水稻RBX1基因cDNA的分离及其过量表达载体对水稻的转化

Isolation of Rice RBX1cDNA and Its Genetic Transformation of RBX1 Over-expression Vector

RBX1蛋白是基于Cullin蛋白的E3复合体中的一个亚基,对于基于Cullin蛋白的E3复合体结合E2及Cullin蛋白的活化具有重要作用.利用RT-PCR分离到水稻RBX1cDNA,序列比对分析显示,水稻RBX1与拟南芥、人、果蝇及酵母中该同源蛋白的氨基酸序列一致性分别为80%、74·4%、72%和51·2%.酵母双杂交实验显示,水稻RBX1与水稻Cullin4蛋白有着相互作用.将水稻RBX1cDNA插入植物表达质粒pHB,利用农杆菌介导法将构建好的植物表达载体导入水稻,获得植株38株.以基因组DNA为模板对潮霉素抗性基因片段进行扩增,初步确定其中32株为转基因植株.

RBX1 protein, a common catalytic subunit of Cullin-based E3 ligases, has a function to bind the ubiquitin-conjuga- ting enzyme E2 and makes it into close proximity with the Cullin-based E3 substrate. In addition to its direct role in Cullinbased E3 ligases activity, RBX1 can promote the RUB/Nedd modification of Cullins and probably has the function as an E3 ligase in the RUB/Nedd pathway. Rice RBXlcDNA was isolated by reverse transcription-PCR, and its sequence alignments showed that the deduced amino acid sequence of rice RBX1 shared 80% , 74.4% , 72% and 51.2% identity with that of its homologues from Arabidopsis, human, Drosophila and yeast, respectively. Also, the yeast two-hybrid test suggested the interaction between rice RBX1 and Cullin4. After the rice RBX1 eDNA was inserted into pHB plasmid and introduced into rice by Agrobacterium-mediated transformation, 38 plants were obtained. After hygromycin gene fragments were amplified by using genomic DNA as template, 32 plants of the 38 were identified to be transgenie.