Study On Expression Vectors For Taxane 14β-hydroxylase And Genetic Transformation Of Taxus

Taxol, first isolated from Taxus Brevifolia Nutt, is one of the most efficacious anticancernatural products hitherto which possess excellent activity against ovarian cancer, breast cancer,etc. It is the low yields of taxol in the plant and the slow growth of taxus plant that make thefurther utilization of taxol to be an important issue. Due to the same precursor in biosynthesiswith taxol and the higher yields in plant, the C-14 oxygenated taxane compounds could bereduced to enhance the production of C-13 oxygenated taxanes including taxol through generegulation.The technologies of antisense RNA and RNAi are the effective methods of studying thefunction of plant genes. Especially, RNAi is the most important one at present. In this study, wetried to silence taxane 14β-hydroxylase(14OH) by using the technology of antisense RNA andRNAi and to study its function because it exist in the branch of taxol biosynthetic pathway. Atthe same time, basing the previous experiments, we optimized the culture condition of taxuscell lines and regeneration system and we also studied the genetic transformation of taxus. Themain results are summarized as follows:(1) Taxus genome DNA extraction method was optimized by using an improved methodin order to get rid of interfering compounds such as polysaccharides and polyphenols.(2) A 915bp DNA fragment of 14OH was cloned from the genomic DNA of Taxuschinensis vat. mairei and this fragment had 98.3％identity comparing with the relevantsequence of reported 14OH in Taxus cupidata.(3) Taxus cell lines were cultured on fresh medium succeedingly at regular intervals andantioxidant such as PVPP was added into culture medium in order to prevent browning of taxuscultured cells.(4) Explant was found an important factor for somatic embryogenesis of Taxus. Theimmature embryos play important role in inducing somatic embryos. At the same time, it wasdiscovered that it was difficult to regenerate taxus plant through organogeny.(5) Detection and analysis of transgenic cell lines of taxus showed that the expression of 14β-hydroxylase could be suppressed by the technology of antisense RNA and RNAi. Thestudy demonstrated that the technology of RNAi is better than antisense RNA in silencing14OH gene. Compared with expression vector pZ14a, the vector pZ14b was more efficient insilencing target gene due to its high specificity.(6) Compared with agrobacterium-mediated gene transfer method, taxus genetictransformation using plasmid DNA directly had better result. The latter almost silence targetgene completely.(7) The function of 14 13-hydroxylase was confirmed by analysis of the chemistrycomponents of taxus transgenic and non-transgenic cell lines. The C-14 oxygen substitutivetaxanes in taxus were reduced when 14β-hydroxylase was knocked down.