薄皮甜瓜果实ACC合成酶cDNA克隆及反义表达载体的构建  

Molecular cloning of a muskmelon cDNA fragment encoding an amino-cyclopropane-1-carboxylie acid(ACC) synthase and construction of antisense expression vector

作　　者：郭庆勋[1] 秦智伟[1] 丁国华[1] 李景鹏[2] 

机构地区：[1]东北农业大学园艺学院 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《东北农业大学学报》2006年第1期22-27,共6页Journal of Northeast Agricultural University

基　　金：黑龙江省科技攻关项目(GB01B402-01)

摘　　要：从成熟的薄皮甜瓜(Cucumis melo cv.Qitian I)果肉组织中分离总RNA,经RT-PCR扩增得到0.7 kb的cDNA片段,将其克隆到质粒载体pGEM-T Easy。测序表明,该基因为777 bp,编码258个氨基酸,与Gen-Bank中甜瓜1-氨基环丙烷-1-羧酸合成酶(1-aminocyclopropane-1-carboxylate synthase,ACS)基因比对,核苷酸同源性为99%,氨基酸同源性为98%。克隆片段经双酶切消化,反向插入到植物表达载体pCAM2301的E8启动子和NOS终止子之间,构建成E8调控下ACS基因反义表达载体。通过冻融法将携带反义cDNA的植物表达载体质粒转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。A cDNA fragment of an amino-cyclopropane-l-carboxylie acid （ACC） synthase was amplified by Reverse Transcription Polymerase Chain Reaction （RT-PCR） from sarcocarp tissue of mature nmskmelon （Cucumis melo cv. Qitian I） and cloned into a vector pGEM-T Easy. DNA sequencing showed that the cloned fragment shared highly homogeneity to the cDNAs of ACC synthases from other Cucubitae plants. The cloned eDNA of ACC synthase was further introduced into binary vector pCAM2301 in a reverse orientation with its upstream promoter （E8）, giving an expressing plasmid containing the antisense ACC synthase gene.

关 键 词：薄皮甜瓜 ACC合成酶基因 反义基因 反义表达载体 

分 类 号：S652.2[农业科学—果树学] Q785[农业科学—园艺学]