Molecular Cloning And Functional Study Of PnMYB1 Transcription Factor Involved In Biosynthetic Regulation Of Panax Notoginseng Saponins

Panax notoginseng(Burk.)F.H.Chen(P.notoginseng)belongs to the genus of Panax,Aralianceae family.The root,stem,flower and rhizome of P.notoginseng can be used as herbs for their pharmacological effects of hemostasis,promoting blood circulation,removing blood stasis and prevention of cardiovascular disease etc.P.notoginseng has been widely used as a drug for more than 400 years in China.The main active compounds of P.notoginseng are saponins which are presented in plants in small amounts.Artificial cultivation of P.notoginseng has some limiting factors including strict environment,long growth cycle and low yield of drugs,thus to hinder the development of P.notoginseng.As the structures of P.notoginseng saponins are complex,they are difficult to be synthesized by chemical process up to now.Therefore,studying the saponins synthesis pathway in P.notoginseng and synthesis saponins by homologous and heterologous expression method has great values in basic research and application.Secondary metabolic processes are complex and usually involved in a variety of enzymes.In order to modulate number of key enzyme genes in the saponins synthesis pathway,regulating transcription factors might be a good strategy.Transcription factor can interact with multiple genes promoter sequence to realize "multi-point regulation"of gene expression,thus to control the synthesis of secondary metabolites.In this study,the fragment sequence of PnMYBI gene was screened by JERE cis-element and the full length of PnMYBl gene was obtained by RACE technique.PnMYB1 gene has a 723 bp open reading frame(ORF),encoding 240 amino acids,relative molecular weight is about 27.359 kDa and has a typical R2R3-MYB domain.The results of homologous sequence alignment and phylogenetic tree analysis showed that PnMYB1 belongs to the family of R2R3-MYB transcription factors.PnMYB1 was fused with green fluorescent protein(GFP)gene,and then transiently transfected into onion endothelial cells.The results indicated that PnMYB1-GFP fusion protein was located in the nucleus.pCAMBIA13005-PnMYB1 was obtained by inserting the PnMYBl into the plant expression vector pCAMBIA1300S and introduced into Agrobacterium tumefaciens EHA105 by freezing-thawing method.Finally,pCAMBIA1300S-PnMYB1 was transfected into P.notoginseng.Positive PnMYB1-transgenic cells were screened by antibiotics and molecular detection methods.The results of qRT-PCR showed that the expressions of PnFPS,PnSE and PnDS in PnMYB1-transgenic cells were significantly increased;PnSS and PnCAS expressions did not changed obviously compared with the control.The content of total saponins in PnMYB1-transgenic cells was significantly higher than those in control;the highest yield of total saponins was about 1.73-flod higher than those in control.R1,Rg1,Re,Rbl and Rd were also increased in different degrees.In order to explore the regulating mechanism of saponin synthesis in Panax notoginseng by transcription factor PnMYB1,partial promoter sequence before 5'-terminal of SS,DS and CAS were cloned by chromosome walking,and then portions of SS,DS,CAS and SE promoter sequences were got.Next,the above promoter sequences replaced the promoter sequence of PBI 121 vector to obtain recombinant vectors which fused with GUS.Finally,the recombinant vectors were introduced into Agrobacterium tumefaciens EHA105.The recombinant vectors were introduced into Agrobacterium tumefaciens EHA105 and then transformed in tobacco leaves individually and co-transformed with PnMYB1.The GUS activity of the tobacco leaves which have been co-transformed PnMYBl and PnSE1 promoter fragments was significantly higher than that in the wild tobacco leaves being transformed by PnSEl promoter fragment alone.Promoter fragments PnDSPl and PnSEP1 were co-transfected with PnMYB1 in tobacco leaves,respectively;the GUS activities of those tobacco leaves were stronger than those in only transfected one promoter fragment into tobacco leaves.The GUS activities of tobacco leaves,which co-transfected PnSS1 and PnCAS 1 with PnMYB1,respectively,have little change compare with those in only transfected promoter fragment into tobacco leaves.Over-expression PnMYB1 in P.notoginseng can realize multi-point regulation in saponin synthesis pathway and promote saponins production.Such results have great value for studying and understanding synthesis pathway of saponins in P.notoginseng,which provide an important theoretical reference for saponins synthesis by homologous and heterologous methods.