Study On Cloning, Expression And Regualtion Of Taxane 13a-hydroxylase From Taxus

The nature product Taxol, produced by Taxus species, is a highly effective anticancer drug;Taxol inhibits cell proliferation at the G2-M phase, thereby it can block mitosis effectively. FDA has approved Taxol in the treatment of refractory ovarian and breast cancer in 1992. Despite its outstanding curative effect,the content of Taxol is at quite low level. For example, the commercial isolation of 1 kg Taxol required about 6.7 t of T.brevifolia bark, equivalent to that of 2,000-3,000 trees. To resolve this contradiction, scholars at home and abroad have made efforts such as artificial cultivation, total syntheses and semisynthesis, cell culture etc. However low yields and high costs preclude them from industrialization produce .For the foreseeable future, the metabolic regulation of the Taxol biosynthetic pathway may provide a feasible solution to the sustainable yield problem.A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. 13α-hydroxylase is an important P450-dependent monooxygenases in the synthesis of Taxol. Here we cloned its full-length gene and constructed expression vectors.The main results are summarized as follows:(1) Full-length sequence coding for Taxane 13α-hydroxylase(13OH) was cloned from Taxus cuspidata cDNA library.The DNA was verified by sequencing after it was inserted into pGM-T easy vector. Compared with the nucleotide sequence in the Genbank, the cloned DNA in our paper showed 99.38% identity with the reported taxane 13α–hydroxylase of Taxus cuspidata.(2) The 13OH gene was cloned in to Pcambia1305.1, pcambia1304 and pEGAD to construct three different plant expression vectors with GUS gene, GUS gene and mgfp5, EGFP gene as their report gene respectively.(3) The binary vector with 13OH in pCAMBIA1305.1 was transformed into Agrobacterium tumefaciens GV3101 by electroporation. Plants were regenerated from the infected leaf dics under the selection of hygromycin. PCR ananlysis with 13OH-specific primer and assay of the fusion GUS reporter gene showed that the 13OH gene was successfully intergrated into and expressed in the leaf cells of transgenic tobacco plants.(4) The precurcor of miR160e, miR164e, miR165a, miR166m, miR169d were cloned from the genomic DNA of Arabidopsis thaliana and Oryza sativa, Compared with the nucleotide sequence in the Genbank, the cloned DNA in our paper showed 100%, 99.24%, 100%, 99%, 100% identities respectively.(5) Plant expression vectors for seven miRNA genes were constructed to test the function of the regulators.(6) Analysis of transient transgenic Taxus shots showed that the expression of 13α-hydroxylase could be suppressed by the technology of miRNA .Compared with miR164e and miR165a ,miR169d was more efficient in silencing target gene due to its high specificity.(7) The result of real-time PCR showed that miR165a has a little function on the expression of 13α–hydroxylase.