参基因的表达稳定性；qPCR检测法呢基焦磷酸合酶(farnesylpyrophosphatesynthase，FPS)基因的表达模式。结果分离到铁皮石斛ACT-1、ACT-2、EF-1α、GAPDH、β-TUB、18SrRNA、26SrRNA等7个候选内参基因片段，引物扩增效率分别为2。12、1。98、2。03、2。08、1。97、2。18和2。05；geNorm分析表达稳定性大小为EF-1α/18SrRNA﹥ACT-1﹥ACT-2﹥GAPDH﹥β-TUB﹥26SrRNA；以EF-1α/18SrRNA为标准分析FPS基因的相对表达量大小为种子>；根>；原球茎>；茎>；叶。结论确定珍稀濒危兰科药用铁皮石斛qPCR分析的最佳内参基因，为基因表达研究提供依据。
Abstract
OBJECTIVE To select reference gene for real time quantitative PCR (qPCR) analysis of Dendrobium officinale. METHODS Homology cloning and RT-PCR techniques were used to isolate candidate reference genes. qPCR analyses were employed to determine the primer amplification efficiency for each gene, along with their relative quantities. The expression stability of each reference gene was analyzed by geNorm software, followed by validation of the expression pattern of the farnesyl pyrophosphate synthase gene(FPS) using qPCR. RESULTS A total of seven candidate reference genes′ fragments including ACT-1, ACT-2, EF-1α, GAPDH, β-TUB, 18S rRNA, and 26S rRNA, were identified from D. officinale. Their primer efficiencies were 2.12, 1.98, 2.03, 2.08, 1.97, 2.18, and 2.05, respectively. The expression stability calculated by geNorm analysis was ranked as follows: EF-1α/18S rRNA﹥ACT-1﹥ACT-2﹥GAPDH﹥β-TUB﹥26S rRNA. The transcriptional level of FPS gene normalized by EF-1α/18S rRNA was in the order of seed>root>PLB>stem>leaf. CONCLUSION The most stable reference gene for qPCR analysis was characterized in the endangered orchid D. officinale, which will be useful for further gene expression study.
关键词
铁皮石斛 /
内参基因 /
定量PCR /
基因表达
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Key words
Dendrobium officinale /
reference gene /
real time quantitative PCR /
gene expression
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中图分类号：
R931
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参考文献
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脚注
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基金
国家自然科学基金资助项目(31070300, 31101608)；陕西省青年科技新星计划项目(2012KJXX-44)
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