Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant Vigna angularis
Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant Vigna angularis
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DOI：
10.1105/tpc.11.11.2075
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发表时间：
1999-11-01
期刊：
影响因子：
11.6
通讯作者：
Brown, RM
中科院分区：
文献类型：
--
作者：
Kimura, S;Laosinchai, W;Brown, RM
纤维素合成酶的催化亚基被证明与维管植物中假定的纤维素合成复合物（玫瑰花结末端复合物[TC]）相关。以水稻表达序列标签（D41261）为引物，构建特异性引物，以花后24天棉花纤维cDNA文库为模板，进行聚合酶链反应，克隆棉花纤维素合成酶催化亚基结构域，在大肠杆菌中表达棉花纤维素合成酶催化亚基，制备多克隆抗血清，胶体金偶联羊抗兔二抗，在透射电镜下观察纤维素合成酶催化区，用冷冻断裂复制标记技术，抗体特异性定位于P-断裂面质膜上的玫瑰花结TC，抗体不特异性标记E-断裂面上的任何结构。值得注意的是，更多数量的免疫探针标记了玫瑰花结TC（即，金颗粒比免疫前的探针更接近于玫瑰花结TC的边缘）。这些实验证实了长期以来的假设，即纤维素合酶是维管植物中玫瑰花结TC的组分，证明酶复合物存在于1980年冷冻断裂首次描述的结构中。此外，本研究提供了独立的证据，Ce/A基因实际上是一个基因的纤维素合成酶在维管植物。
The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template, The catalytic region of cotton cellulose synthase was expressed in Escherichia coli, and polyclonal antisera were produced, Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy, With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face, Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes, These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the Ce/A gene is in fact one of the genes for cellulose synthase in vascular plants.