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  

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

作　　者：齐靖[1] 董祯[2] 张玉星[3] 

机构地区：[1]河北经贸大学生物科学与工程学院,河北石家庄050061 [2]河北女子职业技术学院,河北石家庄050091 [3]河北农业大学园艺学院,河北保定071001

出　　处：《植物分类与资源学报》2014年第5期622-628,共7页Plant Diversity

基　　金：河北省科技计划资助项目(11220103D-9);河北省高等学校科学技术研究指导项目(Z2012065)

摘　　要：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831 bp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。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.

关 键 词：梨 ACC氧化酶 RT-PCR 反义表达载体 遗传转化 

分 类 号：S661.2[农业科学—果树学] Q943.2[农业科学—园艺学]