《植物生理学报》 2012, 48(11): 1050-1056

采用酵母双杂交技术研究OsRUS1与OsRUS2.1之间的相互作用

潘家强¹, 侯学文^1,2,*

华南农业大学生命科学学院1植物逆境生物学研究中心, 2植物功能基因组与生物技术重点实验室, 广州510642

通信作者：侯学文；E-mail: hxw1969@scau.edu.cn；Tel: 020-85287961

摘　要：

为了研究OsRUS1 (ROOT UV-B SENSITIVE 1)与OsRUS2.1 (ROOT UV-B SENSITIVE 2.1)之间是否具有相互作用以 及通过何种结构域相互作用, 采用酵母双杂交技术开展了如下工作。根据对OsRUS1与OsRUS2.1的DUF647结构域分布的 分析, 分别采用分子克隆技术将OsRUS1的4个不同片段[OsRUS1 (1~1 782)、OsRUS1 (1~504)、OsRUS1 (510~1 282)和Os- RUS1 (1 188~1 782)]克隆到诱饵载体pGBKT7上, 并将OsRUS2.1的4个不同片段[OsRUS2.1 (1~1 317)、OsRUS2.1 (1~138)、 OsRUS2.1 (139~879)和OsRUS2.1 (880~1 317)]克隆到猎物载体pGADT7上, 酶切和测序结果表明诱饵和猎物载体构建成功, 读码框正确。将所构建的4个OsRUS1诱饵载体和4个OsRUS2.1猎物载体质粒依次转化酵母AH109, 研究转化AH109菌落在 营养缺陷型培养液SD-Trp-DO或SD-Leu-DO上的生长情况, 以及对报告基因ADE、HIS、MEL1和LacZ的激活情况, 表明它 们均没有对酵母菌株AH109产生毒性和自激活活性。再将4个OsRUS1诱饵载体和4个OsRUS2.1猎物载体的16种组合分别 共转化酵母AH109, 共转化菌株能在二缺营养缺陷板SD-Trp-Leu-DO上生长良好, 但不能在四缺营养缺陷板SD-Trp-Leu- Ade-His-DO上生长, 也不能激活MEL1和LacZ报告基因。这些结果表明OsRUS1与OsRUS2.1之间没有直接的相互作用。

关键词：OsRUS; 酵母双杂交; 相互作用; 自激活

收稿：2012-09-17   修定：2012-10-16

资助：国家自然科学基金(30971709)。

Study of Interaction between OsRUS1 and OsRUS2.1 by Yeast Two-Hybrid Technology

PAN Jia-Qiang¹, HOU Xue-Wen^1,2,*

¹Research Center of Plant Stress Biology, ²Key Laboratory of Plant Functional Genomics and Biotechnology, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China

Corresponding author: HOU Xue-Wen; E-mail: hxw1969@scau.edu.cn; Tel: 020-85287961

Abstract:

Yeast two-hybrid technology was applied to study whether OsRUS1 interacts with OsRUS2.1, and they interact through which domain. The four different length of OsRUS1 fragments [OsRUS1 (1–1 782), OsRUS1 (1–504), OsRUS1 (510–1 282), OsRUS1 (1 188–1 782)] were cloned into bait vector pGBKT7, and four different length of OsRUS2.1 fragments [OsRUS2.1 (1–1 317), OsRUS2.1 (1–138), OsRUS2.1 (139–879), OsRUS2.1 (880–1 317)] were cloned into prey vector pGADT7, according to the distribution of DUF647 domain in OsRUS1 and OsRUS2.1. These constructed vectors were confirmed by enzyme digestion and sequencing. The four OsRUS1 bait vectors and four OsRUS2.1 prey vectors were transformed into yeast strain AH109, respectively. The self-activation and toxicity of the plasmids to host yeast AH109 were analyzed by the expression of ADE, HIS, MEL1 and LacZ reporter genes and by culturing in auxotroph medium SD-Trp-DO or SDLeu-DO. Results showed that the eight constructed vectors had no self-transcriptional activity and not toxicity to yeast strain AH109. Then 16 combinations of four OsRUS1 bait vectors and four OsRUS2.1 prey vectors were cotransformed into yeast strain AH109, respectively. The transformed AH109 could grow well in auxotroph medium SD-Trp-Leu-DO, but could not grow in auxotroph medium SD-Trp-Leu-Ade-His-DO, and both the reporter gene MEL1 and LacZ could not be activated. All these results showed that there was no direct interaction between OsRUS1and OsRUS2.1.

Key words: OsRUS; yeast two-hybrid; interaction; self-activation