目的 筛选适宜狭叶柴胡Bupleurum scorzonerifolium组织表达分析的内参基因，分析柴胡皂苷合成关键酶基因的组织表达模式。方法 选取Actin、α-tubulin、β-tubulin、Cyclophilin、EF-1α 5个常用的内参基因作为候选，分别使用Delta CT、BestKeeper、NormFinder、geNorm软件对各候选内参基因的稳定性进行评价，并对候选内参基因的稳定性进行验证，利用实时荧光定量PCR技术，以筛选得到的内参基因分析狭叶柴胡根、茎、叶、果中柴胡皂苷合成关键酶基因HMGR、IPPI、FPS、SS、β-AS基因的组织表达模式。结果 候选内参基因平均表达稳定性由高到低排序为β-tubulin > Cyclophilin > Actin > EF-1α > α-tubulin，β-tubulin是狭叶柴胡基因组织表达分析的适宜内参基因。HMGR基因表达量由高到低为根 > 茎/果 > 叶，IPPI基因表达量由高到低为根 > 茎 > 果 > 叶，FPS基因表达量由高到低为叶 > 根 > 茎 > 果，SS基因表达量由高到低为叶 > 果 > 根 > 茎，β-AS基因表达量高低为叶 > 根 > 果 > 茎，其中HMGR与IPPI及FPS与β-AS基因的表达显著呈正相关（P < 0.05）。结论 筛选得到了狭叶柴胡不同组织基因表达分析的最适内参基因为β-tubulin，为狭叶柴胡基因组织表达分析奠定方法学基础。柴胡皂苷合成关键酶基因有不同的组织表达模式，可能参与调控狭叶柴胡不同组织中柴胡皂苷合成与积累的流向。

Objective To select suitable references genes of Bupleurum scorzonerifolium for tissue expression analyses, and study the tissue expression characteristics of the key enzyme genes of saikosaponins biosynthesis. Methods Five candidate reference genes including Actin, α-tubulin, β-tubulin, Cyclophilin, and EF-1α were chosen. The stability of these candidate reference genes was investigated by using four softwares (Delta CT, BestKeeper, NormFinder, and GeNorm). The stability of these candidate reference genes was tested and verified by real-time quantitative PCR. Used the stable reference gene, the tissue expression characteristics of the saikosaponins biosynthesis key enzyme genes (HMGR, IPPI, FPS, SS, and β-AS) was analyzed by qRT-PCR. Results The average expression stability of the five candidate reference genes from high to low was β-tubulin > Cyclophilin > Actin > EF-1α > α-tubulin. β-tubulin was the most suitable reference gene for tissue expression analysis in B. scorzonerifolium. HMGR expression level was roots > stems and fruits > leaves, IPPI expression level was roots > stems > fruits and leaves, FPS expression level was leaves > roots > stems and fruits, SS expression level was leaves > fruits > roots > stems, β-AS expression level was leaves > roots > fruits > stems. HMGR was significant positive correlated with IPPI, and FPS was significant positive correlated with β-AS (P < 0.05). Conclusion β-tubulin gene was confirmed as the most suitable reference gene in different tissues of B. scorzonerifolium. It provided a methodological basis for the tissue expression analysis on the functional genes of B. scorzonerifolium. The expression pattern of five key enzyme genes of saikosaponins biosynthesis in different tissues had obvious differentiation, which might be involved in regulating the flow of saikosaponins synthesis and accumulation in various tissues of B. scorzonerifolium.

国家中药材产业技术体系项目（CARS-21）