香石竹ACC氧化酶基因的克隆与植物表达载体构建

Cloning of ACC Oxidase Gene of Carnation and Construction of Its Plant Expression Vectors

根据已发表的ACO核苷酸序列 ,设计一对引物 ,以香石竹品种‘American’基因组DNA为模板 ,扩增出香石竹ACC氧化酶基因 ,将其连接到pMD18加T质粒载体上 ,通过中间载体pBluescriptSK ,构建了ACO基因正向和反向插入的植物表达载体 ,并将其导入了农杆菌。酶切检测表明该片段已插入表达载体 ,序列测定结果显示所克隆基因片段含 3个外显子 ,2个内含子 ,其中

A pair of primers were designed according to the 1 aminocyclopropone 1 carboxylic acid (ACC) oxidase gene of carnation and the primers were used to amplify the genomic DNA fragment of about 1.2 kb by polymerase chain reaction (PCR)by taking genome DNA from 'American' carnation leaves as template. The PCR product was cloned into T tailing pMD18 vector. Sequencing indicated that the ACC oxidase gene included three exons interrupted by 2 introns with identical positions as they are in tomato. ACC oxidase gene were respectively cloned into plant expression vector pMOGMON in sense and antisense orientation. Recombinant expression vectors were identified by restriction enzyme and PCR analysis. PCR indicated plant expression vectors were transferred into A. tumefaciens .