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.

鉴定在细胞水平上感知植物激素的部位是迈向隔离和鉴定受体本身的必要步骤。出于这种目的，大麦种子的钙细胞提供了理想的生物学系统，因为它们以直接且易于检测的方式对吉布雷素（GA）或脱甲酸（ABA）反应。为了在实时模式下研究这两种激素的感知，我们准备并使用了荧光标记的生物活性GA（FGA）或ABA（FABA）。将荧光官能团引入GA或ABA分子中的最重要点是保持植物激素的内在活性。最后，Faba和FGA可以准备并进行下一个实验。通过流动仪分析FabA或FGA_4与麦芽龙原生质体的结合。结果表明，Faba或FGA_4与原生质体结合，而Faba的结合在某种程度上通过添加了ABA淬灭，但FGA_4与原生质体的结合并未通过添加Ga_3淬灭原生质体。荧光素本身是Faba和FGA_4的荧光官能团，即与细胞结合，即Faba分子中的ABA部分，而FGA4分子中的Ga部分则起着结合的决定性作用。用细胞扫描类型显微镜进行分析表明，即使在治疗后60分钟，FABA就在质膜上，而FGA_4很容易掺入细胞质中。该结果与流经术获得的结果一致。上面描述的方法用于分析Faba或FGA_4与受体的结合将使您可以根据存在核龙原生质体存在的条件来检测脱敏或外观和消失。