白芷细胞外钙调素结合蛋白（ECBP21）免疫组化及金标定位分析

ECBP21是我们从白芷悬浮培养细胞外纯化及cDNA克隆的一种钙调素结合蛋白(CaMBP)，亦是植物中首次报道的细胞外CaMBP。本实验以大肠杆菌表达的重组ECBP21蛋白为抗原，制备了高效价特异性抗体；随后，利用免疫组化及金标定位技术，研究了ECBP21蛋白组织特异性分布及亚细胞定位。免疫组化结果表明：ECBP21在白芷各组织中均有分布，但在叶、花、花序轴中较多，而在根中较少，并且ECBP21在细胞中多分布于细胞壁区域；免疫胶体金电镜定位结果显示：在白芷花序轴细胞中金颗粒主要分布在细胞壁，表明ECBP21蛋白主要定位于细胞壁区域，从而首次为细胞外CaMBP(ECBP21)的胞外存在提供了直观证据，并为进一步研究其在植物生长发育中的功能提供了初步信息。

Immunohistochemical analysis and immuno-gold localization of ECBP21 in Angelica dahurica]

ECBP21 is a calmodulin binding protein (CaMBP) purified from extracellular extracts of suspension-cultured cells of Angelica dahurica, and it is the first reported extracellular CaMBP in plant kingdom. We have recently cloned the full-length cDNA for ECBP21. In this work, using recombinant ECBP21 we prepared rabbit antiserum with high specificity and high titer against ECBP21, and investigated the organ-specific distribution of ECBP21 in Angelica dahurica. ECBP21 was found in all organs examined, particularly abundant in the leaves flowers, and raches, and less in the roots. It was also found in all cells examined, and particularly enriched in the cell wall. These data support the notion that ECBP21 is specifically localized extracellularly, and imply that it may be involved in plant growth and development. In addition, using immunogold transmission electron microscopy method, we studied the subcellular localization of ECBP21 in rachis cells of Angelica dahurica. The results indicated that the ECBP21 was mainly localized in cell wall; this provided a direct evidence of the extracellular existence of ECBP21.