Cloning And Functional Characterization Of Lithospermum Erythrorhizon ABC Transporter Genes Involved In The Transport Of Secondary Metabolites

Plants produce a wide array of secondary metabolites that enable them to adapt to their environment.These metabolites have diverse chemical structures and various biological activities.Some metabolites have medicinal applications.For example,morphine is used as an analgesic and vinblastine as an anticancer drug.Stable production of these metabolites in plants requires both biosynthetic enzymes to catalyze metabolic reactions and transport proteins to carry them to various organelles and tissue,e.g.,just as nicotine is accumulated in leaves of tobacco,and berberine is accumulated in the rhizome.The transmembrane transport of plant secondary metabolites is a newly developing research area,and it has been found that there are ATP-binding cassette(ABC)transporters in some higher plants,which were identified as central part of a detoxification process in which they sequester conjugated xenobiotics from the cytoplasm into the central vacuole,and are involved in the cellular and intercellular movement of secondary metabolites.So ABC transporters are among the important research areas for plant metabolic engineering.The roots of Lithospermum erythrorhizon contain red naphthoquinone pigments-shikonin derivatives with antibacterial,anti-inflammatory and antioxidant activities,and recent extensive research has confirmed their antitumor properties as well.They have important values for the study of plant secondary metabolism.In order to understand how ABC transporters are involved in transport and accumulation of secondary metabolites at the molecular level.LeMDR gene of Lithospermum erythrorhizon was cloned,and the functional characterization of ABC transporter in the transport of secondary metabolites was also investigated.A novel full-length cDNA encoding an ABC transporter-LeMDR was cloned from L.erythrorhizon by rapid amplification of cDNA ends(RACE).This gene,a member of MDR subfamily,encodes a full ABC protein and has the common structural characteristics of all functional MDR-type ABC transporter proteins.The domains analysis showed that LeMDR possessed two transmembrane domains(TMDs)and two nucleotide binding domains(NBDs),which are arranged in"TMD1-NBD1-TMD2-NBD2" direction.NBDs have three characteristic motifs common to ABC transporter family,"Walker A","Walker B" and C motif.We isolated and cloned the promoter sequence of LeMDR gene,which was analyzed for putative cis-acting elements using the PLACE database.The analysis revealed that there are several known cis-acting elements related to biotic-stress responses,such as WBOXATNPR1 and MYBCORE element.To determine the localization of LeMDR transporter,we induced the transgenic hairy roots of L.erythrorhizon and performed transient expression of this gene in onion epidermal cells.The results showed that LeMDR transporter is located in the plasma membrane.To analyze that the function of LeMDR transporter as a shikonin transporter.The LeMDR gene was expressed in the yeast strain AD12345678 lacking eight major yeast ABC transporter genes that confer multidrug resistance.Yeast cells expressing LeMDR accumulated lower shikonin than the control yeast cells in the same concentration solution.Results suggested that LeMDR expressed in yeast remained as functionally active as a shikonin efflux transporter.To examine that the LeMDR transporter was highly specific for the transport of shikonin compared with other shikonin derivatives.The accumulation levels were assayed for 5,8-dihydroxy-naphthoquinone(DNQ)and Shikonin lipoic acid ester derivatives(SED)in yeast cell.The results indicated that,whereas DNQ and SED were accumulated to the similar levels as EV yeast cell,only shikonin was exported by LeMDR-expressing yeast.A full-length cDNA encoding an ABC transporter-LeMRP was cloned from L.erythrorhizon by RACE method.Sequence analysis and prediction suggested that leMRP should belong to MRP subfamily of ABC transporter.By inducing the transgenic hairy roots of L.erythrorhizon and using Confocal laser scanning fluorescence microscopy(LSFM),EGFP-LeMRP fusion protein was showed to be localized in tonoplast in the hairy roots.To examine whether LeMRP function as a shikonin transporter,we isolated intact vacuoles from the hairy roots.These isolated vacuoles were incubated with shikonin in an assay buffer,a time course analysis showed that the shikonin content in LeMRP transformants was consistently higher than that of control.The expression of LeMRP was knocked down in L.erythrorhizon by using RNA interference(RNAi)-based approach and vacuoles of LeMRP-RNAi hairy root contained the lower shikonin accumulation levels compared to EV.The results suggested that the shikonin was transported across the tonoplast by LeMRP transporter.In conclusion,by studying the function of the transport of secondary metabolites in L.erythrorhizon and investigating the major mechanism of ABC transporters in plants and their relationship to accumulation of secondary metabolites,we provided not only the possible theoretical explanation about the role in the shikonin metabolism of ABC transporter but also a guide to increase the production of secondary metabolites by genetic engineering.