中国红豆杉羟化酶基因TcCYP725A22的亚细胞定位及过表达作用分析

Sub-cellular localization and overexpressing analysis of hydroxylase gene TcCYP725A22 of Taxus chinensis

抗癌药物紫杉醇生物合成途径中羟化酶的发现是当今研究的热点和难点。文中利用前期分析得到的1个新的中国红豆杉羟化酶基因TcCYP725A22(GenBank登录号:MF448646.1),构建了亚细胞定位载体pCAMIBA1303-TcCYP725A22-EGFP,瞬时侵染洋葱表皮细胞,激光共聚焦显微镜观察结果发现该基因编码蛋白定位在细胞膜。进一步构建了植物表达载体pBI121-TcCYP725A22,经根癌农杆菌AgrobacteriumtumefaciensLBA4404介导转化到中国红豆杉细胞中过表达后,利用荧光定量PCR (qRT-PCR)和液质联用(LC-MS)分析TcCYP725A22过表达对紫杉醇生物合成的影响。结果显示,瞬时转化pBI121-TcCYP725A22的红豆杉细胞中TcCYP725A22基因的转录水平明显高于pBI121空载体对照组。随着TcCYP725A22基因过表达,几个已知的紫杉醇合成关键酶基因的表达量也都有不同程度的上调,且细胞中各紫杉烷的含量普遍提高。这些结果说明羟化酶基因TcCYP725A22极有可能参与紫杉醇的生物合成路径。

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research.In this study,a new hydroxylase gene TcCYP725 A22(GenBank accession number:MF448646.1)was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725 A22-EGFP to get the transient expression in onion epidermal cells.Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane.Furthermore,the recombinant plant expression plasmid pBI121-TcCYP725 A22 was constructed.After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404,qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725 A22 overexpression on the synthesis of taxol.The results showed that,in the TcCYP725 A22 overexpressed cell line,expression levels of most defined hydroxylase genes for taxol biosynthesis were increased,and the yield of taxanes were also increased.It was concluded that the hydroxylase gene TcCYP725 A22 is likely involved in the biosynthetic pathway of taxol.