Cloning And Functional Identification Of The Transporter Gene CrMATE1 In Catharanthus Roseus

Vinblastine and vincristine have high antitumor activity,which are main components of anticancer drugs in clinical therapy.Catharanthus roseus is the only source for vincristine and vinblastine production.However,their low accumulation in C.roseus limits supplying of vinblastine and vincristine,which are main pharmaceutical ingredients in the market.Recently,all pathway genes involved in catharanthine and vindoline biosynthesis have been identified and overexpression of pathway genes has been testified for increasing catharanthine and vodoline production.Although several stratagies have been applied to promote alkaloids accumulation,the production of these compounds are still unefficient.At present,it is widely recognized that the transport efficiency of intermediates in the pathway is very important for secondary metabolites biosynthesis,which points out a novel direction for regulating alkaloids biosynthesis in C.roseus.In this paper,we have identified CrMATE1 gene,which is clustered with the key pathway genes,Strictosidine Synthase(STR)and Tryptophan Decarboxylase(TDC),for monoterpenoid indole alkaloids biosynthesis in C.roseus.Further analysis showed that CrMATE1 belongs to Multidrug And Toxic Compound Export Transporter(MATE)family.Using virus induced gene silencing(VIGS)technique,CrMATE1 was successfully silenced in Catharanthus leaves by Agrobacterium infiltration and HPLC analysis revealed that the accumulation of vindoline and vincristine in leaves decreased by 49% and 47%,respectively,in comparison with that in control leaves.Yeast cells transformed with pDR196,pDR196-CrMATE1,pDRGAL and pDRGAL-CrAMTE1 were grown on a SD(Synthetic Dropout Medium without Leucine,SD)and SG(Synthetic Galactose Minimal Medium without Leucine,SG)plates containing different concentrations of tryptamine for examining transport activity of CrMATE1.Results showed that CrMATE1 could import tryptamine for inhibiting cell growth in comparison with control cells,since tryptamine is toxic to yeast cells according to our previous examination.In order to investigate subcellular localization of CrMATE1 in plant cells,vector pBI121GD-CrMATE1-GFP was constructed for transforming onion epidermal cells to express CrMATE1-GFP fusion protein.Results showed that CrMATE1 localized at plasma membrane by examining GFP signal.In this work,CrMATE1 was successfully identified according to its transporting tryptamine activity.Silencing of CrMATE1 greatly decreased the accumulation of catharanthine and vindoline,which indicated the importance of tryptamine transport on alkaloids biosynthesis in C.roseus.