Molecular Cloning And Characterization Of ABC Transporter Genes From Medicinal Plants

Plants produce a large number of secondary metabolites of important medicinal values, such as vinblastine and vincristine in Catharanthus roseus, taxol in Taxus, flavonoids and ginkgolides in Ginkgo biloba. Vinblastine, vincristine and taxol are highly effective anti-tumor medicines, while flavonoids and ginkgolides are well-known for their preventive and therapeutic functions against cardiovascular and cerebrovascular diseases. But most of these secondary metabolites are produced at very low levels in the natural plants and many of the natural plants are threatened with extinction due to overharvest. Other methods such as plant cultivation, chemical synthesis, plant cell and tissue culture are not suitable for commercialization of the secondary metabolites because of the high cost and low yield. As one of the most promising approaches, metabolic engineering has been developed to improve the product composition and increase the product yield in plants and cultured cell systems. However, the biosynthetic pathways of the medicinal secondary metabolites and the detailed molecular regulation of their accumulation are yet to be fully elucidated. ABC transporters are among the important research areas for plant metabolic engineering because of their capabilities of transporting the toxic secondary metabolites produced by plant cells either out of the cells or into the organelles so as to reduce the toxicity to the cells themselves.Taxus,Catharanthus roseus and Ginkgo biloba are important medicinal plants which can produce valuable secondary metabolites and they are important materials for the study of plant secondary metabolism. In order to further understand the biosynthetic pathways and the accumulating molecular basis of the valuable secondary metabolites in the medicinal plants, gene cloning, expression profiles and a series of work of ABC transporter genes were performed and reported in this study as follows:1. A novel full-length cDNA encoding an ABC transporter protein (named as Tcmdr1; GenBank accession number: DQ660357) was cloned from Taxus cuspidata by rapid amplification of cDNA ends (RACE) for the first time. This gene,a member of MDR subfamily,had a total length of 4385 bp with an open reading frame (ORF) of 3951 bp encoding a predicted polypeptide of 1316 amino acids. Sequence analysis showed that TcMDR1 had high similarity with other plant MDRs. The domains analysis showed that TcMDR1 possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranged in"TMD1-NBD1-TMD2-NBD2"direction, consistent with MDR-type ABC transporters. Within NBDs three characteristic motifs common to all ABC transporters,"Walker A","Walker B"and C motif, were found. Expression patterns analysis revealed that Tcmdr1 expressed at high levels in the root, stem and leaf. Southern blot analysis showed that Tcmdr1 belonged to a low-copy gene family. These results indicated that TcMDR1 was a MDR-type ABC transporter protein that might be involved in the transport and accumulation of secondary metabolites in T. cuspidata. The expression construct pDR196-Tcmdr1 containing Tcmdr1 was constructed and transformed into mutant yeast strain AD12345678. The detection of TcMDR1 protein expression and further substrate analysis will disclose the exact function of TcMDR1, which may help us to increase the yield of certain medicinal secondary metabolites through genetic engineering.2. A novel full-length cDNA encoding an ABC transporter protein (named as Crmdr1; GenBank accession number: DQ660356) was cloned from Catharanthus roseus by RACE for the first time. This gene, belonging to MDR subfamily, had a total length of 4395 bp with an ORF of 3801 bp encoding a predicted polypeptide of 1266 amino acids. The CrMDR1 shared high identity with other plant MDRs. The domains analysis showed that CrMDR1 possessed the common structural characteristics of all functional MDR-type ABC transporter proteins. Expression pattern analysis revealed that Crmdr1 was enriched in the root and stem, but low in the leaf. Southern blot analysis showed that Crmdr1 belonged to a low-copy gene family. These results indicated that CrMDR1 was an MDR-type ABC transporter protein that might be involved in the transport and accumulation of secondary metabolites in C. roseus.3. A new full-length cDNA encoding an ABC transporter protein (named as Gbmdr1; GenBank accession number: DQ779968) was cloned from Ginkgo biloba by RACE for the first time. This gene, which was included in MDR-type ABC transporters, had a total length of 4275 bp with an open reading frame of 3840 bp encoding a predicted polypeptide of 1279 amino acids. The GbMDR1 possessed high homology with other MDRs. Amino acid sequence analysis showed that GbMDR1 had the common structural characteristics of all functional MDR-type ABC transporter proteins. Expression pattern analysis revealed that Gbmdr1 was enriched in the stem and leaf, but very low in the root. Southern blot analysis showed that Gbmdr1 belonged to a low-copy gene family. These results indicated that GbMDR1 was an MDR-type ABC transporter protein that might be involved in the transport and accumulation of secondary metabolites in G. biloba.4. MDR-type ABC transporter proteins from medicinal plants were compared for the first time. The sequence comparison showed that CrMDR1 from C. roseus, CjMDR from C. japonica and TcMDR1 from T. cuspidata shared higher homology among each other (60%), while GbMDR1 from G. biloba shared lower homology with the other three (about 40%). In addition,it was found that GbMDR1 and TcMDR1 possessed the coiled-coil structure on N-terminus which was the common structural characteristics of the inward-uptaking AtPGP4 and CjMDR. Therefore, it was supposed that GbMDR1 and TcMDR1 might also possess the function of inward uptake. Phylogenetic analysis showed that plant MDR proteins were grouped generally into three classes. Together with inward-uptaking AtPGP4, CrMDR1,CjMDR and TcMDR from medicinal plants were classified into Class I. Expression patterns analyses revealed great differences among the four MDR genes from medicinal plants. The function determination of these MDR-type ABC transporters is needed to illustrate the main action styles of MDR-type ABC transporter proteins from medicinal plants and the relationship between the MDR proteins and the accumulation of medicinal secondary metabolites.5. The study of ABC transporter genes involved in the accumulation of Taxol requires the detailed acquaintance of the biosynthetic pathway of Taxol. In this study a new full-length cDNA encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (designated as Tmmecs, GenBank accession number: DQ286391), the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis, was isolated from T. media by RACE for the first time. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp ORF encoding a deduced protein of 247 amino acid residues. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. The homology-based 3D structural modeling of TmMECS was analyzed, which showed TmMECS had a similar 3D structure with the E.coli MECS. Besides, the three highly conserved amino acid residues of MECS family were also found in TmMECS including Asp98, His100 and His132, correspondingly, suggesting that TmMECS had a similar biological function with other MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tested tissues including roots, stems and leaves. Furthermore, overexpression of Tmmecs in the E.coli pushed forward the metabolic flux ofβ-carotin synthesis and led to the increase ofβ-carotin accumulation, confirming that TmMECS had enzymic activity.In this paper three MDR-type ABC transporter genes were cloned from medicinal plant T. cuspidata,C. roseus and G. biloba and, meanwhile,their structural and bioinformatic analyses were carried out, which helped to establish a significant experimental basis for the isolation and functional research of plant ABC transporters of MDR subfamily,the largest full-molecule subfamily. The structures and tissue expression of four MDR-type ABC transporter genes from four medicinal plants were compared and further functional analysis would be helpful to illustrate the main action styles of MDR-type ABC transporter proteins from medicinal plants and the relationship between the action styles of MDR proteins and the accumulation of medicinal secondary metabolites, which would be beneficial to the increase of target secondary metabolites by genetic engineering.Tmmecs, a gene involved in Taxol biosynthesis, was isolated from T. media and its function was confirmed, which enriched the gene data of Taxol biosynthesis and helped further study of ABC transporter genes related with the accumulation of Taxol in Taxus.