果实特异启动子调控薄皮甜瓜ACC合成酶cDNA反义表达载体的构建  

Construction of reversal expression vectors of ACC synthase cDNA regulated by fruit specific promoter (E8) in oriental melon

作　　者：郭庆勋[1] 秦智伟[2] 李景鹏[2] 

机构地区：[1]吉林大学,长春130062 [2]东北农业大学,哈尔滨150030

出　　处：《果树学报》2006年第3期411-414,共4页Journal of Fruit Science

基　　金：黑龙江省科技攻关项目(GB01B402-01)。

摘　　要：应用反义RNA技术抑制甜瓜成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决甜瓜延熟保鲜难题的可行新方法。根据GenBank中甜瓜、黄瓜ACC合成酶基因氨基酸保守序列设计引物,从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总RNA,经RT-PCR扩增得到约0.7kb的ACC合成酶cDNA片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为777bp,编码258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cDNA反义表达载体,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。Improving keeping quality of oriental melon is an important program in research. Application of reversal RNA strategy to reduce ethylene biosynthesis is a new feasible method for breeding oriental melon cultivars with long keeping quality. A cDNA fragment of an amino-cyclopropane-1-carboxylie acid （ACC） synthase was amplified by RT-PCR from sarcocarp tissue of Qitian No.1 and cloned into a pGEM-T Easy. The result of sequencing and amino acid sequence inference of this recombinant plasmid showed： the fragment had 777bases, encoding 258 amino acid. A DNA fragment of an E8 was amplified by polymerase chain reaction （PCR） from leaf tissue of Dongnong 706 and cloned into a pGEM-T Easy vector. The result of sequencing and amino acid sequence inference of this recombinant plasmid showed： the fragment had 2192 bases. The cloned cDNA of ACC synthase and DNA of E8 gene was further introduced into binary vector pCAM2301 in a reverse orientation with its upstream specific promoter （E8）, giving an expressing plasmid containing the anti-reversense ACC synthase gene.

关 键 词：薄皮甜瓜 反义ACC合成酶基因 特异启动子 植物表达载体 

分 类 号：S652[农业科学—果树学]