Cloning And Characterization Of Genes Encoding DXR Gene And MDS Gene From Dioscorea Zingiberensis C.H Wright

Diosgenin is a key compound in synthesis of numerous steroidal drugs. Dioscorea zingiberensis C.H.Wright is specific to China and its rhizomes contain high level of diosgenin and has been the most important material used for production of this compound.Diosgenin is an isopentenyl secondary compound, and isopentenyl diphosphate (IPP) is the crucial precursor in its biosynthesis. It is accepted that diosgenin is synthesized in cytosol of plant cells and the precursor IPP is formed from acetyl coenzyme A via mevalonate pathway (MVA). However, IPP can be synthesized in plastids of higher plants via2C-methyl-D-erythritol4-phosphate pathway (MEP), too. So far, there have no reports on whether MEP involves in diosgenin biosynthesis or not. In order to address this question in future, the full-length cDNA genes encoding1-Deoxy-D-xylulose5-phosphate reductoisomerase (DXR) and2-C-Methy-D-erythritol2,4-cyclodiphosphate synthase (MDS) in MEP pathway were cloned from D. zingiberensis by RT-PCR and RACE-PCR and their functions were verified in this study. The main results are followings:1. The full-length cDNA of DXR of D. zingiberensis is designated as dzDXR. dzDXR is1643bp, containing a42bp5’untranslated regions (5’UTR), a188bp3’untranslated regions (3’UTR) and a1413bp coding sequence (open reading frame, OFR). The deduced protein has470amino acid residues with a molecule weight of50.9KD and an isoelectric point of5.98. Like other DXRs, the protein dzDXR contains two1-deoxy-D-xylulose-5-phosphate binding motifs LPADSEHSAI and NKGLEVIEAHY, two NADPH binding motifs (GSTGS(I/V)GT and (LAAGSN(V/I)), and proline-rich motif PPPAWPGRA at the N-terminal region. Blast analysis reveals that the sequence of dzDXR has a high identity (higher than82%) to those of DXRs from Lilium longiflorum, Oryza sativa, Zea mays, Nicotiana tabacum and Arabidopsis. Evolutionary tree analysis shows the relationship of dzDXR protein is closest to Lilium longiflorum DXR.2. The full-length cDNA of MDS of D. zingiberensis (designated as dzMDS) is896bp, containing a46bp5’UTR、a133bp3’UTR and a717bp OFR. The deduced protein has233amino acid residues with a molecular weight of25.1KD and an isoelectric point of6.54. The protein dzMDS contains two4-diphosphocytidyl-2-C-methylerythritol2-phosphate binding motifs DIG and KAKTHEK, and also include aspartic acid and two histidine sites which consist of MDS protein molecule cavity, and other typical active site of MDS protein. Blast analysis reveals that the sequence of dzMDS has a high identity (higher than73%) to those of MDSs from Rauvolfia verticillata, Ginkgo biloba, Arabidopsis thaliana, Zea mays and Nicotiana tabacum. Evolutionary tree analysis shows the relationship of dzMDS protein is closest to Zea mays MDS.3. Expression vectors of dzDXR and dzMDS in Escherichia coli, designated as pTrc-dzDXR and pTrc-dzMDS, respectively, were constructed using plasmid pTrc-AtIPI. pTrc-dzDXR and pTrc-dzMDS were co-transformed into E. coli together with pAC-BETA. The E. coli clones harbouring pTrc-dzDXR and pAC-BETA or pTrc-dzMDS and pAC-BETA are orange, while those having pAC-BETA or pTrc and pAC-BETA are yellow, indicating that the biosynthesis and accumulation of carotenoid were obviously enhanced in the former clones. These results proves that dzDXR and dzMDS are functional genes in MEP pathway of D. zingiberensis.