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c DNA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p GBKT7,构建重组诱饵载体p GBKT7-OsRPK1-OD。[结果]对pGBKT7-OsRPK1-OD进行毒性检测和转录自激活鉴定,酵母生长结果发现,含有pGBKT7-OsRPK1-OD载体的Y2H Gold细胞,在SD/-Trp、SD/-Trp/X-α-gal营养缺陷型固体培养基上生长出白色菌落,在SD/-Trp/X-α-gal/Ab A固体培养基上生长受到抑制;在SD/-Trp生长出含有pGBKT7-OsRPK1-OD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OsRPK1-OD对酵母生长无毒性作用。[结论]OsRPK1-OD无法参与激活转录酵母中报告基因HIS3,ADE2,MEL1和AUR1-C,该片段可以作为诱饵基因筛查水稻c DNA文库,寻找与之相互作用的蛋白质。
[ Objective ] The aim was to construct leucine rich repeat receptor protein kinase OsRPK1 extraceUuar domains＇ s coding gene as yeast two hybrid bait vector of Oryza sativa L. [ Method ] Using Oryza sativa L. as research material, OsRPK1-OD, extracelluar domains＇ s coding gene of a leucine rich repeat receptor protein kinase OsRPK1 was amplified byPCR with rice roots cDNA as template （969 bp）. The fragment was in- serted into bait vector pGBKT7 which was used for yeast two hybrid to construct recombinant bait vector pGBKTT-OsRPK1-OD sucessfully, pG- BKT7-OsRPK1-OD was transformed into Y2H Gold competence cells to do toxic test and autoaetivition identification. [ Result ] The results of strain Y2H Gold showed Y2H Gold cells containing pGBKTT-OsRPKI-OD could grow up to the white colonies on SD/-Trp, SD/-Trp/X-α-gal plates, and grown inhibited on SD/-Trp/X-α-gal/AbA plates. The colonies of Y2H Gold containing pGBKT7-OsRPKI-OD showed the same size with the colonies of Y2H Gold containing pGBKTT, which meant the inserted OsRPK1-OD had no toxic effect on yeast growing. [ Conclusion] OsRPK1-OD couldn＇t activate the transcription of report genes HIS3 ,ADE2 ,MEL1 and AUR1-C in yeast two hybrid system. It would make the OsRPK1-OD become the bait gene to screen the rice cDNA library and find the interaction protein.
出处
《安徽农业科学》
CAS
2015年第31期45-46,144,共3页
Journal of Anhui Agricultural Sciences
