對稻米抗淹水機制蛋白Sub1A-1之生物物理特性研究

Abstract

由於全球暖化，近年來氣候變遷導致的洪水與旱災比過往更加頻繁並且對全球糧食安全產量造成了嚴重的威脅。稻米餵養世界將近一半的人口，因此保護水稻免於受到天災成為一項重要的課題。多數種植的水稻品種遇到完全滅頂的情況下皆活不過一個禮拜但某些稻米種卻擁有絕佳對抗洪水的能力。藉由基因體的分析，目前已經了解SUBMERGENCE(Sub1)這個位點水稻具有抗淹水能力而其產物為一關鍵的轉錄因子Sub1A-1。然而目前對於Sub1A-1如何活化其他下游基因等分子機制仍未了解。Sub1A-1屬於第七群Ethylene Responsive factor (ERFVII) 的一員，這群蛋白具有一AP2 domain 結合GCC box (AGCCGCC) 。但水稻的基因中具有超過3000個以上的基因其啟動子區域具有GCC box。以傳統基因的方式一一篩選幾乎是不可能的而且也缺乏細胞能及的Submergence assay來檢測。因此我們想出了利用Fluorescence Polarization (FP) assay去減少可能的DNA序列。藉由FP assay及protein truncation 我們發現了AP2 domain本身可能並不能提供完整的DNA結合能力，必須要有N-terminal domain一起結合DNA。這項發現可增加我們對於ERFVII家族蛋白的了解。同時了解這些資訊也可為將Sub1A-1與DNA共結晶鋪路。目前我們已經發現某些條件下能形成小微晶。微調這些條件也許能解出結構，為我們的發現提供切實的證據。

Due to the global warming, recent climate changes result more frequent flood and drought than the pass and becomes a serious threat to the global food security. Rice feeding over half of the population in the world and it is an emerging issue to protect rice production from weather damage. Most domesticated rice cultivars die within a week of complete submergence (flooding). However, some wild rice has a remarkable ability to withstand flood. By Genome-wide studies, it is shown that SUBMERGENCE 1 (SUB1) locus is response for the submergence resistant and the transcription factor, Sub1A-1 coded in SUB1 locus is the key factor. However, the molecular mechanism of Sub1A-1 remains unknown. Sub1A-1 is a member of group VII ethylene responsive factors (ERFVIIs) which contain an AP2 domain for GCC box (AGCCGCC) binding. More than 3,000 genes in rice genome have GCC box in their promoter region. It is virtually impossible to screen them by genetic approaches. Not to mention that there is no cell-based submergence assay. In order to narrow down its downstream targets, we have developed a binding assay by Fluorescence Polarization (FP). In addition, we have discovered that AP2 domain itself may not provide sufficient binding ability. Only with the N-terminal domain could AP2 domain of Sub1A-1 bind to DNA. This new finding could enlarge our knowledge about ERFVII family. Currently, our knowledge about ERFVII family is limited to containing one AP2 domain for DNA binding. Moreover, knowing more about Sub1A-1 protein can facilitate our Sub1A-1 and DNA co-crystallization work. Currently, we have found out several conditions which could form small crystallite. Refining these conditions may solve the 3D-structure of Sub1A-1/DNA complex which could provide solid evidence for our new finding.