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当前位置: 首页 > 文章 > 砂梨果实ACC氧化酶cDNA克隆及其反义表达载体构建 园艺学报 2008 (6) 799-804
Position: Home > Articles > Cloning of cDNA for ACC Oxidase from Fruit of Pyrus pyrifolia Nakai and Construction of Its Corresponding Antisense Expression Vector Acta Horticulturae Sinica 2008 (6) 799-804

砂梨果实ACC氧化酶cDNA克隆及其反义表达载体构建

作  者：
乔玉山;宋长年;渠慎春;胡钟东;熊爱生;姚泉洪;章镇
单  位：
南京农业大学园艺学院;上海市农业科学院生物技术研究所
关键词：
砂梨;ACC氧化酶;cDNA;克隆;反义表达载体
摘  要：
利用RT-PCR和RACE技术从‘早生新水’砂梨成熟果实cDNA中克隆出ACC氧化酶基因保守片段及其5′和3′端,拼接后获得砂梨果实ACC氧化酶cDNA全长。该cDNA全长1225bp,将其命名为Pyp-ACO,GenBank登录号为EF451060。Pyp-ACO核苷酸序列有一个945bp的开放读码框,5′端非翻译区为63bp,3′端非翻译区为217bp,与西洋梨和苹果的编码区核苷酸序列有较高的同源性,分别为98.3%和98.1%。Pyp-ACO推导编码314个氨基酸,该序列具备非血红素二价铁离子依赖型的氧化/加氧酶类的12个保守氨基酸和催化活性所需的3个氨基酸。将Pyp-ACO编码区序列反向插入pYPX145载体,构建了由双35S启动子所控制的双元表达载体,同时已成功将表达载体导入根癌农杆菌菌株LBA4404,为耐贮藏转基因梨选育奠定了基础。
译  名：
Cloning of cDNA for ACC Oxidase from Fruit of Pyrus pyrifolia Nakai and Construction of Its Corresponding Antisense Expression Vector
作  者：
QIAO Yu-shan1,SONG Chang-nian1,QU Shen-chun1,HU Zhong-dong1,XIONG Ai-sheng2,YAO Quan-hong2,and ZHANG Zhen1(1College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;2Biotechnology Research Institute,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China)
关键词：
Pyrus pyrifolia Nakai;ACC oxidase;cDNA;clone;antisense expression vector
摘  要：
A conserved fragment of ACC oxidase cDNA,and its corresponding 5'-and 3'-end sequences were successfully obtained from sand pear(Pyrus pyrifolia Nakai 'Zaosheng Xinshui')by RT-PCR and RACE.They were spliced into a 1 225 bp full length cDNA which designated as Pyp-ACO.Its open reading frame is 945 nucleotides in length,and its upstream and downstream are 63 bp of 5'-UTR and 217 bp of 3'-UTR.The sequence was deposited in GenBank database,accession number EF451060.The nucleic acid of Pyp-ACO shares 98.3% and 98.1% sequence identity with those of P.communis L.and Malus domestica Borkh.The deduced amino acid sequence of Pyp-ACO is 314 residues and contains twelve conserved residues belonging to non-heme iron(Ⅱ)dependent family of oxygenases/oxidases and three residues essential for emzyme activation.A binary antisense expression vector with Pyp-ACO coding region was constructed by inserting the target fragment in reverse orientation in pYPX145.The expression of the gene is controlled by a double 35S promoter.The vector was successfully introduced into Agrobacterium tumefaciens strain LBA4404 for further transformation to develop transgenic pear for fruit with longer shelf life.

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