稻米抗淹水機制蛋白Sub1A-1與ERF67之生物物理與結構特性研究

Abstract

全球暖化與氣候變遷日益加劇，使得全球洪水跟旱災頻繁且難以預期。在這個情勢之下，確保作物能抵抗逆境且穩定地產出是各國糧食安全的重要課題。現今大多水稻在七天淹水後即凋亡，而印度南部卻發現一水稻品種能表達轉錄因子Sub1A-1降低生長與代謝速率，進而能在長達14天缺氧逆境後存活。近期經由基因體分析發現，轉錄因子ERF67受Sub1A-1正向調控，且在缺氧下才得以穩定存在於植體中，為目前發現遵守N-end rule，對調控抗淹水逆境可能有著重要地位。Sub1A-1與 ERF67同屬於第七群乙烯反應因子(Group VII ethylene response factors, ERF-VIIs)，擁有可以辨認GCC box (GCCGCC)的APETALA 2 (AP2) domain。然而稻米基因組中有近10,000 個GCC boxes，ERF67 和 Sub1A-1 的 AP2 domain序列也十分相似。在淹水逆境中，他們如何精準地調控各自的下游還不清楚。除此之外，先前證據指出Sub1A-1的 N-domain對 DNA有很高的親和力。然而這段序列是藉由怎樣的作用力與DNA結合，其機制尚不清楚。為了剖析這個議題，我們從結構角度出發，嘗試培養出ERF67 和 Sub1A-1的protein/DNA複合晶體。為了增加獲得晶體的機率，我們將蛋白結構鬆散的coil去除，並與硫氧還蛋白 (Thioredoxin) 接在一起。藉由偏極化螢光試驗 (Fluorescence Polarization assay) 檢測發現，這些改變並不顯著影響複合體形成。目前我們已經找到某些條件能促成ERF67/DNA與Sub1A-1/DNA形成微小結晶或phase separation。進一步微調這些條件或許能獲得更大、排列更規則的晶體，最終解開他們的分子結構。

Global warming is a serious issue that increases the chances of flooding in Asia. Due to this situation, maintaining stable crop production, especially rice, is challenged. However, there was a rice strain found in south India that could resist up to 14 days submerged. In this rice, a crucial transcription factor, Sub1A-1 was upregulated to decrease the speed of steam elongation and energy consumption. In addition, our recent studies showed that ERF67, a transcription factor that follows the N-end rule of targeted proteolysis, is the direct downstream signaling molecule of Sub1A-1 and might play an important role in oxygen sensing. Sub1A-1 and ERF67 both belong to group VII ethylene response factors (ERF-VIIs). All the ERF-VII family members possess a conserved DNA binding domain, Apetala 2 (AP2) to interact with a cis-regulatory element, the GCC box. However, GCC boxes were found in 1Kb promoter region of 10,000 encoded rice genes. Since ERF67 and Sub1A-1 possess conserved AP2 domain, how the DNA binding specificity is determined still remains unknown. Our previous study showed that Sub1A-1’s N-domain may also interact with DNA. Whether this domain involved in DNA binding specificity remains unknown. To investigate this issue, we rely on protein crystallography to obtain structural information of Sub1A-1/DNA complex. At first, the protein’s coil regions were truncated to increase the conformational uniformity of complex. Besides, thioredoxin, a solubility tag that may increase crystal packing area, was fused with target proteins. Confirmed by the fluorescence polarization assay, these protein constructs for protein crystallization did not apparently influence DNA binding affinity. After screening with more than 75 96-well crystallization trays, protein-rich phase separation or small crystalline were observed in some of the conditions. Future refinement of these conditions may yield well-diffracted crystals to obtain Sub1A-1/DNA complexes at atomic resolution. With structural information, the molecular mechanism of DNA binding specificity will be elucidated.