Osrab5A, a rab5A gene cloned from Rice (Oryza sativa L.), encodes a putative Rab protein with high similarity to mammalian Rab5A, which was localized on the early endosome and in the cytoplast. Rab5A was identified in mammalian cells as a rate-limiting factor for homotypic endosome fusion. The complete coding sequences of Osrab5A cDNA clone were amplified and inserted into the prokaryotic expression vector pGEX4T1. The resulting recombinant plasmid with correct reading frame, pG-5AE, was introduced into E.coli. BL21 (DE3),The GST tagged fusion protein, GST-OsRab5A was induced by IPTG induction and purified on a GSTrapTM column. The GTP-binding assay of this protein showed the GST tagged OsRab5A protein has GTP-binding ability in vitro.

Small GTP-binding proteins (SGPs) are involved in regulating intracellular ve sicular transport by activation of vesicle formation and fusion of inner membran e. By means of screening a rice panicle cDNA library with one map position known genomic BAC clone as probe, an EST encoding a fragment of a SGP was obtained. B ased on the sequence of this EST, the electronic cloning was performed with anot her 3 ESTs. According to the result, the primers were designed and a cDNA clone was got, named Osrab5B, by means of RT-PCR. Osrab5B contains 1016 bp an d shows high similarity to the rab5B genes from Mesembryanthemum crytallinum and L otus j aponicus, all of which belong to a new subfamily of rab family. Further more, ne w primers were designed and the genomic sequence of Osrab5B was obtained. Ou r analysis indicates that it includes at least 7 introns and 8 exons.

The suppressive subtraction hybridization (SSH) technique was used to isolate the cDNA fragments,which showed differential expression between the rice mutant dwarf69 and wild type strain,TGA-2(Oryza sativa, indica).Two differential expressing cDNA libraries were constructed with the cDNAs of dwarf69 as driver and the cDNAs of TGA-2 as tester, vice versa. These two cDNA libraries indicated the differential expressing cDNA fragments in TGA-2 and dwarf69. By reverse Northern blots, ten positive clones were obtained.

利用抑制性消减杂交(Suppressive subtraction hybridization, SSH)分离本实验室获得的一株水稻矮化突变体dwarf69与其野生型对照品系特光矮_2(Oryza sativa,TGA_2 indica)间表达有差异的cDNA片段.分别以dwarf69作为驱赶子,TGA_2为检测子,以及以dwarf69 作为检测子,TGA_2为驱赶子建立了两个差异表达cDNA文库,分别代表在TGA_2和dwarf69中特异表达的cDNA.经反式Northern检测这两个文库共得到10个阳性克隆.