蓝光诱导气孔开放过程中Phot1受体与MAPKKKa-MKKb关系的研究

In the signal network of blue light-induced stomatal opening, the kinases that catalyze the phosphorylation of proteins are not clear. Our premilinary results showed that mutation of MAPKKKa and MKKb genes, which belong to the mitogen-activated protein kinases family and do not affect stomatal developments, led to the reduced stomatal apertures in response to blue light, and MAPKKKa interacted with MKKb. In addition, blue light receptor Phot1 showed interaction with MAPKKKa, and possibly phosphorylated MAPKKKa. In this project, by utilizing cell biological, genetic and biochemical techniques, we will further study: (1) the phosphorylation of MKKb by MAPKKKa, and the up-down stream relationship between MAPKKKa and MKKb during stomatal opening, (2) confirm whether Phot1 phosphorylated MAPKKKa, the possible phosphorylated sites and the biological function of the phosphorylated sites in MAPKKKa, and the effect of Phot1 on the kinse activity of MAPKKKa in vitro; (3) how Phot1 affects the kinase activity of MAPKKKa in plants. The results from this project will reveal a possible regulatory pathway including Phot1-MAPKKKa-MKKb in blue light-induced stomatal opening. The conclusion from this research will enrich signal transduction mechanism in the guard cell during stomatal opening, and will provide useful information for the cultivation of drought-resistant plants with genetic manipulation.

蓝光诱导气孔开放的信号转导中，催化蛋白磷酸化的激酶的作用研究得还不是很清楚。本项目前期结果显示：促分裂原活化的蛋白激酶级联中MAPKKKa与MKKb基因（不影响气孔发育）突变后蓝光下气孔开度比野生型小，且两种蛋白之间有相互作用；此外，蓝光受体Phot1与MAPKKKa有相互作用，且可能将MAPKKKa磷酸化。本项目将利用细胞生物学、分子生物学、生物化学等技术进一步研究：（1）MAPKKKa-MKKb在调控气孔开放过程中的磷酸化及上下游关系；（2）确定Phot1能否将MAPKKKa磷酸化，探究磷酸化位点及功能， 以及体外Phot1对 MAPKKKa活性的影响；（3）植物体内Phot1对MAPKKKa活性的影响；从而揭示在蓝光诱导气孔开放过程中可能存在Phot1-MAPKKKa-MKKb这一调节方式。本项目的研究结果将丰富气孔光调控机制，也为利用基因工程技术培育耐旱植物提供有价值的信息。

蓝光诱导气孔开放时，首先激活蓝光受体Phot1/2，经过一定的信号传递过程，引起质膜上质子泵的激活、离子及水分的进入，最后引起气孔开放。但从保卫细胞蓝光受体Phot1/2感受蓝光到质子泵活化之间的信号传导还需要进一步探究。本项目用植物生理学、生物化学、分子生物学、遗传学等方法探究了MAPK激酶级联的MAPKKK14及下游的MKK7在蓝光诱导气孔开放过程中的作用，以及与蓝光受体Phot1之间的关系。我们按照项目计划开展了实验，结果如下：通过载体构建和转基因技术得到MAPKKK14pro:GUS或MKK7pro:GUS转基因株系，结合组织化学染色技术，确定MAPKKK14和MKK7在保卫细胞都有较强的表达。在白光、红光、红光加蓝光下进行气孔开度分析的结果表明，MAPKKK14和MKK7在蓝光诱导气孔开放的过程中起正调控作用。在酵母、体外pull down体系和植物体内检测了蛋白间的相互作用，发现在三个体系中蓝光受体Phot1与MAPKKK14、MAPKKK14和MKK7都有相互作用。此外，在体外磷酸化体系，检测到Phot1能将MAPKKK14磷酸化，MAPKKK14能将MKK7磷酸化。在植物体内，确定光照后MAPKKK14和MKK7都能被磷酸化，且在光照后的30-60分钟磷酸化程度较强，MAPKKK14的磷酸化依赖蓝光受体Phot1，MKK7的磷酸化依赖MAPKKK14。通过遗传转化、杂交等技术得到双突、及在不同背景下的超表达株系，经气孔开度和气孔导度分析，明确Phot1与MAPKKK14、MAPKKK14与MKK7在遗传学上为上下游关系。从以上结果我们得到以下结论：在蓝光诱导气孔开放的过程中，存在着Phot1-MAPKKK14-MKK7这一信号传导方式。该项研究结果对深入了解气孔运动机理有重要意义，也为作物节水高产方面提供了依据。