Chinese Journal OF Rice Science

Chinese Journal of Rice Science ›› 2013, Vol. 27 ›› Issue (2): 198-202.DOI: 10.3969/j.issn.10017216.2013.02.013

• Short Communications • Previous Articles     Next Articles

Construction and Highefficient Screenings of a Yeast TwoHybrid cDNA Library from the Droughtstressed Roots of Rice

FU Jian 1 , Linkun GU 2, GUO Yiqing 1 , Liyuan ZHANG 2, WANG Linxian 1 , LI Dingqin1, WANG Bo1, Jeff Qingxi SHEN 2,* , CHENG Zaiquan 1,*   

  1. 1 Institue of Biotechnology and Germplasm Resources， Yunnan Academy of Agricultural Sciences, Kunming 650223, China; 2  School of Life Sciences, University of Nevada Las Vegas, NV 89154, USA；
  • Received:2012-07-10 Revised:2012-09-20 Online:2013-03-10 Published:2013-03-10
  • Contact: Jeff Qingxi SHEN2,*, CHENG Zaiquan1,*

干旱胁迫的水稻根高效酵母双杂交体系建立

付坚1,Linkun GU2 ,郭怡卿1,Liyuan ZHANG 2,王玲仙1,李定琴1,王波1 ,Jeff Qingxi SHEN  2,*  ,程在全 1,*   

  1. 1 云南省农业科学院 生物技术与种质资源研究所， 云南 昆明 650223；2 内华达大学 拉斯维加斯校区 生命科学院, 拉斯维加斯 89154, 美国；
  • 通讯作者: Jeff Qingxi SHEN2,*,程在全1,*
  • 基金资助:

    云南省自然科学基金重点项目（2012FA005）； 中央级公益性科研院所基本科研业务专项（PZS00104）； 转基因生物新品种培育重大专项（2011ZX08001002）；云南省高端人才引进项目（2010CI120）。

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Abstract: By using the SMART technique, a yeast twohybrid cDNA library of rice roots was constructed and utilized to study proteinprotein interactions induced by drought. An optimized high efficiency membrane mating method, substituting for liquid mating method, was developed for library screenings. As results, there are about 4.9 ×106 clones in the library with the averaged size of the insert fragments to be about 800 base pairs. To verify the efficiency of the membrane mating method, OsWRKY71, a wellstudied gene encoding a transcriptional repressor of gibberellin signaling, was used as a bait to screen this library.  The mating efficiency was shown to be as high as 6.9%, which was six times higher than that of the liquid mating method. This high efficient mating method is simple, costeffective and suitable for large scale yeast hybrid analyses.

Key words: rice root, drought stress, yeast twohybrid cDNA library, filter membrane, high efficient mating system

摘要： 采用SMART技术在酵母菌株AH109中构建了干旱胁迫下水稻根部全长cDNA文库，采用改进的滤膜杂交方法建立了高效酵母杂交体系，以抗旱相关水稻转录调控因子OsWRKY71基因作为诱饵对该方法进行检验。实验获得的酵母杂交文库容量为4.9×106 ，平均插入片段为800 bp，达到了基因分离和克隆等后续研究的标准。同时采用滤膜杂交法将文库的杂交效率提高到了6.9%，是液体杂交法的6倍以上。该方法不需要特殊的设备，且具有低消耗和高效率的特点，可用于高通量酵母双杂交分析。

关键词: 水稻根部, 干旱胁迫, 酵母双杂交文库, 滤膜, 高效杂交体系

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