Ca^<2+> dynamix in self-incompatibility system of plants

为了阐明Ca~(2+)在十字花科植物花粉-柱头互作中的作用，必须准确地研究授粉过程中花粉和乳突细胞中Ca~(2+)和Gt~(2+)随时间的动态变化。通常，通过向细胞内注入离子敏感染料来分析细胞中的离子运动，但显微注射通常会对细胞造成物理损害，而且很难将染料注入花粉粒，因为花粉粒上覆盖着坚硬的外壳，如外壁。结合分子生物学靶向和荧光读数的优点，研制了钙离子荧光蛋白指示剂“黄色骆驼”(YC)。本研究首先将含有Act1启动子区域和SLG启动子区域的两种YC3.1嵌合基因转化拟南芥。获得了两种分别在花粉粒和乳突细胞中表达YC3.1的转基因植株。用显微操作仪将花粉粒授粉到乳头细胞后，随时间监测Ca~(2+)&Gt~(2+)的动态变化。萌发前，Ca~(2+)~(2+)在花粉粒与乳突细胞接触的部位积累。在萌发的花粉管中，Ca~(2+)和Gt~(2+)的运动速度比体外萌发的花粉管快。另一方面，Ca~(2+)在乳突细胞中的运动慢于在花粉粒中的运动。接下来，将YC3.1嵌合基因转化到白菜中。获得了在花粉粒中表达YC3.1的转基因植株。结果表明，只有在异花授粉时才能观察到Ca~(2+)~(2+)在花粉粒中的动态移动。利用YC转基因植株，可以对授粉过程中花粉粒和乳突细胞内Ca~(2+)和Gt~(2+)随时间的动态变化进行监测。

In order to clarify the role of Ca^<2+> in pollen-stigma interaction of Brassicaceae, dynamic movement of Ca^<2+> in pollen grains and papilla cells during pollination process should be examined precisely and with time. Typically, ion movements in a cell have been analyzed by injecting ion sensitive dye into the cell, but the microinjection often causes physical damage to the cell and it is difficult to inject the dye to a pollen grain because pollen grain is covered with hard coats, such as exine layer. The fluorescent protein indicator for Ca^<2+>, "yellow cameleons" (YC) has been developed to combine the advantages of molecular biological targeting and fluorescence readout. In this study, first two kinds of YC3.1 chimeric genes containing Act1 promoter region or SLG promoter region were transformed into Arabidopsis thaliana. Then two kinds of transgenic plants, which expressed YC3.1 in pollen grains or papilla cells, respectively, were obtained. After the pollen grain was pollinated to the papilla cell using micromanipulator, Ca^<2+> dynamics were monitored with time. Before germination, Ca^<2+> accumulated at the site, which the pollen grain contacted to the papilla cell. In the germinated pollen tube, faster movement of Ca^<2+> was observed, compared with that of in vitro germinated pollen tube. On the other hand, Ca^<2+> movement in the papilla cell was slower than that in pollen grains. Next the YC3.1 chimeric genes were transformed into Brassica rapa. The transgenic plant, which expressed YC3.1 in the pollen grains, was obtained. As a result, dynamic movement of Ca^<2+> in the pollen grains was observed only in cross-pollination. Dynamic movement of Ca^<2+> in pollen grains and papilla cells during pollination process could be monitored precisely and with time using transgenic plants, which expressed YC genetically.