The Function Research Of Key Enzyme Gene OpG10H、Op10HGO、OpSLS In Iridoid Way Of Camptothecin Synthesis

Camptothecin(CPT),an organic chemical of the terpene indole alkaloid(TIA),is the only natural plant active ingredient that exerts cytotoxicity through specific inhibition of topoisomerase I.It is thought to be the three major anti-cancer drug discoveries in 1990 s.Its clincal need is quite large in the world.The Camptotheca acuminata Decne needs a long growth cycle,which makes the lack of drug sources.Recent studies have found that the Ophiorrhiza pumila can also produce camptothecin.The Ophiorrhiza pumila has become an important medicine source plant.We cloned the key enzyme genes of endogenous ring iridoid pathway in the Ophiorrhiza pumila,OpG10 H,Op10HGO and OpSLS,and constructed constitutive over expression vector,inducible over expression vector,interference vector and knockout vector respectively.We studied the function of OpG10 H,Op10HGO,OpSLS in the iridoid pathways synthesis of CPT in Ophiorrhiza pumila hairy roots.For the first time,we simultaneously transformed the constitutive over expression vector harbored OpSLS and the inducible expression vector harbored OpG10 H into it to obtain the transgenic hairy root lines of OpSLS and OpG10 H over-expression,and then comparatively analyze the function of OpSLS and OpG10 H.According to our study,the average content of CPT in OpG10 H over expression transgenic lines reached 1.56 mg/g,compared to that of control group(1.4 mg/g),increased by 11%,and the highest content in transgenic lines reached 2.40 mg/g,and CPT average yield increased to 945.02 μg/bottle,15%-93% higher than that in control group.The content of CPT in induced inducible OpG10 H over-expression transgenic lines reached1.93mg/g,27.96% higher than that of without induced,and the yield increased to some extent.However,CRISPR knockout and RNAi interference OpG10 H gene in the hairy roots actually result to a sharp reduction of CPT.These findings proved that OpG10 H gene is indispensably involved in the CPT biosynthesis in Ophiorrhiza pumila.The loganin and split ring loganin in OpSLS knock-out strains decreased more than 60%,and the content of CPT in which sharply decreased by 90%,on the other hand,the average content of CPT in OpSLS over-expression strains reached 2.36 mg/g,increased by 68.8% compared with the control group,and the average yield of which reached 1478.10 μg/bottle,90.48% higher than that of the control group.These findings showed that over expression of OpSLS plays a significant role in the synthesis CPT in Ophiorrhiza pumila.All these data showed that OpSLS plays an important role in the synthesis of CPT.By analyzing the CPT content and yield in SLS and G10 H co-expression transgenic lines,we concluded that SLS and G10 H are key enzymes in the synthesis of CPT,but SLS may have more important role than the G10 H.Over expression of 10 HGO promoted slightly in CPT synthesis,while the CPT content increased significantly in 10 HGO knock-out lines,take 8HGO isozyme into consideration,10 HGO gene may be in a secondary position in CPT biosynthetic pathway,and gene 8HGO may occupy a more important position in CPT biosynthetic pathway in short Ophiorrhiza pumila.By selecting crude extract of high CPT content lines and performing anti-tumor cell activity detection by MTT assay,we found that the same concentration of the CPT crude extract almost have the same lethality to cancer cells,and the anticancer effect of CPT is better.This would provide technical support for the use of genetic engineering to improve the resources of medicinal plants and ease the contradiction between clinical demand and resource shortage.