【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。更多还原

【Abstract】 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.更多还原