Isolation and expression profile analysis of a new cdna encoding 5-alpha-taxadienol-10-beta-hydroxylase from Taxus media

A full-length cDNA encoding 5-alpha-taxadienol-10-beta-hydroxylase (designated as Tm10BH), which catalyzes the second cytochrome P450-dependent hydroxylation step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of Tm10BH had a 1494 bp open reading frame (ORF) encoding a protein of 497 amino acid residues. The deduced protein had an isoelectric point (pi) of 8.91 and a calculated molecular weight of about 57 kD. Sequence comparison showed that Tm10BH had high homology with hydroxylases reported previously. Tissue expression pattern analysis revealed that Tm10BH expressed strongly in leaves, weak in stems and no expression could be detected in fruits. Expression profiles of Tm10BH under different elicitor treatments such as methyl jasmonate, silver nitrate and ammonium eerie sulphate were also investigated for the first time, and the results revealed that expression of Tm10BH was all induced by the tested three treatments, implying that Tm10BH was an elicitor-responsive gene. This study provides useful information to further understand induction expression and molecular regulation mechanism of genes encoding related enzymes involved in Taxol biosynthesis.