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园艺学报 ›› 2008, Vol. 35 ›› Issue (6): 799-804.

• 果树 • 上一篇    下一篇

砂梨果实ACC氧化酶cDNA克隆及其反义表达载体构建

乔玉山1;宋长年1;渠慎春1;胡钟东1;熊爱生2;姚泉洪2;章 镇1*
  

  1. 1南京农业大学园艺学院,南京210095;2上海市农业科学院生物技术研究所,上海 201106)
  • 收稿日期:2007-12-21 修回日期:2008-05-13 出版日期:2008-06-25 发布日期:2008-06-25
  • 通讯作者: 章 镇

Cloning of cDNA for ACC Oxidase from Fruit of Pyrus pyrifolia Nakai and Construction of Its Corresponding Antisense Expression Vector

QIAO Yu-shan1, SONG Chang-nian1, QU Shen-chun1, HU Zhong-dong1, XIONG Ai-sheng2, YAO Quan-hong2, and ZHANG Zhen1*
  

  1. (1College of Horticulture, Nanjing Agricultural University, Nanjing210095, China; 2Biotechnology Research Institute, Shanghai Academy of Agricultural Sciencse, Shanghai 201106, China)
  • Received:2007-12-21 Revised:2008-05-13 Online:2008-06-25 Published:2008-06-25
  • Contact: ZHANG Zhen

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1 225 bp,将其命名为Pyp-ACO,GenBank登录号为EF451060。Pyp-ACO核苷酸序列有一个945 bp的开放读码框,5'端非翻译区为63 bp,3'端非翻译区为217 bp,与西洋梨和苹果的编码区核苷酸序列有较高的相似性,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。

关键词: 砂梨, ACC氧化酶, cDNA, 反义表达载体

Abstract:

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 was 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.

Key words: Pyrus pyrifolia Nakai, ACC oxidase, cDNA, antisense expression vector

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