甘藷中SUMO基因表現及其訊息傳遞路徑

Abstract

真核生物中蛋白的功能會被一些小的polypeptide，像是ubiquitin（Ub）或是ubiquitin-like（Ubl）proteins等利用共價鍵結的方式所調控，這種調控方式稱做post-translation modification。這些小蛋白會以C端（Gly-Gly motif）並利用isopeptide bond的方式和其目標蛋白上的Lys進行鍵結，而本研究的主角則為此類蛋白中的其中一種，稱做SUMO（small ubiquitin-like modifiers）。此蛋白是我們以二維電泳系統分析經過NO處理的甘藷葉片（Ipomoea batatas cv. Tainong 57）所誘導出來蛋白的其中一個，將這蛋白分離回收後經過比對發現和阿拉伯芥的SUMO2具有較高的性似性，因此我們從其他物種已知序列中設計出Degenerate引子，分別以Inverse PCR (iPCR) 和RACE (Rapid amplification of cDNA ends) 兩種方法找到基因的3’端和5’端，並命名為IbSUMO2。首先從RNA的層次以北方墨點法分析IbSUMO2基因的表現量，發現在分別處理NO（Nitric oxide）、機械性傷害和H2O2的時候，IbSUMO2基因的表現量都會增加；另外在處理NO和H2O2的抑制劑之後再進行機械性傷害，則發現IbSUMO2的基因表現量有減少的現象發生，可以推測在機械性傷害之後IbSUMO2 mRNA的產生是經由NO和H2O2兩條路徑。從內生的IbSUMO2蛋白層次來看，在處理 NO後以西方墨點法對甘藷內生的IbSUMO2蛋白質含量進行分析，結果發現在第四天的時候IbSUMO2蛋白量會增加。另外在IbSUMO2蛋白功能探討方面，在進行傷害十分鐘後，即可發現甘藷內生SUMO的結合態明顯增加，這可以推測SUMO所負責的蛋白修飾作用在植物受到傷害的時候是很早期的一個防禦反應。從免疫沈澱反應中，以傷害24小時的甘藷葉片蛋白和SUMO1的抗體進行沈澱，在45 KDa的地方可以發現兩個明顯的條帶，推測此兩個條帶即為有和IbSUMO結合的蛋白。再來我們也利用洋蔥短暫表現系統來偵測帶有GFP的IbSUMO2在NO刺激下是否有位移的現象出現，不過利用此系統無法發現在NO的處理下，帶有GFP的IbSUMO2有任何的改變發生。從以上結果發現我們可以知道在不同刺激之下，IbSUMO2的確具有其特定的生理功能。

Small ubiquitin-like modifer (SUMO) is a member of the superfamily of ubiquitin-like polypeptides that play a role in post-translational modification, but its functions are different from those of ubiquitin. SUMOs attach target proteins by covalent bond, and alter their function and location. The regulation of SUMO2 expression in sweet potato (Ipomoea batatas) was studied here. We had isolated a nitric oxide (NO)-induced IbSUMO2 protein from two-dimensional electrophoresis system, and also observed a significant increasing of IbSUMO2 mRNA under the treatment of NO or H2O2 induced by mechanical wounding. The increasing of IbSUMO2 protein under NO treatment was also observed by western blotting analysis. On the contrary, the application of NO or H2O2 inhibitors can reduce the expression of IbSUMO2 mRNA induced by mechanical wounding, suggesting IbSUMO2 protein induced by mechanical wounding through NO and H2O2 signal pathway. We also detected the level of IbSUMO2 conjugation under mechanical wounding and NO treatment. The wound-induced conjugate accumulation could be detected within 10 minutes, suggesting that IbSUMO2 conjugation is one of the early events for plant to response to mechanical wounding. On the other hand, immunoprecipitation of IbSUMO2 conjugated proteins were performed with SUMO antibodies, and two major bands around 45 KDa were observed. Furthermore, in transient expression system, we confirm the localization of IbSUMO2 protein within cell under NO stimulations. Conclusively, IbSUMO2 protein conjugated to its target proteins has multiple physiological functions in plant cells.