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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800bp,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。
The full-length cDNA library of rice roots under drought stress was constructed by using SMART technique in yeast strain AH109. A high efficient yeast hybrid system was established by improved filter-hybridization. The drought-associated rice transcriptional regulator OsWRKY71 gene was used as bait to test the method . The yeast hybridization library obtained in this experiment has a capacity of 4.9 × 106 and an average of 800 bp, which meets the criteria for subsequent studies such as gene isolation and cloning. At the same time, the hybridization efficiency of the library was increased to 6.9% by the filter hybridization method, which was more than 6 times of the liquid hybridization method. This method requires no special equipment and is characterized by its low consumption and high efficiency for high-throughput yeast two-hybrid analysis.