Analysis of signal transduction pathway during seed germination

1.研究了异源三聚体G蛋白α亚基在水稻种子萌发和开花过程中的表达模式。种子萌发后第2 ~ 5天，α亚基的转录量迅速增加，随后在生长过程中逐渐增加。用地高辛标记的RNA探针原位杂交分析表达RGA 1 mRNA的细胞，所述探针以RGA 1 cDNA的部分作为模板制备。在萌发过程中，该基因在根尖、胚囊、韧皮部中有表达，而在根冠和木质部中没有表达。在成熟期的小穗轴、珠心和副果皮中也检测到。这些结果表明，RGA 1可能参与细胞分裂和/或物质转运。2.在大肠杆菌中合成了一个重组蛋白，其cDNA编码G蛋白的α亚基。纯化的重组蛋白（rGricealpha）显示出45 kDa的表观分子量，并且以0.36的表观缔合常数（k_）结合鸟苷5 '-（3-O-硫代）三磷酸<app>。rGricealpha被活化的霍乱毒素ADP-核糖基化。针对rGricealpha的单克隆抗体与位于水稻幼苗质膜中的45 kDa多肽反应。质膜中的45-kDa多肽以及rGricealpha被活化的霍乱毒素ADP-核糖基化。所观察到的性质与动物中异源三聚体G蛋白的α亚基的报道相似，这表明推定的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.