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Plant Diversity ›› 2014, Vol. 36 ›› Issue (05): 622-628.DOI: 10.7677/ynzwyj201413225

• 研究论文 • 上一篇    下一篇

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

齐靖1, 董祯2, 张玉星3   

  1. 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
  • 收稿日期:2013-11-20 出版日期:2014-09-25 发布日期:2014-02-20
  • 基金资助:

    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)

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

QI Jing-1, DONG Zhen-2, ZHANG Yu-Xing-3   

  1. 1 College of Biology Science and Engineering, Hebei University of Economics and Business, Shijiazhuang 050061, China; 2 Hebei Women′s Vocational College, Shijiazhuang 050091, China; 3 College of Horticulture, Agriculture University of Hebei, Baoding 071001, China
  • Received:2013-11-20 Online:2014-09-25 Published:2014-02-20
  • Supported by:

    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)


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。

关键词: 梨, ACC氧化酶, RT-PCR, 反义表达载体, 遗传转化


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.

Key words: Yali pear, ACC oxidase, RT-PCR, Antisense expression vector, Genetic transformation