Cloning, Identification And Characterization Of TcDXS And TcDXR Involved In The MEP Biosynthetic Pathway Of Taxus Chinensis

Taxol is one of the terpenoid extrated from the Taxus species, which is a kind of more effective anti-cancer drug. For a long time, the rescource of taxol is mainly procured from the extraction of the bark of the Taxus species. However, Taxus species plant is nationwide-protected and with slow growth rate and low taxol content. The more and more large demand for taxol contracts with the limited availability. So a lot of methods were developed to solve this contradiction, such as chemical synthesis. But these ways always have a lot of defects. In contrast, genetic engineering technology lights a bright way to solve this problem. For it is a foundation carrying out the molecular biology research of taxol biosynthesis pathways, which needs to be understood sufficiently.Different from the traditional views, a lot of experiments confirm that Taxol mainly originates from the recently unveiled MEP pathway. So as to define the biosynthesis of taxol precursors, the deep comprehension at the level of molecular genetics is necessary and important to base the subsequent research. A new key gene involved in the MEP pathway was cloned through the method of RACE, characterized and functionally identified, Which is named TcDXS((the gene encoding 1-deoxy-D-xylulose 5-phosphate synthase from Taxus chinensis). The coding sequence of another key gene (TcDXR,a gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase) was cloned to construct the plant expression vector. FQ-PCR(real-time flurescent quantitive polymerase chain reaction) was used to analyze the expression of two genes in eight tissues. Both genes expressed recombinant protein in Rosetta(a kind of E.coli that was used to prokaryotic expression of foreign gene).In the nonmevalonate terpenoid pathway of Taxol biosynthesis,1-deoxy-D-xylulose 5-phosphate synthase(DXS) is the first enzyme for catalyzing isopentenyl diphosphate biosynthesis. The full-length cDNA of TcDXS was 3031bp, containing 5’,3’untranslated regions and a poly A tail. The encoding sequence of TcDXS was 2229bp, encoding a peptide of 742 amino acid residues. The calculated molecular mass of this peptide was 79.4 kDa and calculated isoelectric point was 7.99.TcDXS principally expressed in green tissues and barkt through the FQ-PCR analysis. The color complementation assay experiment proved that TcDXS could make the host bacteria over-express theβ-carotene. The recombinant protein coincide with the calculated molecular mass was expressed.All the basic work of TcDXS above will promote to understand more about the important role of DXS involved in the Taxol biosynthesis at the molecular level.1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) is the second enzyme in the methylerythritol phosphate (MEP) pathway of Taxol biosynthesis that catalyze the DXP into MEP.A lot of prior experiments confirm that DXR is a key gene in the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis. Therefore it is necessary to construct plant expression victor to develop genetic transformation to authenticate the important effect of TcDXR in Taxol biosynthesis. As well, the recombinant protein coincide with the calculated molecular mass was expressed. Like the TcDXS, the result based on FQ-PCR analysis discovered that the DXR gene in Taxus chinensis expressed highly in the green tissues and bark.