大腸桿菌中 Grx4 蛋白質特性及其與 ClpYQ 的交互關係

Abstract

大腸桿菌中的 glutaredoxin-4 (Grx4) 編碼基因為 grxD，為目前唯一在其中發現的單巰 glutaredoxin (僅具 CGFS 的活性區域)。本篇研究利用 grxD-lacZ 融合基因及染色體中 Grx4-3xFLAG 觀察 grxD 的調控，當 small RNA大量表現時，FnrS 負向調控 grxD-lacZ 達原本約 1/2 的表現量，在 Grx4-3xFLAG 蛋白質層面，則看到更明顯的抑制。RyhB 亦負向調控 grxD-lacZ 約 20% 的表現。除此之外，轉錄因子缺失狀態下，發現 NsrR 參與 grxD 的正向調控，nsrR 缺失造成表現量下降近 1/2 ，而 Grx4-3xFLAG 蛋白質層面則影響更鉅。因此 grxD-lacZ 的表現受到如：氧、鐵、一氧化氮相關因子等的調控，也在 Grx4-3xFLAG 對生長產生的影響指出這部分的精細調控的重要性。 除了基因調控的研究之外，在 E. coli 基因工程中 P1 噬菌體扮演重要的地位，必須要用於建構上述各種突變株，故本篇論文亦克服之前 P1 容易失活的問題，利用新的增殖方式使 P1 在基因工程操作上運作順利。最後，藉由之前對 SulA、ClpYQ 的深度研究，我們開發了一套「新穎之轉形及轉殖效率增加與抑制細胞分裂技術」，也期望能夠應用在基因工程、微生物族群分析等研究上。

The glutaredoxin-4 (Grx4)-encoding in grxD gene in Escherichia coli, which is the only monothiol glutaredoxin (only active region with CGFS) found in it. In this study, grxD-lacZ fusion gene and Grx4-3xFLAG in chromosome were used to observe the regulation of grxD. When small RNA was expressed in a high level, FnrS negatively regulated grxD-lacZ to about 1/2 of the original expression, at the level of Grx4-3xFLAG protein the more obvious repression happend. RyhB also negatively regulates the performance of grxD-lacZ by ~20%. In addition, in the absence of transcription factors, NsrR was found to be involved in the positive regulation of grxD, and nsrR deletion caused a decrease in performance by nearly 1/2, while the Grx4-3xFLAG protein level was more influential. Therefore, the performance of grxD-lacZ is regulated by factors such as oxygen, iron, and nitric oxide related factors, and the influence of Grx4-3xFLAG on growth indicates the importance of fine regulation of this part. In addition to gene regulation studies, P1 phage plays an important role in E. coli genetic engineering and be used to construct these various mutants. Therefore, this paper also overcomes the problem of easy inactivation of P1 before, and utilizes new proliferation, the way to make P1 work well in genetic engineering operations. Finally, with the previous in-depth study of SulA and ClpYQ, we have developed a set of 'new enhance cloning and transformation efficiency method and cell division inhibition technology' and expected to be applied in genetic engineering, microbial population analysis and other research.