Effect Of Overexpression Of Panax Notoginseng PnFPS Gene On The Content Of Terpenoids In Tomatoe

Panax notoginseng(Burk)f.H.chen is a perennial herbal medicinal plant of Araliaceae Panax genus.It is one of the rare traditional Chinese medicinal herbs.It serves a variety of functions,such as dispersing blood,stopping bleeding,reducing swelling,and calming pain.It can be used in coronary heart disease,angina pectoris,myocardial ischemia,cerebral infarction,sequelae of cerebral hemorrhage,local congestion,swelling and pain.The saponins in P.notoginseng are mainly damaranetype tetracyclic triterpene saponins,which are derived from the terpenoid synthesis pathway.Isopentenyl pyrophosphate(IPP)and dimethylallyl diphosphate(DMAPP)were first synthesized by mehydroxyvaleric acid(MVA)pathway and methylerythritol4-phosphate(MEP)pathway.Then,Damalenediol is produced step by step from geranyl pyrophosphate synthase(GPS),Farniki pyrophosphate synthase(FPS),squalene synthase(SS),squalene oxidase(SE),Damalenediol synthase(DS)and other synthases.At the last stage,two aglycans,protopanaxatriol(PPT)and protopanoxadiol(PPD),were synthesized under the catalysis of cytochrome enzyme(CYP450),and then the aglycans were modified by different glycosyltransferase(UGT)to obtain each monomer saponin.However,the industrial development of P.notoginseng has been affected by its limited cultivation range,long growing years,and low levels of many rare saponins,which are difficult to extract.Tomato(Solanum lycopersicum)is an annual herb belonging to the genus Solanaceae.It is a potential biosynthetic reactor for panax notoginseng saponins and has a pathway for the synthesis of terpenoids.FPS is an important ring-limiting enzyme in the panax notoginseng saponins synthesis pathway.In this paper,we intend to overexpress PnFPS gene in tomato,and explore the use of tomato as a bioreactor to synthesize panax notoginseng saponins or its precursor substances.In this paper,we use bioinformatics to analyze FPS protein sequences of 45 medicinal plants including P.notoginseng and construct phylogenetic trees.It was found that the FPS protein sequences of different medicinal plants are highly conserved with similar physicochemical properties and 45 lipid-soluble FPS proteins,all of which are hydrophilic proteins with no signaling peptides.Subcellular localization in the cytoplasm is predicted.Various potential phosphorylation and glycosylation sites have been predicted on FPS proteins of 45 medicinal plants,suggesting that phosphorylation and glycosylation may affect FPS protein function.The above predictions and analysis set the stage for further studies on the function of FPS enzymes.In this study,total RNA was extracted from the leaves of four-year-old P.notoginseng and cDNA was obtained by RT-PCR.The PnFPS gene in P.notoginseng was amplified by PCR,and a PnFPS-Pearleygate 303 overexpression vector was constructed,which was transformed into a tomato plant by Agrobacterium tumefaciens mediated transcription,and the regenerated plant was induced by plant tissue culture techniques..According to the identification,11 of the 23 induced regeneration tomato plants were positive OE-PnFPS transgenic tomato,and PnFPS gene was successfully overexpressed in tomato.The content of PnFPS metabolite FPP in transgenic tomato was determined,and it was found that the content of FPP in transgenic tomato was increased by 1.4-2.7 times,which proved that the over-expression of PnFPS in tomato can effectively improve the biosynthesis of panax notoginseng saponin precursor.The chlorophyll and carotenoid content of products downstream of the terpenoid synthesis pathway were also significantly increased in this study,further demonstrating that overexpression of PnFPS promotes terpenoid biosynthesis.In conclusion,in this paper,PnFPS,one of the important ring-limiting enzyme genes in the panax notoginseng saponins synthesis pathway,was transformed into tomato,which promoted the biosynthesis of terpenoids and increased the content of FPP by 1.4-2.7 times.The research results laid an important foundation for constructing a complete panax notoginseng saponins synthesis pathway in tomato and obtaining high yield of panax notoginseng saponins...