Identification Of HD86-9 Strains Of Taxol And Cloning Of Related Enzymes

Nowadays, Taxol is mainly isolated from the bark of yew tree. But for the method isolated from the yew tree, whith is relative scarcity, slow growing rate and extremely low content of Taxol in the taxus, the supply of Taxol is very limited. Therefore, scholars all over the world exploit many ways to replace the yew found in fungi as a source of drug Taxol, paclitaxel production by microbial fermentation was the source of the shortage of drugs to solve one of the effective ways.In this study, a total of 213 isolates of endophytc fungus were isolated from Taxus cuspidate. It was confirmed that one strain(HD86-9) produced Taxol by ways of thin layer chromatography(TLC),high performance liquid chromatography(HPLC) and mass spectrum(MS). HD86-9 isolated endophytic fungi was observed by morphology and analysis, and carried out the identification of fungi. Through a series of results in Morphological and molecular biology, the HD86-9 was a new identified for variant of Aspergillus niger and named Aspergillus niger var. taxi D. P. Zhou, K. Zhao et W. X. Ping, var. nov. Using RT-PCR technology, HD86-9 of the cDNA as a template amplified geranyl geranyl pyrophosphate synthase gene. In accordance with paclitaxel GenBank key enzyme known conservative region gene primers were designed to HD86-9 of the cDNA as a template for PCR amplification of synthetic geranyl geranyl pyrophosphate synthase gene. As a result, the 371bp of gene fragment was acquiered, GenBank using the Blast sequence comparison revealed that the sequences and geranyl geranyl pyrophosphate synthase homology reached 99%. This study established substantial background for building high product taxol genrtic engineering strains.