甘藍黑斑病菌芥子酶基因選殖與功能特性之分析

硫配醣體-芥子酶 (glucosinolate-myrosinase)系統，利用水解硫配醣體產生具毒性的化學物質，作為植物的一種非專一性化學防禦機制。芥子酶活性主要存在於十字花科植物中，目前已有許多物種被報導具有芥子酶活性，然而僅在少數植物及蚜蟲發表芥子酶基因之選殖。本研究利用阿拉伯芥及蚜蟲的芥子酶基因之蛋白質序列，在甘藍黑斑病原菌 (A. brassicicola) 基因庫進行序列比對，得到3個候選基因：AB03581，AB00050及 AB10392。進一步分析芥子酶胺基酸序列的親緣關係，發現AB03581與AB10392與阿拉伯芥非典型芥子酶PEN2及蚜蟲芥子酶的親緣較為相近，而AB00050則位於芥子酶親緣類群之外。利用生物資訊學工具進行蛋白質結構模擬分析，AB03581和AB00050都具有與芥子酶酵素相似的 (α/β)8筒狀蛋白質結構。而AB10392序列上的缺失，則造成筒狀結構的缺陷，推測AB10392 不具有功能。在黑芥子硫酸鉀 (sinigrin) 誘導實驗發現，經6小時誘導後，AB03581基因的表現量上升30倍。進一步地，將AB03581基因進行大腸桿菌異源表現，純化出AB03581蛋白質，成功地測得芥子酶活性，並將AB03581命名為AbMyr1基因。將AbMyr1純化蛋白質系列稀釋成1-6 μg/μL，滴在含有鋇及黑芥子硫酸鉀的培養基中，3 μg/μL 以上的蛋白質濃度皆可造成白色的沉澱。同時亦發現，額外添加Zn2+ 可以促進蛋白質的活性，而添加抗敗血酸 (ascorbate) 則對酵素沒有影響。

關鍵字

硫配醣體；芥子酶甘藍黑斑病病原真菌； AbMyr1基因；蛋白質異源表現

並列摘要

Glucosinolate-myrosinase system is a well-studied nonspecific chemical defense mechanism of plant by hydrolyzing glucosinolates to produce toxic substances. Myrosinase activities are widely found and studied in cruciferous plants, and also reported in some aphids, fungi, bacteria, and intestinal bacteria of mammals. However, myrosinase genes were identified only in plants and aphids. In this study, we utilized the sequences of Arabidopsis thaliana TGG gene family and PEN2, and Brevicoryne brassicae BMY1 protein to search the putative myrosinase genes in Alternaria brassicicola genome database. Three candidate genes including AB03581, AB00050 and AB10392 were identified. Phylogenetic analysis showed that AB03581 and AB10392 are more closely related to an atypical myrosinase PEN2 gene and aphid myrosinase BMY1 gene, whereas AB00050 fell out of the myrosinase major clade. Molecular modeling revealed that AB03581 and AB00050 were similar to other myrosinase proteins which display the (α/β)8-barrel structure when using protein structure simulation analysis. In contrast, due to truncation of N-terminal part, AB10392 lost the catalytic site of typical β-glucosidase, implying that AB10392 may be not functional. Gene expression studies showed that the expression level of AB03581 increased about 30 folds after 6 hours under sinigrin inductive condition. Finally, AB03581 gene was heterologously expressed in E. coli expression system and proteins were purified and shown to contain myrosinase activity and we therefore named the gene of A. brassicicola as AbMyr1. Serial dilutions of AbMYR1 purified protein from 1 to 6 μg/μL were spotted onto BSA plate and opaque precipitation was observed when the concentration was higher than 3 μg/μL at 37°C. Addition of Zn2+ enhanced the AbMyr1 enzymatic activity, whereas ascorbate showed no effect.

並列關鍵字

glucosinolate ； myrosinase ； Alternaria brassicicola ； AbMyr1 gene ； heterologous protein expression

參考文獻

Agerbirk, N., and Olsen, C.E. 2012. Glucosinolate structures in evolution. Phytochemistry 77:16-45.

Andersson, D., Chakrabarty, R., Bejai, S., Zhang, J., Rask, L., and Meijer, J. 2009. Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties. Phytochemistry 70:1345-1354.

Andreasson, E., and Jorgensen, L.B. 2003. Chapter four Localization of plant myrosinases and glucosinolates. Pages 79-99 in: Recent Advances in Phytochemistry, John, T.R., ed. Elsevier.

Bones, A.M., and Rossiter, J.T. 1996. The myrosinase-glucosinolate system, its organisation and biochemistry. Physiologia Plantarum 97:194-208.

Burmeister, W.P., Cottaz, S., Rollin, P., Vasella, A.a., and Henrissat, B. 2000. High resolution x-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. J. Biol. Chem. 275:39385-39393.

國際替代計量

甘藍黑斑病菌芥子酶基因選殖與功能特性之分析

全文下載

主題瀏覽