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Studies on receptor-ligand analysis with fluorescence-labeled plant hormones

荧光标记植物激素受体-配体分析研究

基本信息

批准号：
09660121
负责人：
ASAMI Tadao
金额：
$ 1.86万
依托单位：
The Institute of Physical and Chemical Research (RIKEN)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Identification of the site where plant hormones are perceived at the cellular level is an essential step towards the isolation and identification of the receptors themselves. For such a purpose aleurone cells of barley seeds provide an ideal biological system because they respond to both gibberellin (GA) or abscisic acid (ABA) in a direct and easily detectable way. In order to study the perception of these two hormones by aleurone protoplasts in a real time mode, we prepared and used fluorescence-labeled bioactive GA (FGA) or ABA (FABA). The most important point in introducing fluorescence functional group into GA or ABA molecules is to keep the intrinsic activity of the plant hormones. Finally FABA and FGA could be prepared and subjected to the next experiments. The binding of FABA or FGA_4 to aleurone protoplasts was analyzed with a flowcytometer. The result showed that FABA or FGA_4 bind to protoplasts and while the binding of FABA was quenched to some extent by the addition of ABA, the binding of FGA_4 to protoplasts was not quenched by the addition of GA_3. Fluorescein itself, which was the fluorescence-bearing functional group of both FABA and FGA_4, did not bind to cells, i.e., the ABA part in FABA molecule and the GA part in FGA4 molecule play a determinant role for the binding. Analysis with a cell scan type microscope suggested that the FABA was on the plasma membranes even 60 minutes after the treatment, whereas FGA_4 was readily incorporated into cytosol. This result is consistent with that obtained by flowcytometry. The method described above for analyzing the binding of FABA or FGA_4 to receptors will make it possible to detect a desensitization or an appearance and disappearance of receptors depending on the conditions where aleurone protoplasts exist.

识别植物激素在细胞水平上被感知的位点是分离和识别受体本身的重要步骤。大麦种子糊粉层细胞对赤霉素（GA）和脱落酸（阿坝）的反应直接且易于检测，因此是一个理想的生物系统。为了研究糊粉原生质体对这两种激素的真实的时间感知，我们制备并使用了荧光标记的生物活性GA（FGA）或阿坝（FABA）。在GA或阿坝分子中引入荧光功能基团的最重要的一点是保持植物激素的内在活性。最后可以制备FABA和FGA并进行下一个实验。用流式细胞仪分析了FABA和FGA_4与糊粉原生质体的结合。结果表明，FABA和FGA_4与原生质体结合，阿坝对FABA的结合有一定的猝灭作用，而GA_3对FGA_4与原生质体的结合无猝灭作用。荧光素本身是FABA和FGA_4的荧光承载官能团，不与细胞结合，即，FABA分子中的阿坝部分和FGA 4分子中的GA部分对结合起决定性作用。细胞扫描显微镜分析表明，处理后60分钟，FABA仍在质膜上，而FGA_4则很容易掺入胞质中。这一结果与流式细胞术获得的结果一致。上述用于分析FABA或FGA_4与受体结合的方法将使得根据糊粉原生质体存在的条件检测受体的脱敏或出现和消失成为可能。