The Effects Of FPS Overexpression And CAS Expression Interfered On The Biosynthesis Of Saponins In Panax Notoginseng Cells

The application history of Panax notoginseng in China has been more than 600 years,and in addition to the efficacy of stopping bleeding and promoting blood circulation,P.notoginseng also has a special effect to prevent and cure cardiovascular diseases.However,the limited habitat,long growth cycle and crop rotation have made it difficulty to increase the production of P.notoginseng continuously.At present,the gap between supply and demand has been broadened significantly,so it is necessary to strengthen the basic research about P.notoginseng in order to provide the support for solving the shortage of resources by use of biotechnology.P.notoginseng saponins(Panax notoginseng saponins,PNS)are the major active ingredients of P.notoginseng.The biosynthesis pathway of PNS has been clarified basically,thus the pathway can be directly regulated from the gene level,Farnesyl pyrophosphate synthase(FPS)in the PNS biosynthesis pathway can catalyze the isopentenyl pyrophosphate and dimethylallyl pyrophosphate to form farnesyl pyrophosphate which is the common precursor of sesquiterpene and triterpene,and this step of enzymatic reaction has been generally considered as a key rate-limiting reaction in PNS biosynthesis pathway.Although cycloartenol synthase(CAS)in the pathway does not directecly participate in the biosynthesis of PNS,its competition with dammarenedion-? synthase(DS)for(common precursor)2,3-oxidosqualene may result in the decrease of metabolic flow which gives into PNS.Thus CAS has also been thought as one of key rate limiting enzymes.In this study,the over-expression vector of FPS was constructed and integrated into the genome of P.notoginseng cells which had been transferred in RNAi fragment of CAS.The forward and reverse regulations of PNS biosynthesis were realized.The main research results are shown below:1.The FPS sequence with the size of about 1200 bp was obtained from the total RNA of P.notoginseng by RT-PCR.Compared with the FPS gene sequence of P.notoginseng published in NCBI,they were proved to have 100%sequence similarity.Using pCAMBIA1300S as plant overexpression vetor,the FPS fragment was then recombinated into pCAMBIA1300S to construct pCAMBIA1300S-FPS overexpression vetor.2.The FPS was inserted into P.notoginseng genome with CAS RNAi segment by Agrobacterium tumefaciens EHA105.After 5-7 rounds of double antibiotic screening,7 strains of positive cell lines which had been transferred by FPS and CAS RNAi segment at the same time were screened out by screening marker gene(npt ? and hpt ?)PCR detection.5 strains of cell lines with good growth state were selected out for subsequent research,which were numbered T-1,T-2,T-3,T-4 and T-5,respectively.3.The expression levels of FPS and CAS in T-1,T-2,T-3,T-4,T-5 were detected by RT-PCR and qRT-PCR.The results showed that the ordinary RT-PCR and qRT-PCR expression analysis of FPS and CAS in P.notoginseng cell lines were accordant,and three strains(T-3,T-4,T-5),in which FPS was overexpressed and CAS was inhibited,had been achieved.4.The contents of PNS,the important monomer ginsensides and phytosterols of the non-transgenic cell(C)and the cell which had been transferred by CAS RNAi segment(T-Ri),T-3,T-4,T-5 were determined.The results showed that the contents of phytosterols in T-Ri,T-3,T-4 and T-5 were lower than those in non-transgenic control(C),suggesting that the inhibition of CAS expression could decrease the synthesis metabolic flux of the phytosterol branch so that more precursors flowed to the direction of PNS synthesis,and ultimately promoted the accumulation of PNS.In addition,the contents of PNS in T-3,T-4 and T-5 were higher than those in T-Ri,suggesting that overexpression of FPS could help to break the rate-limiting reaction catalyzed by FPS in the biosynthesis pathway of PNS,thus provided sufficient precursors for the biosynthesis of PNS.At the same time,compared with the interference of a single gene,simultaneous interference and overexpression of double genes could further improve the content of PNS and they had the additive effect.