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作物学报 ›› 2011, Vol. 37 ›› Issue (05): 764-771.doi: 10.3724/SP.J.1006.2011.00764

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用磁珠富集马铃薯BAC文库中NBS-LRR类晚疫病抗性基因

徐建飞,金黎平,庞万福,卞春松,段绍光,刘杰,黄三文,屈冬玉*?   

  1. 中国农业科学院蔬菜花卉研究所，北京 10008
  • 收稿日期:2010-11-18 修回日期:2011-03-08 出版日期:2011-05-12 网络出版日期:2011-03-24
  • 通讯作者: 屈冬玉, E-mail: dyqu@mail.caas.net.cn, Tel: 010-82105943
  • 基金资助:

    本研究由国家自然科学基金项目(31000738), 国家科技支撑计划(2007BAD49A01)和“农业部园艺作物遗传改良重点开放实验室”资助。

Isolation of NBS-LRR Late Blight Resistance Genes from Potato BAC Library by System of Magnetic Separation

XU Jian-Fei, JIN Li-Ping, PANG Wan-Fu, BIAN Chun-Song, DUAN Shao-Guang, LIU Jie, HUANG San-Wen, and QU Dong-Yu*   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2010-11-18 Revised:2011-03-08 Published:2011-05-12 Published online:2011-03-24
  • Contact: 屈冬玉, E-mail: dyqu@mail.caas.net.cn, Tel: 010-82105943

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被引次数

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摘要： 栽培马铃薯是高度杂合的四倍体作物，利用传统的基因克隆方式进行晚疫病抗性基因分离难度很大。然而，晚疫病抗性基因具有序列保守性，属于NBS-LRR类基因。本研究中，根据晚疫病抗性基因R3a家族的序列比对结果设计R3a基因家族的保守探针，并将含有R3a基因的BAC SH23G23部分酶切成7~11 kb DNA片段。通过结合保守探针的磁珠系统对上述7~11 kb DNA片段进行R3a基因分离，将磁珠富集的片段克隆到双元载体pBINPLUS上。通过阳性克隆和菌落PCR鉴定表明，含有R3a基因的克隆比率达到82.76%，相对于磁珠系统富集前，提高R3a基因比率近19倍。本研究建立了抗病基因及其同源序列的磁珠分离系统，为分离马铃薯等多倍体作物中具有保守结构的基因提供了实验基础。

关键词: 马铃薯, 晚疫病, 抗性基因, 磁珠富集

Abstract: Cultivated potato (Solanum tuberosum) is an auto-tetraploid crop, which is very difficult to isolate late blight resistance genes. Nevertheless, it is discovered that there is conseved NBS-LRR domain among late blight resistance genes. In this study, conserved probes of R3a late blight resistance genes family were developed by alignment of sequences. And then, the DNA of BAC SH23G23 containingthe R3a gene was partially digested into 7–11 kb fragments. By magnetic separation system combined with conserved probes, the 7–11 kb fragments were enriched and cloned into binary vector pBINPLUS. Through identification of positive clones and colony PCR, the ratio of clones including R3a gene to all positive clones reached 82.76%, which was nearly 19 times higher than enrichment before. The system of magnetic separation for R genes and their analogs established in this study provides a new strategy for conserved domain genes cloning from polyploid crops.

Key words: Potato, Late blight, Resistance genes, Magnetic separation

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