拟南芥AtPUB18的亚细胞定位和酶活性及AtPUB18的表达

通过RT-PCR技术从拟南芥中克隆到AtPUB18全长ORF。利用VectorNTI和MEGA 5.0对蛋白序列进行生物信息学分析结果显示,AtPUB18和AtPUB19蛋白序列相似性为74.9%,一致性为63.5%;构建AtPUB18启动子(1 974bp)融合GUS报告基因载体并转化拟南芥,组织化学染色分析显示,低温和干旱诱导后转基因植株中AtPUB18表达水平显著提高;构建AtPUB18与绿色荧光蛋白基因(GFP)融合的瞬时表达载体并转化原生质体,激光共聚焦显微镜观察发现,AtPUB18-GFP融合蛋白分布在细胞核和细胞质中;构建AtPUB18与麦芽糖结合蛋白基因(MBP)融合表达载体并转化大肠杆菌表达融合蛋白,纯化后的蛋白进行泛素连接酶活性检测结果显示,在小麦泛素激活酶E1和人泛素结合酶E2存在时,AtPUB18具有泛素连接酶活性。研究表明,AtPUB18是一个有功能的泛素连接酶,定位在细胞膜和细胞质中,可能参与拟南芥对非生物胁迫的响应。

Subcelluar Localization and Ligase Activity of AtPUB18 and Its Expression Pattern in Arabidopsis thaliana

The full length ORF of AtPUB18 was cloned from Arabidopsis thaliana by RT-PCR.Informatic analysis manifested that AtPUB18 shared similarity of 74.9% and identity of 63.5% with AtPUB19 using VectorNTI and MEGA5.0;A 1 974 bp promoter fragment of AtPUB18 was fused with GUS gene to generate transgenic A.thaliana.The expression level of AtPUB18 was elevated after drought and cold treatment by histochemical GUS assay.AtPUB18 was fused with green fluorescent protein(GFP) gene to generate a transient expression vector.AtPUB18-GFP fusion protein was expressed in A.thaliana protoplasts and observed using confocal microscope.The result showed that AtPUB18-GFP fusion protein distributed in nucleus and cytosol;AtPUB18 was fused with maltose binding protein(MBP) gene to generated AtPUB18-MBP fusion protein in E.coli for ubiquitin ligase activity assay.The result indicated that AtPUB18 had ubiquitin ligase activity with the presence of wheat E1 and human E2.Our studies implied that AtPUB18 was a functional E3 ligase and localized in nucleus and cytosol,and might be involved in the response to abiotic stress in A.thaliana.