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