Molecular Cloning And Functional Study Of PjFPS/?-AS Key Enzyme Genes Involved In The Biosynthetic Regulation Of Panax Japonicus Saponins

Panax japonicus belongs to the family of Araliaceae,and the roots of P.japonicus have been widely used for a long history in China.P.japonicus is mostly distributed in Yunnan,Guizhou,Sichuan,Shanxi and other places of China,and has the effects on relieving cough,eliminating expectoration,anti-inflammatory,sedative,anti-cancer and anti-oxidation.P.japonicus saponins are the main bioactive components composed by dammarane-type and oleanane-type triterpenoid saponins.So far,most of P.japonicus herbs are in the wild,which have long growth cycle and can be used after6-7 years growth.The gap between supply and demand is becoming large.Therefore,it's necessary to study the biosynthetic pathway of P.japonicus saponins by using modern molecular biology and genetic engineerin,and then carry out the synthetic biology research of P.japonicus saponins.In this study,the total RNA was extracted by the method of guanidinium thiocyanate and the full-length sequence of Pj FPS was successfully cloned?KP684141?by the method of homologous cloning based on the cDNA.The bioinformatics of the gene was analyzed.The cloning vector pGEM-T-PjFPS and plant overexpression vector pCMBIA1300s-Pj FPS was successfully constructed.Pj FPS was transferred into the P.japonicus cells and the?-AS-transgenic P.japonicus cells to get the positive transgenic cells which only included the Pj FPS and included Pj FPS and Pj?-AS at the same time respectively by the method of Agrobacterium tumefaciens transformation.The relative expression levels of Pj FPS,the enzyme activitiy of PjFPS,the contents of P.japonicus saponins and phytosterols in Pj FPS transgenic cell lines were determined by the methods of real-time fluorescence quantitative PCR,colorimetry,and saponification.The relative expression levels of Pj FPS and Pj?-AS the enzyme activities of PjFPS and Pj?-AS,the contents of P.japonicus saponins and phytosterols in Pj FPS/?-AS transgenic cell lines were determined by the same methods.The bioinformatics results showed that the gene of Pj FPS had an 1029 bp open reading frame?O RF?which encod ing 342 amino acids and had five typical FPS conservative domains.The molecular weight of Pj FPS is 39.657 kD with the molecular formula of C₁₈₀₈H₂₈₀₉N₄₅₅O₅₂₂S₁₂.According to homologous sequence alignment and phylogenetic tree analysis,the key enzyme PjFPS protein belongs to the FPS subfamily and come from the same ancestor with P.ginseng and P.notoginseng.The result of subcellular localization prediction showed that PjFPS protein was located in the eukaryotic cytoplasm.The expression level of Pj FPS,the enzyme activity of FPS and the content of total P.japonicus saponins in Pj FPS transgenic cell lines were increased to some extent compared with the wild type.Furthermore,the expressions of relative key enzymes genes were promoted because of the increased flux of saponins.In the best-performing positive cell line,the expression of Pj FPS,the enzyme activity of FPS and the content of total saponins were 12 times,4 times and 3 times of those in control,respectively.Meanwhile,the content of phytosterol in transgenic cell lines was also enhanced.In the Pj FPS/?-AS transgenic cell lines,the relative expressions of Pj FPS and Pj?-AS,the enzyme activity of FPS,the contents of total saponins and phytosterols were also improved to some extent.The results showed that in the best positive cells of the Pj FPS/?-AS transgenic cell lines,the contents of saponins and phytosterols were 3times and 4.5 times of those in the control;furthermore they are 2.0 times and 1.5 times of those in the Pj FPS transgenic cell line,respectively.The relative expression levels of Pj FPS and Pj?-AS in the transgenic cell lines were increased to some extent compared with the non-transgenic cells.This study comfirmed that the saponins biosynthesis could be affected by regulating the expressions of key enzyme genes in the biosynthetic pathway of P.japonicus saponins.Meanwhile,the effect on regulation of two genes was better than only one gene.This study provides a theoretical reference and scientific basis for obtaining highly efficient and stable regulation technology for the synthesis of P.japonicus saponins and the eatablishment the of efficient homologous or heterologous expression system of saponins.