陰道滴蟲ap65基因之啟動子調控分析

Abstract

在過去對陰道滴蟲ap65-1啟動子的研究中，了解ap65-1啟動子的活性會受到生長以及鐵離子的正向調控，而ap65-2啟動子也受到類似的正向調控。而在本研究中將培養液中的鐵離子進一步降低後，對於ap65-1啟動子調控有更深入的認識。 ap65-1啟動子內包含數個核蛋白結合序列，其中MRE-1/MRE-2r和MRE-2f區域對基礎活性與鐵調控都有相當的重要性，而且MRE-1，MRE-2r分別突變有不一致的變化，顯示此區域的調控並非單一啟動子元素所主控。基礎活性可能受MRE-1、MRE-2f、AGTGA區域負向調控，生長活性可能受MRE-1/MRE-2r及AGTGA區域正向調控，鐵活性可能受MRE-2f正向調控以及MRE-2r和AGTGA區域的負向調控。 由近化學平衡培養的實驗中發現，當培養環境較穩定時，ap65-1啟動子的生長調控即消失，故生長調控可能是因為環境變化所誘發出的反應。 另一方面在ap65-2的啟動子調控分析，由啟動子刪短實驗得知影響ap65-2啟動子活性的最高的區域為-112~-160之間，此區域中又以T-rich區域的突變對活性影響最大，故其週遭區域可能有其他重要的調控相關序列存在。

Previous research showed that ap65-1 promoter is positively regulated by iron and growth. And ap65-2 promoter has similar regulation character. In this research, we deplete the iron in the medium, it could help us more deeply to understand the regulation of ap65-1 promoter. Several nuclear protein binding sequences exist in ap65-1 promoter. MRE-1/MRE-2r and MRE-2f regions are very important to basal activity and iron-inducible activity. And individually mutating MRE-1 and MRE-2r result in different activity change. This result show the MRE-1/MRE-2r region is not a single regulation unit. Basal activity might be negatively regulated by MRE-1, MRE-2f and AGTGA regions. Growth-related activity might be positively regulated by MRE-1/MRE-2r and AGTGA regions. Iron-inducible activity might be positively regulated by MRE-2f region, and negatively regulated by MRE-2r and AGTGA regions. According to experiment of the semi-chemostatic culture, when culture environment is stable, the growth-related activity of ap65-1 promoter disappear. Suggesting the growth-related activity might result form environment change. In the analysis of ap65-2 promoter, we discover the most important region to ap65-2 promoter activity is between -112~-160. In this range, the mutation of T-rich region could result to the hugest influence to ap65-2 promoter activity. Suggesting maybe some important regulation regions are located near T-rich region.