热不对称交错PCR克隆bapt基因侧翼序列

摘要

紫杉醇是来源于红豆杉植物的抗癌药物,广泛应用于乳腺癌和小细胞肺癌等临床治疗.巴卡亭Ⅲ-氨基苯基丙酰-13-O-转移酶基因(bapt)的表达是紫杉醇合成的瓶颈,解析该基因侧翼序列是人工调控紫杉醇合成途径的前提.采用同源克隆和热不对称交错PCR技术,扩增获得bapt基因及其侧翼的染色体序列5.6 kb.生物信息学分析表明,bapt基因含有2个外显子和1个内含子,编码区长度1 338 bp;5'端侧冀序列1 604 bp,包含启动子元件;3'端2 582 bp,含有终止子元件242 bp.发现的转录元件将应用于代谢工程改造红豆杉细胞,提高紫杉醇产量.%Taxol is a natural anticancer drug produced from Taxus species,and widely used in the clinical treatment of breast cancer and small cell lung cancer.The expression of baccatin Ⅲ-aminophenylpropanoyl-13-O-transferase (bapt) gene is limited for taxol biosynthesis,and its flanking sequences are prerequisite for regulating taxol production.We cloned bapt gene and its flanking sequences with the length of 5.6 kb by homologous cloning and thermal asymmetric interlaced PCR.Bioinformatic analysis of the sequence showed that bapt gene contains two exons and one intron with the open reading frame of 1 338 bp.5 ‘ Flanking sequence of 1 604 bp contains the promoter and 3 ' flanking sequence of 2 582 bp contains 242 bp long terminator.The regulatory elements discovered in this research would be helpful to improve the production of taxol through metabolically engineering taxus cells.