中国生物工程杂志

A 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 and amino acid sequence in the Genbank, nucleotide of the cloned DNA in our paper showed 99.38% identity with that of the reported taxane 13α-hydroxylase of Taxus cuspidate and its deduced amino acid sequence showed 99.18% identity with that of the reported taxane 13α-hydroxylase of Taxus cuspidata. To construct the plant expression vector pC13OH, the full-length DNA was inserted into pCambia1305.1. PCR and restriction enzyme analysies confirmed the correctness of the construct, then the recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 by electroporation. The transformation of this engineered Agrobacterium strain with tobacco(Nicotiana Tabacu L.) was studied. Plants were regenerated from the infected leaf dics under the selection of hygromycin. PCR ananlysis with 13OH-specific primers shows 4 plants were positive. Among them, 3 palnts were GUS positive after doing the assay of the fusion GUS reporter gene.