Analysis of signal transduction pathway during seed germination

1.研究了一种异源三聚体G蛋白α亚基在水稻种子萌发期和开花期的表达规律。α亚基转录物的数量在萌发后第2 ~ 5天迅速增加，随后在生长过程中逐渐增加。以部分RGA1 cDNA为模板制备地高辛标记RNA探针，原位杂交分析表达RGA1 mRNA的细胞。在萌发过程中，根尖、鳞茎、韧皮部中检测到mRNA，而根冠和木质部中检测不到mRNA。在油菜期小穗、珠心和果皮中均有检测到。这些结果提示RGA1可能参与细胞分裂和/或物质运输。在大肠杆菌中合成了一种具有编码G蛋白α亚基cDNA的重组蛋白。纯化后的重组蛋白rGricealpha的表观分子质量为45 kDa，结合鸟苷5′-（3- o -硫）三磷酸，表观结合常数（k_<app>）为0.36。rgriceα被活化的霍乱毒素adp核基化。抗rGricealpha单克隆抗体与定位于水稻幼苗质膜上的45 kda多肽反应。被激活的霍乱毒素使质膜上一个45 kda的多肽以及rGricealpha被adp核糖基化。观察到的性质与在动物中报道的异三聚体G蛋白的α亚基相似，这表明假定的α亚基确实是α亚基本身。

1.The expression pattern of the putative alpha subunit of a heterotrimeric G protein in rice seeds was investigated during germination and flowering stages. The amount of the transcript for the alpha subunit was observed to increase rapidly from 2nd to 5th day after germination, followed by a gradual increase during growth. The cells expressing the mRNA for RGA1 were analyzed by in situ hybridization with digoxigenin-labeled RNA probes prepared with parts of RGA1 cDNA as the templates. The mRNA was detected in the root tip, scutellum, phloem but not in the root cap and xylem during germination. It was also detected in rachilla, nucellus, and paricape during rapening stage. These results suggest that RGA1 may take part in cell division and/or material transport.2.A recombinant protein with the cDNA that encodes the putative alpha subunit of G protein was synthesized in Escherichia coli. The purified recombinant protein (rGricealpha) showed an apparent molecular mass of 45 kDa and bound guanosine 5'-(3-O-thio) triphosphate with an apparent association constant (k_<app>) of 0.36. The rGricealpha was ADP-ribosylated by activated cholera toxin. Monoclonal antibodies raised against rGricealpha reacted with a 45-kDa polypeptide localized in the plasma membrane of rice seedlings. A 45-kDa polypeptide in the plasma membrane, as well as rGricealpha, was ADP-ribosylated by activated cholera toxin. The properties observed are similar to those reported for the alpha subunits of heterotrimeric G proteins in animals, suggesting that the putative alpha subunit is truly the alpha subunit itself.