Molecular Cloning And Tissue Expression Analysis Of Saikosaponin Biosynthesis-Related Genes In Bupleurum Chinense DC

Bupleurum chinense DC.(umbelliferae,bupleurum) is one of the main sources of medicinal bupleurum.The main effective component saikosaponin is oleanane-type triterpene saponin.At present, there were little research on the saikosaponin biosynthesis,cDNA fragments,encoding HMGR,IPPI,FPS andβ-AS which were involved in saikosaponin biosynthesis pathway in B.chinense were amplified by RT-PCR with the total RNA as a template,cDNA fragments of HMGR,IPPI,FPS andβ-AS gene were 470bp,532bp,466bp,1349bp in length encoding 157,177,155,449 amino acids respectively.The results of 4 genes sequence analysis(including blast analysis,conservative domain analysis,enzyme digestion sites analysis,southern hybridization analysis,phylogeneic tree analysis) indicated that the most conservative core fragments of 4 genes were cloned,cDNA fragments of six housekeeping genes were cloned by RT-PCR for the first time,the cDNA fragments were 101bp(18S rRNA),96bp(Cyclophilin), 128bp(EF1α),121bp(L2),101bp(β-tubulin),99bp(Actin) in length respectively.After the real time RT-PCR,expression stability of 18S rRNA,Cyclophilin,EF1α,L2,β-tubulin,Actin were analyzed with C_T value and genorm,the result indicated that the most stable gene isβ-tubulin,on the contrary,is 18S rRNA.Using theβ-tubulin as the internal reference standard,HMGR,IPPI,FPS,β-AS gene were amplified by real time RT-PCR.The data were analysed by 2^{-ΔΔCT}.HMGR gene far from end product andβ-AS gene nearby end product showed the highest expression level on root.Root was the main organ for saikosaponin biosynthesis probably.The result of tissue expression of saikosaponin biosynthesis-related genes showed that saikosaponin can be synthesized in every organs from B. chinense.Four of the saikosaponin biosynthesis-related genes were all expressed in roots,stems,leaves, flowers and fruits from B.chinense.The highest expression level of IPPI was in stems and the lowest was in flowers.The highest expression level of FPS was in flowers and the lowest was in leaves.