Molecular Cloning And Expressing Of A 5-alpha-taxadienol-10-beta-hydroxylase Gene From Taxus

It is difficult to extract total RNA from Fir plants, because they contain plenty of polysacchrides and polyphenols. Compared with several representative methods, an improved method was established with the agents of acid phenol and PEG in the present study. The improved method is easy to carry out with low cost. And it can effectively eliminate the interferences of polysacchrides and polyphenols in the plant tissues to obtain high-qualified RNA sample. So it is useful for preparing total RNA from those plants that contain the polysacchrides and polyphenols.Total RNA was extracted form Taxus sp. tender leaves as the template. Taxus cuspidata 5-alpha-taxadienol-10-beta-hydroxylase (TDH) gene was amplified with RT-PCR. The amplified TDH DNA was analyzed by restriction enzymes and sequence measure. Then the amplified DNA was ligated with plasmids pPIC9K and pET28a(+) in vitro, and transferred into E. coli. The expression plasmids of pPIC9K-TDH and pET-TDH were obtained from recombinants. The homology of nucleotide acid sequence between Taxus cuspidate TDH gene and the data published in GenBank was above 99%.When the plasmid pET-TDH was transferred into E. coli BL21 (DE3), a new recombination carrying pET-TDH was screened from the transformations of E. coli BL21 (DE3). Induced by IPTG, TDH gene in the host was expressed effectively. A distinguished protein band was observed in SDS-PAGE by comparing with the control. The molecular weight of the protein is approximate 60 KD. It indicates that the clonedTDH gene of Fir plant can be expressed in prokaryote.Two recombinant plasmids established in this present study can be used as a foundation for further exploring the regulation and control of the TDH gene, and producing taxol by gene engineering technique.