Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens
Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens
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DOI：
10.1104/pp.120.2.501
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发表时间：
1999-06-01
期刊：
影响因子：
7.4
通讯作者：
Rasmussen, SK
中科院分区：
文献类型：
--
作者：
Kristensen, BK;Bloch, H;Rasmussen, SK
从大麦（Hordeum vulgare L.）预测了一个341个氨基酸的蛋白质，分子量为36，515。N-和C-末端推定的信号肽，表明液泡的位置的过氧化物酶。免疫印迹和逆转录聚合酶链反应表明，Prx 7蛋白和mRNA积累丰富的大麦胚芽鞘和叶表皮接种白粉病真菌（Blumeria graminis）。从大麦胚芽鞘中纯化了两种同工酶，其等电点分别为9.3和7.3（分别为P9.3和P7.3）。P9.3和P7.3的Reinheitszahl值分别为3.31和2.85，比活性（以2，2 '-连氮基-二-[3-乙基-苯并噻唑啉-6-磺酸]，pH 5.5作为底物）分别为11和79单位/mg。N-末端氨基酸测序和基质辅助激光解吸/电离飞行时间质谱肽分析确定P9.3过氧化物酶活性是由于Prx 7。使用活性染色等电聚焦凝胶和免疫印迹研究Prx 7的组织和亚细胞积累。过氧化物酶活性由于Prx 7积累在大麦叶片接种白粉病孢子后24小时或受伤的表皮细胞。Prx 7主要积累在表皮中，显然是在液泡中，似乎是唯一的病原体诱导的液泡过氧化物酶在大麦组织中表达。这里提供的数据表明，Prx 7负责被称为大麦醇胺的抗真菌化合物的生物合成，大麦醇胺在大麦胚芽鞘中大量积累。
A cDNA clone encoding the Prx7 peroxidase from barley (Hordeum vulgare L.) predicted a 341-amino acid protein with a molecular weight of 36,515. N- and C-terminal putative signal peptides were present, suggesting a vacuolar location of the peroxidase. Immunoblotting and reverse-transcriptase polymerase chain reaction showed that the Prx7 protein and mRNA accumulated abundantly in barley coleoptiles and in leaf epidermis inoculated with powdery mildew fungus (Blumeria graminis). Two isoperoxidases with isoelectric points of 9.3 and 7.3 (P9.3 and P7.3, respectively) were purified to homogeneity from barley coleoptiles. P9.3 and P7.3 had Reinheitszahl values of 3.31 and 2.85 and specific activities (with 2,2'-azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid], pH 5.5, as the substrate) of 11 and 79 units/mg, respectively. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry peptide analysis identified the P9.3 peroxidase activity as due to Prx7. Tissue and subcellular accumulation of Prx7 was studied using activity-stained isoelectric focusing gels and immunoblotting. The peroxidase activity due to Prx7 accumulated in barley leaves 24 h after inoculation with powdery mildew spores or by wounding of epidermal cells. Prx7 accumulated predominantly in the epidermis, apparently in the vacuole, and appeared to be the only pathogen-induced vacuolar peroxidase expressed in barley tissues. The data presented here suggest that Prx7 is responsible for the biosynthesis of antifungal compounds known as hordatines, which accumulate abundantly in barley coleoptiles.