| Diosgenin has been a good material in synthesis of steroid hormonal medicines, its giobal demand is second only to the demand for antibiotics. Dioscorea zingiberensis C.H Wright of dioscoreaceae, is an important medicinal plant growing only in our country by reason that the high contect of diosgenin in its rhizome is one of the most important medical resource plants for steroid hormonal medicines, also has the highest contect of diosgenin among all the species in the word.At present, the research for diosgenin biosynthesis has not yet been fully elucidated, but it has been considered saponin synthesis in the cytoplasm. The isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) form The mevalonate (MVA) pathway in cytoplasm are the important intermediate precursors of synthetic saponin. However there is another2-methyl-D-erythritol-4-phosphate (MEP) pathway in synthesis of IPP and DMAPP in the plastid. whether IPP and DMAPP from the MEP way in D. zingiberensis involve and influce the way diosgenin biosynthesis, there is no relevant reports.4-hydroxy-3-methyl-2-butenyl pyrophosphate reductase (HDR, also called LytB or IspH) catalytic the last step of MEP way to produce IPP and DMAPP. In order to synthesize future research at the molecular level in MEP pathway of D. zingiberensis, is or not involved in the saponin synthesis. The full length cDNA of4-hydroxy-3-methyl-2-butenyl pyrophosphate reductase HDR is cloned and preliminary verified its function. The results of the study are as follows:1. The full length cDNA of HDR were cloned from D. zingiberensis by methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique, named dzHDR. The full-length cDNA of dzHDR is1570bp, including5’non-coding region of22bp,3’non-coding region of162bp and open reading frame (ORF) of1386bp, encoding461amino acids. The deduced protein has calculated molecular weight about54.9kD and an isoelectric point (pI) of5.11. The result of bioinformatic analysis its subcellular may locate in the chloroplast, it belongs to4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase protein family. The transit peptide sequence of34amino acid residues (A1-A34) and multiple conserved functional sites (A117, A208,A262, A345) of plant HDR protein were found in the deduced coding sequence of dzHDR. Blast analysis showed that the predicted proteins of dzHDR were highly homologous with other dzHDR in Oncidium hybrid cultivar, Dendrobium officinale, Oryza sativa Indica Group, Sorghum bicolor, Zea Mays with over77%amino acid identity. through Phylogenetic analysis, the HDR of D. zingiberensis is closest with the HDR of Dioscorea Oncidium, Dendrobium officinale in evolutionary relationship.2. According to the full-length of cDNA sequences of dzHDR gene, primers were designed to amplify ORF, and constructed dzHDR E.coli expression vector PTrc-dzHDR. Then co-transfect it and pAC-BEAT plasmid together into E.coli TOP10F’strains. The results showed that the colonies only containing plasmid pTrc-dzHDR appeared white, colonies only containing plasmids pAC-BEAT appeared yellow, and the colonies containing pTrc-dzHDR and pAC-BEAT double plasmid presented dark yellow to orange. Analysis showed that by the combined action of dzHDR and pAC-BEAT carried several exogenous genes, can promote the metabolism synthesis of MEP pathway in E. coli, indicating that cloned dzHDR in this study is suer the4-hydroxy-3-methyl-2-butenyl pyrophosphate reductase gene of D. zingiberensis C.H Wright. |
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