|
Author:
Date:
2014-09-20
[Abstract] This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis.
Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005). ...
[摘要] 该快速方案允许从瞬时转化以表达感兴趣的表位标记蛋白的本氏烟草（本氏烟草）叶中提取富含叶绿体的蛋白。因此，当它与Western印迹分析结合时，它可以作为研究目标蛋白质的叶绿体定位的工具。使用土壤杆菌介导的转化（Agroinfiltration，Romeis等人，2001）瞬时表达在烟草叶中携带表位标签的蛋白质。在这里，与土壤传播的小麦花叶病毒携带19K的农杆菌菌株的共浸润抑制转录后基因沉默，并因此增加转化效率（Te等人，2005） 。转化的叶子的叶绿体分离基于Romeis等人（2001）的修改，包括细胞壁和膜的机械破坏，通过过滤去除未破坏的组织，通过Percoll层离心分离完整叶绿体。
|