【摘要】 自噬参与了动植物的生长、发育、衰老和逆境胁迫响应等过程。NBR1是选择性自噬的底物受体之一,其识别泛素化的特定蛋白底物并通过与自噬膜上的ATG8互作引导底物进入自噬降解过程。在前期克隆了两个小麦NBR1基因的基础上,本研究通过常规的分子克隆技术构建了两个基因的原核表达载体并将其转化到大肠杆菌中,利用SDS-PAGE方法鉴定了两个NBR1基因在大肠杆菌中的表达情况。结果表明,导入大肠杆菌的两个小麦NBR1均能够被IPTG诱导表达;表达重组蛋白两端含有His(6)标签,其表观分子量与理论分子量基本一致;重组蛋白在诱导后2 h即表现较高的表达量,并在诱导后4 h达到最高的表达量。研究结果为后续小麦NBR1蛋白的纯化、抗体的制备以及该蛋白的互作性质、表达特征等功能研究工作奠定了基础。更多还原

【Abstract】 Autophagy plays an important role in the growth, development, aging and stress responses of plants and animals. NBR1 functions in selective autophagy as a substrate receptor, which recognizes specific ubiquitinated proteins and presents them to the autophagic degradation process through its interaction with ATG8 on autophagic membranes. Previously, two wheat NBR1 genes were cloned in Tianjin key laboratory of animal and plant resistance. In this study, prokaryotic expression vectors for the two wheat NBR1 s were constructed and transformed into E. coli. Results from SDS-PAGE showed that wheat NBR1 s could express efficiently in E. coli through IPTG induction. The expressed recombinant proteins had N-and C-terminal His(6) tags, and their molecular weights were consistent with the predicted values. High levels of recombinant proteins were detected at as early as 2 h after IPTG induction, and the highest levels were reached at 4 h after IPTG induction. These results laid a foundation for the preparation of recombinant wheat NBR1 proteins and their antibodies for use in assays on the interaction and expression features of wheat NBR1 s.更多还原