甘藷 IbSUMO 結合蛋白之功能分析

Abstract

蛋白質的轉譯後修飾作用在調節蛋白質功能上扮演非常重要的角色，small ubiquitin-related modifier（SUMO）為其中一種可修飾蛋白質的小分子蛋白質。SUMO 藉由共價類胜肽鍵（isopeptide bond）的方式與目標蛋白質結合，進而調控目標蛋白質的活性、穩定性或於細胞內的位置，此一過程稱為 sumoylation。SUMO 與 ubiquitin 具有相似的蛋白質結構，並且 sumoylation與 ubiquitination具有相似的作用步驟。目前研究中已經知道 sumoylation 可以調控植物的生長發育及對逆境的反應等。在實驗室先前的研究中，藉由二維電泳分離甘藷葉片（Ipomoea batatas cv. Tainung 57）處理一氧化氮（nitric oxide，NO）前後的差異性蛋白，經由液相層析質譜儀（LC/MS/MS）分析發現一個和阿拉伯芥 SUMO2 胺基酸序列具高相似度之蛋白質，將之命名為 IbSUMO2。接著建構出大量表現 GFP-IbSUMO2 融合蛋白之阿拉伯芥，在處理過氧化氫（Hydrogen peroxide，H2O2） 後利用免疫沉澱（immunoprecipitation，IP）分離在 H2O2 逆境下可能與 IbSUMO2 結合之蛋白質進行，再利用 LC/MS/MS 分析比對，釣取出四個可能與 IbSUMO2 結合之蛋白質，分別為luminal-binding protein 1（BiP1）、heat shock cognate 70 kDa protein（HSC70-1）、putative F-Box protein（PFP）以及 Patellin 1（PATL1）。本篇實驗中，我們分析了這四個目標蛋白基因被 T-DNA 突變的阿拉伯芥種子，篩選 homozygous lines 進行處理H2O2後相關生理性狀分析，從結果得知 bip1、hsc70-1 與 WT 相較，在 H2O2 處理後在根部延長上並無顯著差異。另外使用 bimolecular fluorescence complementation（BiFC）證明這四個蛋白質是否能與 IbSUMO2 結合，可以發現只有 HSC70-1 與 IbSUMO2 在細胞核外圍結合。將 HSC70-1 於資料庫中比對後得到甘藷具有一相似蛋白質 IbHSP70，利用 BiFC 可觀察到 IbSUMO2 會與 IbHSP70 結合，作用位置遍布在細胞核與細胞質。另外為了確認IbSUMO2在阿拉伯芥系統中是否可代替AtSUMO1及AtSUMO2，並且研究IbSUMO2在阿拉伯芥中的功能，我們期望可構築出背景為atsumo1/atsumo1/atsumo2/atsumo2且大量表現GFP-IbSUMO2的阿拉伯芥轉殖株，不過經由大量篩選後仍然挑取不到預想中之轉殖株，推測IbSUMO2無法恢復atsumo1/atsumo1/atsumo2/atsumo2造成胚胎致死性的性狀。未來進一步的研究可以利用 in vitro及 in vivo 的方式更加證明目標蛋白質會被 sumoylation；也可建立大量表現 GFP-IbSUMO2 之甘藷，處理 H2O2 後釣取甘藷中會與 IbSUMO2 結合之蛋白質。

The post-translational modification of proteins by SUMO is important for plant growth, development, and stress responses. In the previous study, the leaves of sweet potato (Ipomoea batatas cv. Tainung 57) were treated with nitric oxide (NO), and the NO-induced proteins was analyzed by 2D electrophoresis. IbSUMO2 identified by LC/MS/MS is one of the NO-induced proteins. Immuneprecipitation and 2D electrophoresis were further used to isolate the putative proteins modified by IbSUMO2 in Arabidopsis treated by hydrogen peroxide (H2O2). After identified these proteins by LC/MS/MS, four candidate proteins, luminal-binding protein 1 (BiP1), heat shock cognate 70 kDa protein (HSC70-1), putative F-Box protein (PFP) and Patellin 1 (PATL1)1, might be sumoylated by IbSUMO2. The T-DNA insertion mutant lines of BiP1, HSC70-1, PFP, and PATL1 were obtained by ABRC. The physiological properties of their homozygous lines were studied. Results indicated that the root lengths of the homozygous bip1 and hsc70-1 were similar to those of wild type. Via BiFC, HSC70-1 interacted with IbSUMO2 around nuclei, while other proteins showed no interaction with IbSUMO2. To confirm IbSUMO2 can complement the function of AtSUMO1/2 in Arabidosis, I also attempted to construct a transgenic which is atsumo1/2 and overexpressing GFP-IbSUMO2. However, the expected transgenic Arabidopsis can not be obtained due to the uncomplement of IbSUMO2 in atsumo1/2 genetic backgroung. For further study, in vitro and in vivo approaches should be used to confirm that target proteins will be sumoylated by IbSUMO2. A sweet potato that overexpressing GFP-IbSUMO2 should be constructed so that the IbSUMO2-interacted proteins would be study in sweet potato.