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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48791

Title: 研究植物中可辨認PR-1並切出具有系統性免疫功能胜肽CAPE1之蛋白酶
Study of the Protease for the Production of CAPE1 from PR-1 to Trigger Systemic Immune Response in Plant
Authors: Chen, Kuan-Chia
陳冠嘉
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 病原相關蛋白;CAPE1 胜肽;半胱胺酸蛋白酶
PR-1 protein;CAPE1 peptide;caspase-1 protease
Date: 2017
Issue Date: 2018-08-22T06:31:21Z
Abstract: 植物系統性免疫反應 (systemic acquired resistance, SAR) 的主要特徵是透過植物荷爾蒙水楊酸啟動下游反應基因,以及伴隨著病原相關蛋白 (pathogenesis-related protein 1, 簡稱PR-1) 蛋白的大量表現。在我們實驗室先前的研究中首次發現,番茄在組織受傷後,會將 PR-1 的 C端斷出一段具有免疫調節功能的小片段胜肽 CAPE1。同時也發現 CAPE1 能引發植物賀爾蒙茉莉酸及水楊酸,進而啟動植物抵抗病蟲害的反應。然而,啟動PR-1截切產生 CAPE1 的關鍵機制仍然未明,找出這個能截切 PR-1 的蛋白酶,將有利於我們對於植物免疫調控機制有更深入的了解。 經由序列比對發現 PR-1 及其 C 端15個胺基酸序列(CNYx.PxGNxxxxxPY) 在其他物種也具有高度保留性。其中除了 CAPE1 的十一個胺基酸外,在其切位前三個胺基酸也明顯被保留,推測此序列可能是蛋白酶辨認的區域。運用 (drug repositioning and adverse drug reaction via chemical-protein interactome, DRAR-CPI) 軟體針對 PR-1 化學結構預測蛋白質交互作用種類,分析出能與此區域作用的,以人類的半胱胺酸蛋白酶 (caspase-1) 可能性最高。推測可能是植物中類似 caspase-1 的蛋白酶可與 PR-1作用。為了找出這個 caspase-1 相似蛋白酶,在本研究中先以人類 caspase-1 作為目標,建立可以追蹤酵素活性以及純化酵素的研究工具。 本研究在綠螢光蛋白 (green fluorescent protein, 簡稱 GFP) 的 C 端融合了一段遮蔽胜肽 (quenching peptide, 簡稱 QP),並將caspase-1 蛋白酶的切位序列或是 PR-1 切位序列加在遮蔽胜肽與 GFP之間。一旦蛋白酶將 GFP 與 QP 胜肽切開分離,即可觀察到 GFP 螢光的回復。由實驗結果顯示,人類 caspase-1 可以成功截切帶有 YVAD 的蛋白,並產生螢光回復的現象。而我們也首次發現,與植物 PR-1 同一家族的人類 GLIPR-2 蛋白的 EVAD 序列也可以被 caspase-1 截切。但是這樣截切的現象有何生理意義仍待研究。至於植物 PR-1 的 CNYD 序列則無法被人類 caspase-1 截切。但本研究已經成功建立一系列 caspase-1 蛋白酶作用基質,未來可以利用這些蛋白質來找尋植物中可與 PR-1 作用的蛋白酶。 關鍵詞: 病原相關蛋白、CAPE1 胜肽、半胱胺酸蛋白酶
Systemic acquired resistance (SAR) has been known to display and be activated through the regulation of salicylic acid, a plant defense hormone for biotrophic pathogens. Salicylic acid in plants can greatly increase the expression of pathogenesis-related protein 1 (PR-1). Hence, PR-1 has been recognized as an important protein marker to activate SAR. However, the principal molecular function of PR-1 still unclear. According to our previous studies, a small immune-regulating peptide CAPE1 was identified to be processed from the C-terminal of PR-1 in the wounded tomato. Moreover, we also confirmed that CAPE1 has the ability to activate the defense response of plants to resist biotic threats pesticide by triggering the plant hormones, salicylic acid and jasmonic acid. Therefore, knowing which type of protease can recognize PR1 and process it to produce CAPE1 is important for having a mechanistic insight of the plant immunity system. According to the sequence alignment of PR-1, we found that the C-terminal 15 amino acids (CNYx.PxGNxxxxxPY) of PR-1 are highly conserved among different species. In addition to CAPE1, three amino acids before the cleavage site are also highly conserved. Using the DRAR-CPI software to predict the type of protein interaction of PR-1 protein structure, it was found that human caspase-1 was the most likely to interact with this region. It is speculated that caspase-1-like proteases in plants may interact with PR-1 protein. In order to identify this caspase-1-like protease, human caspase-1 was first used as a target to establish a research tool that tracks protease activity and purified proteases. In this study, a quencher peptide (QP) was fused at the C-terminus of green fluorescent protein (GFP), and the cleavage site of caspase-1 protease or PR-1 cleavage sequence was added between QP and GFP. Once the protease cleaves the GFP from the QP leucine, the recovery of the GFP fluorescence is observed. From the experimental results, human caspase-1 can successfully cleavaged the protein with YVAD, and produce fluorescence recovery phenomenon. We also found for the first time that the EVAD sequence of human GLIPR-2 protein in the same family as plant PR-1 can also be cleavaged by caspase-1. However, the physiological significance of this phenomenon is still to be studied. As for the CNYD of plant CNYD the sequence can not be cleavage by human caspase-1. However, a series of caspase-1 proteases have been successfully established in the present study. In the future, these proteins can be used to search for proteases that can interact with PR-1 in plants. Keywords: PR-1 protein, CAPE1 peptide, caspase-1 protease
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010236031.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48791
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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