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標題: | 單胞綠藻SUMO E3黏合酶Siz1之基因選殖與蛋白質表現 Molecular cloning and protein expression of Siz1 SUMO E3 ligase in Chlamydomonas reinhardtii |
作者: | Ya-Ling Tseng 曾雅伶 |
指導教授: | 張世宗 |
關鍵字: | SUMO E3黏合酶,基因選殖,熱休克, SUMO E3 ligase,molecular coloning,heatshock, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | SUMO E3黏合酶參與SUMO化 (sumoylation) 反應的最後一個步驟。而SUMO E3黏合酶,除了能夠加速SUMO化反應的進行及增加SUMO對於目標蛋白質的專一性外,亦可調控細胞內許多不同的生理功能。細胞中有非常多能被SUMO修飾的受質,但目前已知且較常被探討的SUMO E3黏合酶只有三種,因此相關的研究仍有很大的發展空間,尤其對單胞綠藻 (Chlamydomonas reinhardtii) 之SUMO E3黏合酶的研究的研究仍未見。
本論文先利用阿拉伯芥的SUMO E3黏合酶AtSiz1的胺基酸序列,以BLAST方法找出單胞綠藻中與其相似的序列,並找到了一個功能未知的蛋白質序列。此序列與AtSiz1擁有相似的保守性區域,如N端之SAP (scaffold attachment factor A/B/Acinus/PIAS) domain,分子中間的PHD (plant homeodomain) domain及RING (really interesting new gene) domain,因此推測這個預測的蛋白質可能為一種SUMO E3黏合酶,本論文將其暫時命名為CrSiz1。目前已成功選殖到此段序列,及表現各個保守性區域之重組蛋白質,值得一提的是CrSiz1之C端含有一段特有之胺基酸序列,並未見於其他Siz1之序列中。有研究指出阿拉伯芥於熱休克處理後,AtSiz1可以增加受質的SUMO化以抵抗逆境。然而利用CrSiz1之專一性抗體觀察單胞綠藻於熱休克處理後之CrSiz1表現情形,並未偵測出其差異性,因此目前尚無法確定CrSiz1是否具有SUMO E3黏合酶之生化性質與功能。 The SUMO E3 ligase involves in the last step of sumoylation. In addition to faciliate sumoylation and increase specificity for the targets, SUMO E3 ligases modulate a wide range of physiological functions. There are many sumoylated proteins in the cells, but only three kinds of SUMO E3 ligases have been shown in the literatures. Notably, the study of SUMO E3 ligase in Chlamydomonas reinhardtii has not been reported. In this study, using the Arabidopsis SUMO E3 ligase, AtSiz1, as the template for BLAST search, a gene encoding for a predicated protein with unkown function has been remarked. This predicated protein has similar conserved domains with AtSiz1, such as N-terminal SAP (scaffold attachment factor A/B/Acinus/PIAS) domain, PHD (plant homeodoamin) and RING (really interesting new gene) domain in the middle of the sequence. Based on the sequence observation, this predicated protein was herein named CrSiz1. The partial sequences of CrSiz1 have been expressed and purified from the Escherichia coli. It is noted that CrSiz1 cotains a unique C-terminal sequence which has not been found in other Siz1 proteins. It has been reported that AtSiz1 can inscrease sumoylated proteins under the heat shock condition. By using the antibodies specific to CrSiz1 for western blotting against the C. reinhardtii samples obtained from the cells cultured at 42 |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23304 |
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