鸭梨ＡＣＣ 氧化酶基因ｃＤＮＡ 片段的克隆及农杆菌介导的反义遗传转化

研究根据ACC氧化酶基因的保守序列设计一对特异性引物，以鸭梨果实为试材，借助RT PCR方法扩增得到一条长度为831bp的鸭梨ACC氧化酶基因cDNA片段，该片段编码276个氨基酸残基，与其它梨品种ACC氧化酶基因序列同源性均在94%以上。将此片段反向插入真核表达载体pBI121的CaMV 35S启动子和NOS终止子之间，构建了鸭梨ACC氧化酶基因的反义表达载体，并在农杆菌LBA4404的介导下实现对鸭梨组培苗的遗传转化。经PCR鉴定证实共有4株鸭梨组培苗中外源基因得到成功转化，Southern杂交显示在这4株转基因鸭梨中除有1株外源基因呈双拷贝外，其余3株中外源基因均以单拷贝形式存在。

Cloning of ACC Oxidase Gene from Yali Pear and Tranformation of Its Antisense Expression Vector with Agrobacterium mediated Method

In this study, a partial ACC oxidase (ACO) gene like cDNA sequence was obtained through homology based cloning from Yali (Pyrus bretschneideri cv. ‘Yali’) plant. Primers were designed according to the highly conserved regions of published ACO gene sequences, and RT PCR cloning was conducted by using Yali fruit cDNA. The obtained ACO like cDNA fragment contains 831 base pairs which encodes 276 predicted amino acid residues, and shares no less than 94% nucleotide sequence identity with all published ACO genes. We further inversely inser ted the ACO like cDNA fragment into pBI121 expression vector, and transformed it into tissue cultured Yali plants by using Agrobacterium LBA4404. Finally, 4 independent transgenic lines harboring the anti sense ACO like fragment were obtained and validated by PCR analysis. Southern blotting assay revealed 3 transgenic lines containing single copy of the foreign gene, and 1 line with 2 insertion copies.