稻米Sub1A-1如何脫逃N-end Rule Pathway之研究

Abstract

極端氣候會造成稻米產量下降，進而危及全球糧食安全，其中乾旱與洪水是兩大主因。依據氣候變遷模型預測，這兩種危害的發生頻率將會上升。帶有屬於第七群乙烯反應因子Sub1A-1基因的稻米品種，能夠於完全淹水的環境下，撐過超過兩週的時間。Sub1A-1除了能抵抗淹水逆境外，還能在稻米不缺氧的情況下，調控其抗旱、抗長期黑暗與抗水退的反應，這要歸功於Sub1A-1並不適用於N-end rule pathway的特性。N-end rule pathway屬於泛素-蛋白酶體系統(UPS)中的一部份，具有偵測植物體內平衡的功能，以及調控包含第七群乙烯反應因子在內，會受缺氧誘發的轉錄因子。有趣的是，Sub1A-1是唯一不會被N-end rule pathway降解得第七群乙烯反應因子。本論文的目的是藉由生物化學與生物物理實驗，來探討Sub1A-1是如何逃離N-end rule pathway。 對此目前有一種假設與兩種可能的原因；N-end rule pathway是受到空間障礙的干擾，導致相關酵素無法接近Sub1A-1位於N端的MCGG序列進行反應。第一種可能是，Sub1A-1具有穩固且特殊的結構能保護其N端，防止酵素接近。能藉由圓二色光譜儀與核磁共振光譜儀來判斷，其N端區域(domain)是否有此種特殊結構；又或者是Sub1A-1的二聚體型態具保護功能，所以我們以微量熱泳動儀(MST)與等溫滴定微量熱儀(iTC)測定不同蛋白區域間的結合親和力，以判定二聚體中間介面的位置。第二個可能是有其他植物細胞內的蛋白質參與，Sub1A-1與蛋白質形成能保護Sub1A-1的N端受質的複合體。為了瞭解是否有其他蛋白質參與，目前正在建立體外N-end rule實驗來驗證。

Rice losses as a result of extreme climate put a severe threat to global food security. Drought and flood are the most prevailing climate-related disasters hindering the growth of rice and climate models predict an increased frequency of these two abiotic stresses. Rice cultivars with the ERF-VII (group VII ethylene responsive factor transcription factors), Sub1A-1 can survive more than two weeks of submergence. Apart from inundation, Sub1A-1 also regulates the adaptive response to desubmergence, drought and prolonged darkness in normoxia owing to the ability to escape N-end rule pathway. N-end rule pathway, a part of the ubiquitin-proteasome system (UPS), that acts as a homeostatic sensor and regulates the key hypoxia-response transcription factors including ERF-VIIs in plants. Interestingly, Sub1A-1 is the only known rice ERF-VIIs that can escape N-end rule pathway. My aim is to study how Sub1A-1 escapes the N-end rule pathway by biophysical and biochemical approaches. We have one hypothesis with two possible routes. We proposed that the N-end rule pathway may be disrupted by steric obstacles, so the enzymes cannot approach to the N-terminal MCGG sequence of Sub1A-1. One route is Sub1A-1 with a rigid and special structure to shield N-degron from enzymatic reaction. I studied the secondary structure of N domain by circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy to examine if N-terminus possesses a special structure. The dimerization of Sub1A-1 may protect N-degron from the N-end rule, so we measured the binding affinity between different domains of Sub1A-1 by MST and iTC to determine the dimer interface. The second route is that another protein participates in the Sub1A-1 interaction. The Sub1A-1/protein complex may shield N-degron from N-end rule enzymes. In order to test if there is additionally protein involved, I am developing the in vitro reconstitution of N-end rule pathway assay.