Universität Tübingen, ZMBP, Plant Physiology, Auf der Morgenstelle 1, D-72076, Tübingen, Germany.
Plant Methods. 2012 Jul 12;8(1):25. doi: 10.1186/1746-4811-8-25.
Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed.
理解蛋白质和基因的功能需要使用生化、分子或遗传工具来鉴定相互作用的伙伴。在植物中，寻找新的蛋白质-蛋白质相互作用仅限于蛋白质纯化测定、异源体内系统（如酵母双杂交或突变筛选）。理想情况下，人们应该能够在活的植物细胞中搜索新的蛋白质伙伴。我们证明，从原生质体化的拟南芥植物细胞中的随机文库中筛选新的蛋白质-蛋白质相互作用是可行的，并回收了一些相互作用的伙伴。我们的筛选基于用 FACS 捕捉 YN-bait 融合伙伴和完全随机的 prey YC-cDNA 文库之间 mYFP 的双分子互补。候选相互作用使用体内 BiFC 测定和体内 FRET-FLIM 测定进行了验证。通过这项工作，我们表明，经过充分表征的蛋白钙依赖性蛋白激酶 3（CPK3）与 APX3、HMGB5、ORP2A 和含有 ricin B 相关凝集素结构域的蛋白 At2g39050 相互作用。这是首次成功应用的随机体内筛选之一。
