上海大学学报(自然科学版) ›› 2019, Vol. 25 ›› Issue (4): 604-611.doi: 10.12066/j.issn.1007-2861.1949

• 研究论文 • 上一篇    下一篇

水蜜桃E3泛素连接酶PpARI1基因的克隆及表达载体构建

苑婕1, 马方玮1, 李梦云1, 曹庆1, 郑伟尉2, 万嗣宝1()   

  1. 1. 上海大学 生命科学学院 上海市能源作物育种及应用重点实验室, 上海 200444
    2. 浙江农业大学 农业与食品科学学院 浙江省农产品品质改良技术研究重点实验室, 杭州 311300
  • 收稿日期:2017-05-31 出版日期:2019-08-30 发布日期:2019-09-04
  • 通讯作者: 万嗣宝 E-mail:wansibao@126.com

Cloning and expression vector construction of E3 ubiquitin ligase PpARI1 gene from juicy peaches

Jie YUAN1, Fangwei MA1, Mengyun LI1, Qing CAO1, Weiwei ZHENG2, Sibao WAN1()   

  1. 1. Shanghai Key Laboratory of Bio-energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China
    2. The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, College of Agriculture and Food Science, Zhejiang A & F University, Hangzhou 311300, China
  • Received:2017-05-31 Online:2019-08-30 Published:2019-09-04
  • Contact: Sibao WAN E-mail:wansibao@126.com

摘要:

以新鲜水蜜桃为材料, 根据NCBI中预测的桃E3泛素连接酶ARI1基因序列设计引物, 通过聚合酶链式反应(polymerase chain reaction, PCR)克隆该序列. 利用双酶切定向连接的方法将PpARI1基因开放阅读框(open reading frame, ORF)正向重组到表达载体pCAMBIAy1300的XbaⅠ和BamHⅠ酶切位点之间, 并用热激法转化到农杆菌感受态细胞中. 成功克隆出的长度为1 764 bp的PpARI1基因CDS序列, 与预测基因序列的一致性高达99.83${\%}$. 对重组子测序结果表明, PpARI1基因ORF准确插入pCAMBIAy1300载体的启动子和终止子之间, 表明载体构建成功. PCR检测结果也表明, 该重组载体已成功转入农杆菌中. 这为下一步更深入地研究水蜜桃PpARI1基因的功能奠定了实验基础.

关键词: 水蜜桃, E3泛素连接酶, 克隆, 表达载体构建

Abstract:

Fresh juicy peaches were used as materials, and a novel sequence of E3 ubiquitin ligase PpARI1 was obtained using a polymerase chain reaction (PCR) method with the primers designed according to the predicted gene sequence of ARI1 in NCBI. The open reading frame (ORF) box of PpARI1 gene was forward recombined into pCAMBIAy1300, an efficient expression vector, between Xba I site and BamH I site. The sequencing results showed that the obtained PpARI1 was 1 764 bp with 99.83${\%}$ identity to that of the predicted gene sequence, and the ORF of PpARI1 gene was correctly inserted between the promoter and the terminator. The recombined plasmid was transformed to competent Agrobacterium cells with a heat shock method. The PCR result showed that the vector pCAMBIAy1300-PpARI1 was successfully transferred into Agrobacterium. The results would lay a foundation for further function researches of PpARI1 gene in juicy peaches.

Key words: juicy peach, E3 ubiquitin ligase, cloning, expression vector construction

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