Studies On Taxol Metabolism Regulation In Taxus Chinensis Var. Mairei Cell Cultures

Taxol has unique anticancer activities and Taxus cell culture has become one of the most promising alternative methods for obtaining taxol. Three aspects were studied for regulation of taxol metabolism in Taxus cell culture. Firstly, cell proliferation. Factors affecting the proliferation of Taxus cell were studied and the main results were as follows: 1) Plant growth regulators were very important to cell growth. It was proved that the greatest growth rate of cell, 2.517 was obtained with the combination of 1mg/L NAA and 1 mg/L 6-BA among the choice of 75 phytohormone treatments; 2) The special growth rate of Taxus cell subcultured on logarithmic phase was 1.6 folds of that on stationary phase and the production of taxol was increased by nearly 4 times; 3) The Taxus cell subcultured on logarithmic phase uptook carbohydrate and nitrate earlier than that subcultured on stationary phase. However, the two treatments uptook phosphate and ammonia anions had no significant difference in the studies on kinetics of nutrient uptake. Subculturing on logarithmic phase was good to the accumulation of biomass and the synthesis of taxol. Secondly, taxol metabolism in cell culture. It was suggested that baccatinIII was accumulated and the productivity was 3 times as high as that of taxol in our study of secondary metabolism of Taxus cell cultures. The reason for that were 1) the scarcity of C-13 side chain synthesis, 2) the low expression level of the phenylpropanoyltransferase (BAPT) transfering the side chain directly to baccatinlll. The precursors of C-13 side chain were supplemented for the first reason, but the results of had no difference. So we focus our attention on the second reason..Thirdly, the effects of BAPT overexpressing in Taxus cell on taxol metabolism. The whole sequence of BAPT was cloned from Taxus chinesis van mairei by RT-PCR and the full-length cDNA has 1338 bases by sequence analysis. The result showed the phentylpropanoyltransferase cDNA was highly homologous with that of Taxus cuspidate. We constructed the pBI-BAPT recombinant plasmid, and obtained the transgenic cell suspension cultures via Agrobacterium Hmefaciens GV3101 as a vehicle. And the exogenous gene was identified to be inserted into the genome of Taxus chinesis van mairei via histochemical GUS-staining, PCR, PCR-Southern and Southern blot. The overexpression of BAPT was detected by western blot successfully. The result indicated that the hybridization signal of BAPT transgenic cells was stronger than that of non-transgenic cells. It was suggested by HPLC that the bacIIIcontent of BAPT transgenic cells was reduced by 60% but the taxol content was 0.309mg/g DW, 3 times as high as that of non-transgenic cells.