Reference genes screening and spatio-temporal expression analyses of cytochrome P450 monoxoygenase genes in Polygala tenuifolia by qRT-PCR / 中草药

ABSTRACT

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 Ct 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.