Abstract
Taxadiene 5 alpha hydroxylase (T5h alpha) of Cytochrome P450 family causes the conversion of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-diene-5 alpha-ol which is the first oxygenation step and the third specific step of taxol biosynthetic pathway -a central feature in taxol biosynthesis. A PCR based differential display cloning approach was used to enhance the production of taxol from the Yew (Taxus brevifolia). Total RNA was extracted from the leaves of T brevifolia and it was reverse transcribed into cDNA followed by PCR amplification. The annealing temperature of RT PCR was obtained by performing Gradient PCR. PCR amplified gene product was electophoresed in 2% agarose gel and it was purified using the Quiazen gel elution kit. The purified T5h alpha gene was then cloned into a pTZ57R/ T vectors with the T-4 DNA ligase and the recombinant vectors were transformed into the competent E.coli DH5 alpha cells. The cloning of T5h alpha gene was confirmed by restriction digestion analysis with EcoRI and Hind III and the transformation of vectors were confirmed by the blue-white screening. This study successfully describes the cloning of T5h alpha gene into pTZ57R/T vectors and the transformation of T5h alpha cloned recombinant vectors in competent E. coli DH5 alpha cells for application in taxol production.