Molecular cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase gene from Taxus chinensis / 中草药

ABSTRACT

Objective To obatin the key enzymes of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in taxol biosynthetic pathway from Taxus chinensis (TcDXS), and carry out the bioinformatics analysis, tissue profile, subcellular localization, and functional complementation assay. Methods RACE technologies were used to obtain the full length cDNA of TcDXS for the bioinformatics analysis. Semi-quantitative PCR was used to detect the gene expression levels in different parts of T. chinensis. The localization and function of TcDXS were carried out by subcellular localization and functional complementation assay. Results The full-length cDNA of TcDXS was 3 031 bp containing a coding sequence of 2 229 bp encoding a 742-amino-acid residues which was predicted to have a molecular weight of 79 400 and an isoelectric point of 7.99. The qRT-PCR results showed that the highest expression level of TcDXS was detected in petioles and followed by leaves and barks. However, the expression of TcDXS was very low in roots and stems. What’s more, functional complementation assay results showed that the E. coli, co-transformed with PAC-BETA and pTrcTcDXS, was changed to orange. Conclusion TcDXS was considered to play an essential role in the control of taxol biosynthesis and provided a target for the metabolic engineering of taxol production and plant molecular breeding in T. chinensis.