Optimization Of The Genetic Transformation System Of Taxus Cells And Expression Analysis Of ORCA3 And MET1-RNAi Genes In Transformed Cell Lines

Large-scale culture of taxus is the most promising way to solve the problem of paclitaxel drug source shortage. The taxol content of Taxus cell is low and extremely unstable, which restrict seriously its industrialized process of large-scale culture of taxus in a certain degree. The clarify of taxol biosynthetic pathway and cloning of key 2enzymes and transcription factors made it possible to construct high yield transgenic taxus cell lines using genetic engineering method. Currently the research about genetic transformation system of Taxus cells is relatively rare, Thus the agrobacterium-mediated genetic transformation system of Taxus was optimized in the study and a series of genetically modified cell lines were obtained.The main results are described as follows:1) The construction of the plant expression vector containing ORCA3 or MET1 gene. The ORCA3(Octadecaniod-derivative Responsive Catharanthus AP2-domain protein) and DNA methytransferase MET 1 genes were cloned from periwinkle and arabidopsis cells and then constructed ORCA3 plant expression vector and met1-RNAi interference vector respectively.Then the vectors were transformed into the agrobacterium of EHA105 strains.2) Opitimition of agrobacterium-mediated genetic transformation system of media Taxus. The best genetic transformation conditions are described as follows: using agrobacterium strain of EHA105, Taxus suspension cells in good condition as a receptor material, agrobacterium density OD600=0.6, infect 25 min, co-culture in 21℃for 48 h. The best way to kill agrobacterium and antibiosis concentration of cefotaxime and hygromycin is that the cell were washed by sterile water that contains 100 mg/L cefotaxime for 10 s first, then the cells were cultured to the medium containing 300 mg/L cefotaxime, two weeks later, the cells were transferred to culture medium which contains 150 mg/L kanamycin or 8mg/L hygromycin for 3 cycles.Then the genetic transformed Taxus cells are obtained at last.3) Expression analysis of ORCA3 gene in genetic transformed cell lines. We detect the transformed cells with GUS activity assay, introduced gene PCR and western blot analysis. The results showed that the exogenous gene has been successfully integrated into Taxus genomes and expressed in protein level. The effects of introduced gene to taxol yield are different.The highest yield of taxol of transformed cells is 159.3μg/g dry weight, 2.5 times of the control.4) Expression analysis of MET1-RNAi vector in genetic transformed cell lines. GUS activity assay and introduced gene PCR results showed that the exogenous gene had been successfully intergrated into the purpose yew cells chromosomes. The taxol yield in the transgenetic cell lines are higher than the control group ,the highest yield is 178.3μg/g dry weight, nearly 2.87 times of the control group, which suggested that the RNAi recombinant expression vector had biology functions.