朱晓南

摘要[目的]寻找水稻LRR型蛋白受体激酶（Leucine rich repeat receptor protein kinase）OsRPK1胞外结构域的互作蛋白。 [方法]以日本晴水稻为研究材料，以水稻根部cDNA为模板通过PCR扩增方法得到OsRPK1胞外结构域基因编码片段OsRPK1-OD（969 bp）。将该片段插入酵母双杂交诱饵载体pGBKT7，构建重组诱饵载体pGBKT7OsRPK1OD。[结果]对pGBKT7-OsRPK1OD进行毒性检测和转录自激活鉴定，酵母生长结果发现，含有pGBKT7OsRPK1OD载体的 Y2H Gold细胞，在SD/Trp、SD/Trp/Xαgal营养缺陷型固体培养基上生长出白色菌落，在SD/Trp/Xαgal/AbA固体培养基上生长受到抑制；在SD/Trp生长出含有pGBKT7OsRPK1OD载体的 Y2H Gold酵母菌落大小与转化了空载的酵母菌落大小一致，显示插入的OsRPK1OD对酵母生长无毒性作用。[结论]OsRPK1OD无法参与激活转录酵母中报告基因HIS3，ADE2，MEL1和AUR1C，该片段可以作为诱饵基因筛查水稻cDNA文库，寻找与之相互作用的蛋白质。

关键词水稻受体激酶；OsRPK；酵母双杂交；诱饵载体

中图分类号S188文献标识码

A文章编号0517-6611（2015）31-045-02

Construction on Leucine Rich Repeat Receptor Protein Kinase OsRPK1 Extracelluar Domainss Coding Gene as Yeast Two Hybrid Bait Vector of Oryza sativa L.

ZHU Xiaonan

（Jiangnan University Bioengineering academy，Wuxi， Jiangsu 214122）

Abstract[Objective] The aim was to construct leucine rich repeat receptor protein kinase OsRPK1 extracelluar domainss coding gene as yeast two hybrid bait vector of Oryza sativa L.[Method] Using Oryza sativa L. as research material， OsRPK1OD， extracelluar domainss coding gene of a leucine rich repeat receptor protein kinase OsRPK1 was amplified by PCR with rice roots cDNA as template （969 bp）.The fragment was inserted into bait vector pGBKT7 which was used for yeast two hybrid to construct recombinant bait vector pGBKT7OsRPK1OD sucessfully. pGBKT7OsRPK1OD was transformed into Y2H Gold competence cells to do toxic test and autoactivition identification. [Result]The results of strain Y2H Gold showed Y2H Gold cells containing pGBKT7OsRPK1OD could grow up to the white colonies on SD/Trp， SD/Trp/Xαgal plates， and grown inhibited on SD/Trp/Xαgal/AbA plates. The colonies of Y2H Gold containing pGBKT7OsRPK1OD showed the same size with the colonies of Y2H Gold containing pGBKT7， which meant the inserted OsRPK1OD had no toxic effect on yeast growing. [Conclusion] OsRPK1OD couldnt activate the transcription of report genes HIS3，ADE2，MEL1 and AUR1C in yeast two hybrid system. It would make the OsRPK1OD become the bait gene to screen the rice cDNA library and find the interaction protein.

Key words Rice receptor protein kinase； OsRPK；Yeast two hybrid； Bait vector

植物LRR型受體蛋白激酶是数目最庞大的激酶家族之一[1]，广泛参与植物生长发育、响应外界环境变化等途径。植物中对拟南芥油菜素甾醇类（brassinosteroid，BR）信号通路研究较为全面。BR是拟南芥产生的一类特殊活性成分[2]，对植物的多种生理功能产生作用，主要影响茎的伸长、花粉管生长、木质部分化和叶片卷曲等，且在多种植物中对其功能进行了验证[3]。拟南芥BRI1是第一个分离出的BR受体[4]，其含有25个LRR结构域；BR另一受体BAK1[5]仅含有5个LRR结构域，同属于LRRRLK家族[6]；此外，植物BIN2蛋白表达量在BR存在时表现降低。当植物受到BR诱导刺激时， BRI1与BAK1在细胞膜上形成BRI1BAK1异源二聚体[7-9]，抑制BIN2表达，核内相关酶类去磷酸化并大量积累从而调控BR相关基因的表达[10]。……