阿拉伯芥Di19-2蛋白之功能性研究

Abstract

植物在生長發育中，當環境劇烈改變時，像是乾旱、高鹽和極端溫度，植物會藉由不同的訊息傳遞路徑以適應不同的非生物性逆境。根據前人研究，阿拉伯芥乾旱誘導蛋白AtDi19可能會參與不同的逆境訊息傳遞路徑。目前在阿拉伯芥中共發現七個Di19基因，AtDi19-2 (At1G02750)是其中唯一被預測，不帶有核定位訊號(NLS)和核輸出訊號(NES)的蛋白，而其基因表現會受到高鹽處理所誘導，且不會受到離層酸(ABA)所調控，但目前對於AtDi19-2蛋白的功能還是未知的。本實驗室之前的研究，利用雙重性親合純化(tandem affinity purification)和pull-down分析，發現AtDi19-2會跟14-3-3蛋白和NUTCRACKER (NUC)轉錄因子分別有蛋白質和蛋白質的分子間交互作用。此外，利用雙分子互補螢光系統(bimolecular fluorescence complementation)和石英晶體微天平(quartz crystal microbalance)的方法來驗證AtDi19-2跟14-3-3 omega和NUC之間的交互作用。結果顯示AtDi19-2會在細胞質和細胞核中表現，除了跟14-3-3 omega在細胞質和細胞核內有交互作用外，也和NUC在細胞核內有交互作用。而且，di19-2 di19-5雙重突變株在高鹽處理下種子發芽率會比野生型高，有著對高鹽逆境比較不敏感的外表型。這些結果顯示AtDi19-2或AtDi19-5可能在高鹽逆境反應中扮演重要角色。

Plants as sessile organisms must respond to changes in environmental conditions, such as various environmental stresses including too little water (drought), too much salt (salinity), and extreme temperature. The family of drought-inducible 19 (Di19) contain seven member with unknown functions in Arabidopsis. The AtDi19 family contains two atypical Cys/His zinc finger-like domains which may participate in different stress response and signaling pathways. AtDi19-2 (At1G02750) transcript level increased in response to high-salt stress, but its protein expression pattern and functions remain unknown. Previous study suggested that AtDi19-2 can interact with 14-3-3 proteins and NUC/AtIDD8 by use of tandem affinity purification and pull-down assay. Here, I use Bimolecular fluorescence complementation (BiFC) assays and quartz crystal microbalance (QCM), and confirmed that AtDi19-2 can interact with 14-3-3 proteins in nucleus and cytosol, and interact with NUC in the nucleus. In addition, I found that di19-2 di19-5 double mutant have higher germination rate than wild-type under condition of salt stress. This suggested that AtDi19-2 or AtDi19-5 may play important role in salt stress response.