GFP/DsRED-TaATG8h融合基因的表达载体构建及在小麦原生质体中的表达

Construction of Expression Vectors for GFP/DsRED-TaATG8h Fusion Genes and Their Expression in Wheat Protoplasts

细胞内自噬水平监测方法的建立是深入揭示自噬调控机制和生理功能的重要基础。ATG8蛋白定位于自噬小体膜上,对其荧光蛋白标记分子的荧光观察是监测细胞内自噬发生水平的重要方法。本研究选择小麦ATG8家族的TaATG8h基因,构建了GFP-TaATG8h和DsRED-TaATG8h融合基因表达载体,载体构建的正确性得到酶切和测序结果的确认;以易于转化的小麦原生质体为受体,实现了两个融合基因在原生质体细胞中的高效表达,在表达融合蛋白的细胞中观察到了清晰的荧光。本研究结果为在重要农作物小麦上建立以ATG8为靶标的自噬水平监测技术和深入探索自噬在小麦生长发育中的作用奠定了基础。

The establishment of autophagy monitoring method was important for revealing the regulation mechanisms and physiological functions of autophagy. ATG8 locates on autophagosome membranes and its fluorescent protein-tagged forms have been successfully used to monitor autophagy level in cells. In this study, TaATG8h, one member of the wheat ATG8 family, was selected and GFP-TaATG8h and DsRED-TaATG8h fusion gene expression vectors were constructed. The correctness of constructed vectors was confirmed by results of restriction enzyme digestion and DNA sequencing. Wheat protoplasts, which were easily transformed with genes,were used to express the two fusion genes, and clear fluorescence was observed in the cells expressing fusion proteins. The results of this study laid a foundation for establishing ATG8-based autophagy monitoring technology and for exploring the role of autophagy in the important crop species of wheat.