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。
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.
出处
《园艺学报》
CAS
CSCD
北大核心
2008年第6期799-804,共6页
Acta Horticulturae Sinica
基金
江苏省高技术研究项目(BG2006313)
国家高技术研究与发展计划重点项目(2006AA100108)
关键词
砂梨
ACC氧化酶
CDNA
克隆
反义表达载体
Pyrus pyrifolia Nakai
ACC oxidase
cDNA
clone
antisense expression vector
