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。
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 SHEN2,*,程在全1,*
. 干旱胁迫的水稻根高效酵母双杂交体系建立[J]. 中国水稻科学, 2013
, 27(2)
: 198
-202
.
DOI: 10.3969/j.issn.10017216.2013.02.013
By using the SMART technique, a yeast twohybrid cDNA library of rice roots was constructed and utilized to study proteinprotein 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 wellstudied 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, costeffective and suitable for large scale yeast hybrid analyses.
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