果实特异启动子调控薄皮甜瓜ACC合成酶反义基因植物表达载体的构建

从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总DNA,经PCR扩增得到约0.5kb的ACC合成酶基因的片段,将其克隆到质粒载体pMD18-T中,测序表明,该基因为583bp,编码194个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶反义基因植物表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。

Construction of Reversense Expression Vectors of ACC Synthese Gene Regulating by Fruit Specificity Promotor (E8) in Oriental Melon

A DNA fragment of an amino-cyclopropane-1-carboxylie acid (ACC) synthase was amplified by Polymerase Chain Reaction (PCR) from sarcocarp tissue of Oriental melon Qitian No.1 and cloned into a pMD18-T vector. The result of sequencing and amino acid sequence inference of this recombinant plasmid shows: the fragment has 583 bases, encoding 194 amino acid; A DNA fragment of an E8 was amplified by Polymerase Chain Reaction (PCR) from leaf tissue of Tomato Dongnong 706 and cloned into a pGEM-T Easy vector. The result of sequencing and amino acid sequence inference of this recombinant plasmid shows: the fragment has 2192 bases; The cloned DNA of ACC synthase and DNA ofE8 gene was further introduced into binary vector pCAM2301 in a reverse orientation with its upstream specificity promotor(E8 ), giving an expressing plasmid containing the antireversense ACC synthase gene.