文心兰OnAP1-like基因表达载体构建和遗传转化体系初探  

Construction of Expression Vector of OnAP1-like Gene and Exploration of Genetic Transformation System of Oncidium

作　　者：武振江[1] 刘佳[1] 崔波[1,2] 叶永忠[1] 

机构地区：[1]河南农业大学生命科学学院,河南郑州450002 [2]郑州师范学院生物工程研究所,河南郑州450044

出　　处：《河南农业科学》2014年第5期124-129,共6页Journal of Henan Agricultural Sciences

基　　金：河南省重点科技攻关项目(092102110128);郑州市重大科技专项(112PZDZX030)

摘　　要：根据NCBI网站上文心兰OnAP1-like基因序列设计引物,用RT-PCR法从南茜文心兰花葶中扩增OnAP1-like基因,对扩增产物进行测序。结果表明,获得的南茜文心兰OnAP1-like基因为690bp,与报道序列完全一致。将OnAP1-like基因插入pRI101-ON载体中,经PCR、双酶切及测序鉴定,证实重组表达质粒中含有目的片段,表明成功构建了高效植物表达载体pRI101-OnAP1。对OnAP1-like基因的结构域和所表达蛋白质的亲水性进行了分析,并预测了该基因所表达蛋白的三维结构。结果显示,该基因包含MADS-box和K-box保守域,属于MADS-box基因家族,所表达蛋白为亲水性蛋白。确定了一种文心兰的遗传转化条件,得到筛选抗生素G418的最适质量浓度为40mg/L,为进一步研究文心兰中AP1基因的功能奠定基础。The OnAP1-like gene was amplified from the scape of Oncidium ＇Gower Ramsey＇ with primers designed according to the OnAP1-like gene sequence of Oncidium from NCBI website by RT-PCR method.The PCR product was 690 bp which was in fully accord with the sequence published.The OnAP1-like gene was inserted into pRI101-ON vector.The recombinant plasmid pRI101-OnAP1 identified by PCR,double digestion and sequencing was constructed successfully.The multi-domain of the OnAP1-like gene and the hydrophilia of the protein encoded by the OnAP1-like gene were analyzed.The 3D structure of the protein encoded by the OnAP1-like gene was predicted.The results showed that this protein contained the MADS-box and K-box domain,belonging to MADS-box superfamily.The protein encoded by the OnAP1-like gene was a hydrophilic protein.The transformation system of Oncidium ＇Gower Ramsey＇ mediated by Agrobacterium was established,and the optimum concentration of the antibiotic G418 was 40 mg/L for screening transformants.The results provide theoretical foundation for further research of the function of AP1 gene in Oncidium ＇Gower Ramsey＇.

关 键 词：文心兰 载体构建 OnAP1基因 遗传转化体系 

分 类 号：S682.31[农业科学—观赏园艺]