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首页> 外文期刊>Journal of Experimental Botany >Molecular cloning of a bifunctional β-xylosidase/α-L-arabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme
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Molecular cloning of a bifunctional β-xylosidase/α-L-arabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme

机译：从苜蓿根部克隆双功能β-木糖苷酶/α-L-阿拉伯糖苷酶：在：藜苜蓿中的异源表达和纯化酶的底物特异性

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摘要

Glycoside hydrolases are often members of a multigene family, suggesting individual roles for each isoenzyme. Various extracellular glycoside hydrolases have an important but poorly understood function in remodelling the cell wall during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa (Medicago sativa L.) belonging to the glycoside hydrolase family 3 (β-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the control of the CaMV 35S promoter was expressed in the model legume Medicago truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants exhibited 5–8-fold increased activities towards three p-nitrophenyl (PNP) glycosides, namely PNP-β-D-xyloside, PNP-α-L-arabinofuranoside, and PNP-α-L-arabinopyranoside. An antiserum raised against a synthetic peptide recognized MsXyl1, which was processed to a 65 kDa form. To characterize the substrate specificity of MsXyl1, the recombinant protein was purified from transgenic M. truncatula leaves by concanavalin A and anion chromatography. MsXyl1cleaved β-1,4-linked D-xylo-oligosaccharides and α-1,5-linked L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a multifunctional β-xylosidase/α-L-arabinofuranosidase/α-L-arabinopyranosidase implicated in cell wall turnover of arabinose and xylose, particularly in rapidly growing root tips. Moreover, the findings of this study demonstrate that stable transgenic M. truncatula plants serve as an excellent expression system for purification and characterization of proteins.
机译：糖苷水解酶通常是多基因家族的成员，表明每种同工酶的各自作用。各种细胞外糖苷水解酶在植物生长过程中对细胞壁重塑中具有重要但尚不为人所知的功能。在此，表征了MsXyl1，其是来自苜蓿（Medicago sativa L.）的糖苷水解酶家族3（β-D-木糖苷酶分支）的伴刀豆球蛋白A结合蛋白。在根（特别是根尖），根瘤和花朵中检测到MsXyl1的转录本。 CaMV 35S启动子控制下的MsXyl1在豆科植物苜蓿（Medicago truncatula）（Gaertner）模型中表达。来自转基因植物的伴刀豆球蛋白A结合蛋白对三种对硝基苯基（PNP）糖苷（即PNP-β-D-木糖苷，PNP-α-L-阿拉伯呋喃糖苷和PNP-α-L）的活性增加了5-8倍-阿拉伯吡喃糖苷。针对合成肽产生的抗血清识别为MsXyl1，将其加工为65 kDa形式。为了表征MsXyl1的底物特异性，重组蛋白通过伴刀豆球蛋白A和阴离子色谱法从转基因的M. truncatula叶中纯化而来。 MsXyl1裂解了β-1,4-连接的D-木糖寡糖和α-1,5-连接的L-阿拉伯糖寡糖。阿拉伯糖木聚糖（来自小麦）和阿拉伯聚糖（来自甜菜）是MsXyl1的底物，而木聚糖（来自燕麦球茎）则抗降解。此外，MsXyl1从苜蓿根部分离的细胞壁多糖中释放出木糖和阿拉伯糖。这些数据表明，MsXyl1是一种多功能的β-木糖苷酶/α-L-阿拉伯呋喃糖苷酶/α-L-阿拉伯吡喃糖苷酶，涉及阿拉伯糖和木糖的细胞壁更新，特别是在快速生长的根尖中。此外，这项研究的结果表明，稳定的转基因梅花苜蓿植物可作为纯化和表征蛋白质的出色表达系统。

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