紫杉烷13α-羟基化酶基因的克隆及其转化烟草的研究

本研究利用东北红豆杉(Taxus cuspidata )cDNA文库作为模版，通过PCR技术扩增得到我国东北红豆杉紫杉烷13α-羟基化酶(Taxane 13α-hydroxylase，简称13OH)的全长cDNA，将PCR产物克隆到pGM-T载体后测序结果表明该序列长度为1458bp。同源性比较分析结果表明：其碱基序列与已经报道的东北红豆杉(Taxus cuspidata)的13OH基因的一致性为99.38%，其氨基酸序列同与已经报道的东北红豆杉的13OH氨基酸序列的一致性为99.18%。将获得的cDNA全长序列正向插入到含有GUS报告基因的pCambia1305.1后成功地构建出东北红豆杉13OH植物表达载体pC13OH，通过电击法把pC13OH转入根癌农杆菌GV3101中。利用该工程菌株对普通烟草进行了转化，在潮霉素选择压力下获得了完整的再生植株。利用13OH基因特异引物，通过PCR技术筛选到4株阳性再生植株。在这4株再生植株中，有3株植株GUS报告基因的组织化学染色呈现阳性反应，表明该植物载体表达载体中与13OH相融合的GUS基因成功地得到了表达。本研究为今后深入研究13OH基因在烟草中的表达和开展紫杉烷13α-羟基化酶基因对红豆杉细胞的转化以及研究作用于该基因的小RNA调节子打下了基础。

Cloning of Taxane 13α-hydroxylase from Taxus cuspidata and Its Transformation to Nicotiana tobacum

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.