砂梨ACC合酶cDNA全长克隆及其反义与正义表达载体构建

Cloning of full-length cDNA encoding ACC synthase of Pyrus pyrifolia Nakai and construction of its antisense and sense expression vector

为构建干扰砂梨(Pyrus pyrifolia Nakai)ACC合酶基因表达的遗传转化载体,利用RACE技术从'早生新水'成熟果实cDNA中克隆了ACC合酶的cDNA,该cDNA全长为1939bp,命名为Pyp-ACS,GenBank登录号为EF566865。Pyp-ACS核苷酸序列含有5′末端非翻译区109bp、1488bp完整的开放阅读框和3′末端非翻译区342bp(含25bp的Ploy+(A))。Pyp-ACS编码495个氨基酸,与白梨(Pyrus×bretschneideri Rehd.)、西洋梨(Pyrus communisL.)、中国李(Prunus salicina Lindl.)、桃(Prunus persica(L.)Batsch)、梅(Prunus mume Seib.et Zucc.)、苹果(Malus×domestica Borkh.)和柿(Diospyros kaki Thunb.)有较高的同源性。该氨基酸序列具备ACC合酶7个保守区和组成该酶活性中心的12个氨基酸残基,即SLSKDMGFPGLR,进一步验证了克隆的正确性。以pYPX145载体为基础,分别将Pyp-ACS编码区序列反向和正向插入相应位点构建反义和正义表达载体,并分别命名为pPyp-ACS(-)和pPyp-ACS(+),目的基因由双35S启动子所控制。分别将这2个表达载体导入根癌农杆菌菌株(Agrobacterium tumefaciens)EHA105,为耐贮藏转基因梨的遗传转化提供载体。

For the construction of transformation vector to interfere with expression of 1-aminocyclopropane-1-carboxylate（ACC）synthase gene,the full-length cDNA of ACC synthase from ripening fruit of Pyrus pyrifolia Nakai＇Zaosheng Xinshui＇was cloned by RACE（rapid amplification of cDNA ends）,designated as Pyp-ACS,with the full-length 1 939 bp.Pyp-ACS contains 109 bp of 5＇-untranslated region,an open reading frame（ORF）of 1 488 bp nucleotides,and 342 bp nucleotides of the 3＇-untranslated region having 25 bp Ploy＋（A）tail.The sequence was deposited in GenBank database,with accession number EF566865.The deduced amino acid sequence of Pyp-ACS was 495 amino acid residues,which included seven conserved regions and twelve essential amino acid residues,i.e.SLSKDMGFPGLR,which made up active center of ACC synthase.The amino acid sequence of Pyp-ACS was higher identity with those of Pyrus×bretschneideri Rehd.,Pyrus communis L.,Prunus salicina Lindl., Prunus persica（L.）Batsch,Prunus mume Seib.et Zucc.,Malus×domestica Borkh.and Diospyros kaki Thunb..Binary antisense and sense expression vectors of Pyp-ACS coding region,named as pPyp-ACS（-）and pPyp-ACS（＋）,were constructed by inserting the target fragment in pYPX145.The gene expression was controlled by a double 35S promoter.The pPyp-ACS（-/＋）was successfully introduced into Agrobacterium tumefaciens strain EHA105,which was a useful vector for further transformation to develop transgenic pear for fruit with longer shelf life.