目的 筛选适用于不同生长年限和不同部位的栽培远志实时荧光定量PCR分析（quantitative real time PCR，qRT-PCR）的稳定内参基因，为远志体内各个基因表达分析提供合适内参基因。并对4个细胞色素P450单加氧酶（cytochrome P450 monooxygenase，P450s）基因在上述远志样品中的差异表达进行分析，明确4个P450s基因在栽培远志中的时空表达分布。方法 通过qRT-PCR获取Tubulin 1、Tubulin 2、Elongation 1、Elongation 2、Actin 1、Actin 2、GAPDH和cdc-42 8个候选内参基因在上述远志样品中的熔解曲线与C_t值；利用软件geNorm和NormFinder对候选内参基因的表达稳定性进行评价；通过qRT-PCR分析4个P450s基因CYP709B2、CYP71AP39、CYP88A85、CYP714E38在上述远志样品中的相对表达情况。结果 geNorm分析结果表明，在远志（1~3年生）根、茎、叶、花中稳定表达的为Tubulin 2与Elongation 1；NormFinder结果显示，Elongation 1是最稳定的内参。CYP709B2、CYP71AP39基因在远志茎、叶（1~3年生）中的mRNA表达量最高，在根与花中的表达量则较少。而CYP88A85、CYP714E38基因则在远志根（1~3年生）中的mRNA表达水平较高。结论 Elongation 1适合作为栽培远志体内基因表达分析的内参基因。栽培远志中的P450s基因在远志根、茎、叶、花中的表达趋势不一致。

Objective To provide a suitable expression analysis of Polygala tenuifolia in different growth years and tissues, so as to select the stable internal control genes. The spatial and temporal expression of four cytochrome P450 monooxygenase genes in the above-mentioned samples was analyzed. Methods The soluble curve and C_t value of eight candidate internal reference genes including Tubulin 1, Tubulin 2, Elongation 1, Elongation 2, Actin 1, Actin 2 and cdc-42 in different growth years (1-3 years old) and tissues (root,stem, leaf, and flower) of P. tenuifolia were obtained by real-time quantitative PCR (qRT-PCR). The expression stability of candidate reference genes was assessed by geNorm and NormFinder. Then, the qRT-PCR technique was also used to identify the temporal and spatial expression of four P450 genes including CYP709B2, CYP71AP39, CYP88A85 and CYP714E38 in P. tenuifolia. Results The geNorm results showed that the stable internal reference genes expressed in P. tenuifolia were Tubulin 2 and Elongation 1. The NormFinder results showed that the most stable and suitable internal reference gene for expression analysis was Elongation 1. The mRNA expression levels of CYP709B2 and CYP71AP39 genes in stems and leaves (1-3 years old) were higher than that in roots and flowers. The CYP88A85 and CYP714E38 genes had got a higher mRNA expression level in roots (1-3 years old). Conclusion Elongation 1 is suitable as an ideal internal control gene for qRT-PCR analysis of P. tenuifolia. The expression trend of P450s in roots, stems, leaves and flowers of cultivated P. tenuifolia is inconsistent.

山西省重点研究开发计划（201603D3111003）；国家中药标准化项目（ZYBZH-Y-JIN-34）；地产中药功效物质研究与利用山西省重点实验室项目（201605D111004）；山西省科技创新重点团队（201605D131045-18）；山西省中药现代化振东专项-远志品种选育及配套适应性高产栽培技术研究（2016ZD0106）