Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module
Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module
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DOI：
10.1007/s10126-009-9196-z
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发表时间：
2010-02
影响因子：
3
通讯作者：
Yoshiaki Umemoto;T. Araki
中科院分区：
文献类型：
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作者：
Yoshiaki Umemoto;T. Araki
红藻Bangia atropurpurea的细胞壁由三种独特的多糖（β-1,4-甘露聚糖、β-1,3-木聚糖和紫菜聚糖）组成，与紫菜的细胞壁相似。在本研究中，我们通过使用碳水化合物结合模块 (CBM) 和绿色荧光蛋白 (GFP) 的融合蛋白，对 B.atropurpurea 原生质体再生细胞壁中的 β-甘露聚糖进行可视化。来自弧菌的 β-1,4-甘露聚糖酶 (Man5C) 的甘露聚糖结合家族 27 CBM (CBM27)。将菌株 MA-138 与 GFP 融合，所得融合蛋白 (GFP-CBM27) 在大肠杆菌中表达。天然亲和凝胶电泳显示，GFP-CBM27 保持了与可溶性 β-甘露聚糖的结合能力，而正常 GFP 不能与 β-甘露聚糖结合。使用三种细菌酶从 B.atropurpurea 的叶中分离原生质体。将GFP-CBM27与不同生长阶段的原生质体混合，通过荧光显微镜观察细胞壁再生过程。培养 12 小时后，一些原生质体开始在细胞表面的某些区域分泌 β-甘露聚糖。随着原生质体培养的进行，β-甘露聚糖遍布整个细胞表面。培养 12、24、36、48 和 60 小时后，与 GFP-CBM27 结合的原生质体百分比分别为 3%、12%、17%、29% 和 25%。尽管GFP-CBM27在生长初期与细胞结合，但其与成熟叶的结合尚未得到明确证实。这是第一份关于使用荧光标记的 CBM 可视化 β-甘露聚糖再生藻类细胞壁的报告。
The cell wall of the red algaBangia atropurpureais composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that inPorphyra. In this study, we visualized β-mannan in the regenerating cell walls ofB.atropurpureaprotoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) fromVibriosp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed inEscherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds ofB.atropurpureaby using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.