遗传 ›› 2008, Vol. 30 ›› Issue (6): 771-775.doi: 10.3724/SP.J.1005.2008.00771

• 研究报告 • 上一篇    下一篇

红莲型水稻杂种F1花药噬菌体展示文库的构建

胡朝凤;彭晓珏;周杨永;谭艳平;李绍清;朱英国   

  1. 武汉大学生命科学学院, 武汉大学植物发育生物学教育部重点实验室, 武汉 430072

  • 收稿日期:2007-11-23 修回日期:2008-01-26 出版日期:2008-06-10 发布日期:2008-06-10
  • 通讯作者: 李绍清

Construction of T7 phage display library from the anther of Honglian hybrid line of rice

HU Chao-Feng;PENG Xiao-Jue;ZHOU Yang-Yong;TAN Yan-Ping;LI Shao-Qing;ZHU Ying-Guo   

  1. Key Laboratory of MOE for Plant Development Biology, College of Life Science, Wuhan University, Wuhan 430072, China
  • Received:2007-11-23 Revised:2008-01-26 Online:2008-06-10 Published:2008-06-10
  • Contact: LI Shao-Qing

摘要:

噬菌体展示是研究蛋白质相互作用的重要手段, 为深入研究红莲型水稻不育和育性恢复的分子机理, 以红莲型水稻杂种F1花药为材料, 分离纯化mRNA, 经反转录合成双链cDNA, 在双链cDNA 末端加上定向EcoRⅠ/HindⅢ接头, 再用EcoRⅠ和HindⅢ消化接头, 形成两端分别带有EcoRⅠ和HindⅢ粘性末端的双链cDNA。经Mini Column 纯化后, 收集300 bp 以上的双链cDNA 片段, 将其连接到带有EcoRⅠ和HindⅢ末端的T7 Select 10-3b 载体上, 经体外包装后, 以BL T5403 为受体菌构建了红莲水稻杂种F1花药的噬菌体展示文库。经测定显示, 该噬菌体展示文库容量为1.03×106 pfu/mL, 重组率为100%, 扩增后文库滴度为2.14×1012 pfu/mL。对随机挑取的100 个噬菌斑进行PCR 鉴定, 97%的插入片段大于300 bp。

关键词: 文库构建, 噬菌体展示, 花药, 水稻

Abstract:

Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRⅠ/HindⅢlinkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoRⅠand HindⅢ, which resulted in ds cDNA with EcoRⅠand HindⅢends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoRⅠand HindⅢends. After packaging in vitro, the T7 Select 10-3b vertor was tansformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03×106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14×1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.