Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Hai Dian District, Beijing 100193, China.
Mol Biol Rep. 2011 Nov;38(8):5017-23. doi: 10.1007/s11033-010-0648-3. Epub 2010 Dec 16.
It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.
在使用实时定量 PCR(qRT-PCR)进行基因表达分析时，选择稳定的参照基因至关重要。本研究以柴胡 DC.的 18S、Actin、EF1α、α-微管蛋白、β-微管蛋白、环磷酰胺和细胞质核糖体蛋白 L2 (L2) 等 7 个常用内参基因作为参照，对 5 种组织(根、茎、叶、花和果实)进行组织特异性基因表达分析。结果表明，β-微管蛋白是这 7 个候选基因中最稳定和可靠的内参基因。以β-微管蛋白为内参，在 5 种组织中检测了参与柴胡皂苷(柴胡的药理活性化合物)生物合成的 4 个基因(HMGR、IPPI、FPS 和β-AS)的表达水平。这 4 个基因在 5 种组织中均有表达，表达谱不同，HMGR 表达顺序为根>花，茎和叶>果，IPPI 表达顺序为茎>叶和果，根和花，FPS 表达顺序为花>果>茎和根>叶，β-AS 表达顺序为根>花，茎和果>叶。FPS 和β-AS 基因主要在花和根中表达。本研究可为各种组织的定量基因表达分析提供合适的内参，并深入了解药用柴胡 4 个参与柴胡皂苷生物合成基因的组织表达谱。
