甘藷核醣核酸水解?之生理特性及蛋白質純化分析

Abstract

植物中核醣核酸水解?（ribonucleolytic enzyme; RNase）受外在環境變化而影響其活性，進而調控體內核醣核酸的合成與分解，使植株能抵抗外來因數的改變而生存。本實驗以台農57號甘薯為研究材料，探討甘薯在老化過程及逆境狀況，例如：冷馴化、黑暗處理、缺水、重金屬汞、鎘及荷爾蒙乙烯誘導，和去甲基化藥劑：5-AzaC，ethionine處理，觀察甘薯核醣核酸水解?變化情形。 在正常狀況下A、B、C and D四群RNases於甘藷體內穩定表現，但在老化的植株中group A、B、C、DRNases量減少，卻有一特殊的F2 RNase表現，其分子量約6000 Da左右，且此F2 RNase亦在上述的逆境環境中會被誘導而表現出活性。我們對這一個老化特定表現的核醣核酸水解?在老化及逆境環境過程中所扮演的角色非常感興趣，發現其生化活性方面確實比其他群RNases有不同之處，例如：pH活性範圍從3-9，屬於一廣範圍的RNase，其對必須元素或少量元素的需求亦較其他RNases有更大的離子忍受能力，且SDS、β-mercaptoethanol存在的情況下亦測得到其活性的表現。 在核酸序列水解專一性方面，以一段人工合成的18 base的RNA受質，以此RNA oligomer當作substrate和甘薯的各群RNases作用後，再以電泳分析，在分子層次探討甘薯不同RNases與RNA作用的微觀分析。 利用蛋白質分離系統，以DE52陰離子交換法，及膠體過濾法G75和FPLC的Mono S進一步純化此RNase F，希望能在以後做史進一步探討。 甘薯老化所產生的RNase F，分子量小、活性強卻是前人所沒發現的，然而在其他植物，例如：鏚葉牽牛、馬蹄金、矮牽牛、阿拉伯芥、大豆、日日春、燕麥、水稻的老化葉中依然強烈表現。我們希望借此普遍存在植物老化間的RNaseF作進一步研究，將來能史進一步在植物老化過程中RNA如何被代謝調控上有更深入的探討。

We do the research on the group F RNase of sweet potato. We wish to understand the RNA metabolism during the aging process and stress treatments. The RNase (ribonucleolytic enzyme) activities of plants are affected by the environmental factors. The alteration of RNase activity can control the RNA synthesis and degradation. A serial experiments were carry on sweet potato to observe the alteration of RNase activities under various stress environments and developmental stages including chilling, dark, droughtly, heavy matal ect. In addition, 5-AzaC and ethionine which can demethylate the gene were also included to elucidate the regulation of RNase genes. Four groups: group A, B, C, D RNases express constitutively in sweet potato when it grows in normal condition, but they decrease quickly on the wilting leaves. It is interesting that a vagarious group F RNase, 6 kDa, was specifically induced during the process of withered leaves and various stress condition. We are very interested in the role of group F RNase which shows the activity only in the decrepit organ and stress conditions. The biochemical properties of the F2 RNase are somewhat different from others, e.g. broad pH optiman range, higher tolerance to microelement condition, and activities retaining under SDS and β-mercaptoethanol containing buffer. We synthesize an 18-mer RNA oligomer as a substrate. The 18-mer RNA were digested with sweet potato RNase. We can understand the RNases specific recognition site of RNases and differentiate F2 RNase from the other groups based on the sequence specificity. We purified the F2 RNase by DE 52, G75 and Mono S, wish to get the view on the F2 RNase in detail. The F2 RNase, with a low molecular weight and strong activity, has not been characteryed before. The group F RNase can be detected on many plant aging leaves, e.g. morning glory, Pertunia, Arabidopsis, bean, barly, and rice. The F RNase also shows a strongly activity on the fading leaves of these plants.