Characteristics Of Functional Enzymes In Panax Notoginseng And Molecular Evolution Involved In Plant Triterpoid Biosynthesis Pathway

Triternpenoids are a structurally diverse class of secondary metabolites in abundance widely existing in plant kingdom with very important biological significance In nature,not only triterpenoids occur in monocotyledons,dicotyledons,but also in fungus,ferns,even in animals and marine life,particularly ubiquitous in dicotyledons, such as Asteraceae,Euphorbiaceae,Meliaceae,Celastraceae,Rubiaceae, Burseraceae,Lamiaceae et al.Medicinal plants from Araliaceae,Fabaceae, Campanulaceae,Polygalaceae,Umbelliferae,Ranunculaceae,are abundant in various different structures of triterpenoid saponins with diversal biological activity. Naturally,these triterpenoid saponins play numerous functional roles in defence pathogen and botanic pest in ecological interations.Triterpenoid saponins are major effective compounds in many medicinal plants,which are responsible for biological activities of anticancer,antivirus,lowering cholesterol,elevating immunity,or exert the important pharmacologic actions on cardiovascular system and nervous system, possessing the considerable pharmaceutical and commerce values.The process of biosynthesis of tritepene saponins in various plant species first involves in the formation of MVA,mevalonic acid,to yield squalene(30C)via the isoprene pathway,then 2,3 oxidosqualene cyclization result in the pentacyclic or tetracyclic triterpene backbone,undergoing several kinds of modification,for example oxidation,substitution and glycosylation catalyzed by monooxygenase, glycosyltransferases and other enzymes dependent on cytochrome P₄₅₀.With the completion of mode plant arabidopsis genome and rice genome projects,the anatations to the functional genes in plant species quickly increase,to lead to gradually the acumulation of various enzyme gene cloning sequences involved in triterpenoid biosynthesis.Panax notoginseng,which belongs to Araliaceae,panax perennial herbs,had been one of special local medicinal plants in Guangxi.About 20 kinds of dammarane type of triterpenoid have been isolated from panax notoginseng that the major components are accordance with those in ginseng.Pharmacological actions on the notoginsenosides have been extentively researched no matter internal or external, confirming that notoginsenosides are the main effective components in panax notoginseng,and presenting a good perspective to be developed and utilized.It is the major aim for the project to research systematically the pathway of triterpenoid biosynthesis in Panax notoginseng from Guangxi special local medicinal plants,to analyze the basic characteristics of the key enzymes involved in this pathway in favor of the regulation for the triterpenoid biosynthesis which are effective components in some medicinal plants and to facilitate the synthesis and accumulation of triterpenoid in some medicinal plants.On the other hand,to supply some theory basis for the research of plant phylogenesis on the molecular level through the clustering analysis of enzyme molecules that have been registered in GenBank related to the triterpenoid biosynthesis in some plants.Base on the information about the functional enzyme genes involved in plant isoprenoid and triterpenoid biosynthesis,the cDNAs cloning of FPS,SS,SE,CAS from root of panax notoginseng grown in Jingxi county,Guangxi,were performed with the RT-PCR,3'-RACE and 5'-RACE technology,respectively and integrite open reading frames for these four gene expression have been obtained.All these cDNA squences have been registered in GenBank of NCBI and released publicly Prokaryote expression vectors of notoginseng FPS and SE have been constructed successfully as pET32a(+)-FPS and pET32a(+)-SE,and transferred into BL21(DE3), respectively,which can be induced expression by IPTG validly in the host cells and the recombinant protein's molecular weight expressed are coincidence with the predictation.The alignment results revealed that the identities of the induced amino acid sequences of panax notoginseng FPS,SS,SE,CAS compared with those of ginseng are 98.8%,98.1%,96.7%,90.1%,respectively,which has shownthatthefunctional enzyme genes involved in triterpenoid biosynthesis are high homologous between ginseng and notoginseng.With the formation of squalene(30C),oxidation,cyclization, modification,the molecular weight of enzymes needed for the pathway become gradually larger,for example FPS containing 343 amino acids,SS 415 amina acids, SE 537 amino acids,CAS 758 amino acids.It is presumed that it is needed because the substrate molecules turn to be larger and their structures become more complex with the process of the pathway in advance.Near to the end of the pathway,the differences of enzymes gradually increase accoding to the alignment analysis involved in triterpenoin biosynthesis in panax notoginseng compared with those in ginseng.Panax notoginseng SS transctrition expression analysis were performed with SYBR Green I Real-Time RT-PCR technology using notoginseng GAPDH as internal control and recombinant plasmid pMT18-GAPDH fragmant and pMT18-SS serve as standard.Transcription expression of SS in root,rootstock and stem of one year growth notoginseng were analyzed.Meanwhile the total amount of notoginsenoside in root,rootstack,stem and leaves tissues were messured,respectivley.The messurement for the total saponin in root,rootstock,stem and leaves tissues are 2.73%,11.86%,1.09%,5.96%in order.ANOVA analysis was performed through comparison of any two of several samples,P=0.000 for each comparison and showed that the differences are distinct statistically,the saponin contents are different for any two groups.Transcription analysis and statistic results reveals that the SS expression level in notoginseng root is higher than in stem and rootstock.The amount of total triterperoids in rootstock is higher than in leaf,root,stem in order but it is still difficult to determine whether SS expression are different between in rootstock and in stem.The results show that SS gene play an important role during the process of notoginseng saponin biosynthesis.Perhaps it involves in the expression differences of other key enzymes after SS on the triterpenoid synthesis pathway or/and the directional transportation and accumulation of triterpenoid in different tissues and organs of plants.It is needed to do further research to thoroughly reveal the relationship between SS expression and notoginsenoside contents.After the cDNA clones of notoginseng FPS,SS,SE and CAS involved in triterpenoid biosynthesis were obtained,phylogenetic tree were constructed with the enzymes registered in GenBank related to triterpenoid saponin or sterol biosynthesis in plant species to probe into the evolution rules of triterpenoid biosynthesis enzymes genes.The results reveal that no matter FPS,SS,SE or CAS,there are more close cluster relationship between ginseng,notoginseng in Araliaceae and Centalla asiatica, Bupleurum chinense in Umbelliferae,suggesting nearer genetic relationship evolutionally between Panax and Cenllata,Bupleurum.This is coincidence with the genetic relationship between Araliaceae,Panax and Umbelliferae,Cenllata, Bupleurum in phytotaxonomy,offerring a new evidence for the research on the evolution process of plant species and genetic relationship on molecular level.It is further certificated that the functional enzyme genes of triterpenoid biosynthesis pathway present quite high homologous in the same family and genus plants or in the plants which contain similar triterpenoid components,correlated to the plant evolution process.It could be used to offer some suggestions for digging out some medicinal plant resource containing triterpenoid structure similar.