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eDNA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CaMV35S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 homologybased cloning from Yali （ Pyrus bretsehneideri 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 eDNA. The obtained ACO-like eDNA 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 inserted the ACO-like cDNA fragment into pBI121 expression vector, and transformed it into tissue cultured Yali plants by using Agrobacterium LBA4404. Finally, 4 independent transgenie 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.