RSSG58基因在水稻精细胞中的表达

RSSG58 Gene Expression in Rice Sperm Cells

RSSG5 8是利用抑制差减杂交技术从水稻精细胞文库中筛选到的在精细胞中优势表达的基因 ,推测其编码的蛋白质与拟南芥的肌球重链蛋白有一定的同源性 (4 6 % ) ,并具有肌球蛋白特色的结构域。把RSSG5 8基因开放编码框连接到表达载体pQE30上 ,重组质粒在E .coliM15中表达出N端融合了 6×His的融合蛋白。SDS PAGE分析表明 ,表达产物的分子量约为 6 6kD ,其表达量占菌体总蛋白的 8.6 %。分离纯化融合蛋白来免疫家兔 ,制得了高效价、高特异性的多克隆抗体。Western杂交显示 ,在分离的精细胞内该基因编码的蛋白表达量很高 ,而成熟花粉和二细胞中只有微弱表达 ,单细胞花粉、花粉母细胞没有杂交信号 ,表明RSSG5 8基因在精细胞中优势表达。

RSSG58 gene, had been cloned from the cDNA library of rice ( Oryza sativa L.) sperm cells by SSH using dominantly expressed clone as probe in subtractive clone. The protein encoded by the open reading frames showed 46% homology to the myosin heavy chain protein of Arabidopsis thaliana (Fig.1). In order to study its function in development of sperm cells, the coding region of RSSG58 cDNA was cloned into expression vector pQE30. After induction with IPTG, RSSG58 gene protein was expressed in E.coli strain M15(pREP4) consisting of 6×His fused to the N terminal. The protein product accounted for 8.6% of the total bacterial protein. SDS PAGE analysis confirmed that the molecular weight of recombinant protein was 66 kD (Fig.3A). The expression product was purified by Ni NTA resins to a higher than 90% purity. The purified recombinant protein was used to immunize rabbits get antiserum. Western blotting showed that the strong hybridization signal was detected in the sperm cell of pollen. A weak signal was detected at mature pollen and bicellular pollen, no detectable signal was observed at microsporocyte and microspore stages (Fig.4B). These results suggest that the RSSG58 encoded protein is abundantly expressed in rice sperm cells. The next goal is understanding the function of the gene in the process of fertilization of higher plants.