拟南芥BIPP1调节油菜素甾醇受体活性的遗传和生化机制

Brassinosteroids (BRs) are a class of plant hormones on the growth and development of plants. The study of BR signaling pathway is a hot topic. We are interested in exploring the molecular mechanism of BRI1 deactivition of in Arabidopsis. We previously contructed the yeast library expressing Arabidopsis catalytic subunits of protein phosphotases. To get the new phosphotases interacting with BRI1, yeast two hybrid screening was performed using BRI1-JKC as bait. A new BRI1 interacting protein phosphotase, shortly named as BIPP1, was identified. Further studies showed that BRI1 was dephosphorylated by BIPP1.The genetic and biochemical analysis of the knock-out mutant bipp1 showed that bipp1 mutation resulted in the enhancing output of brassinosteroids signaling. So we hypothesized that BIPP1 maybe play a negative regulator role in brassinosteroid signal-transduction pathway. To prove the hypothesis, four experiments need to do: 1) The dephosphorylation of BRI1 by BIPP1 will be determined using a continuous spectrophotometric assay and phosphospecific antibody assay. The new dephosphorylated sites of BRI1 are further analyzed by LC/MS.2) The genetic analysis that BIPP1 regulates BR signal pathway through BRI1 will be performed. 3) The relationship between BR hormone and BIPP1 will be analyzed. 4) The new interacting proteins of BIPP1 will be isolated by LC/MS. This study will not only fill in a gap of brassinoteroid signal-transduction pathway, but also it will be possible to apply it to studies of other receptor kinases and helpful to understand the biological function of other receptor kinases.

油菜素甾醇(BRs)是一类在调控植物生长发育和环境适应中发挥重要作用的植物激素。细胞表面的受体BRI1感受并转导BR信号，虽然BRI1的磷酸化及其激活机制取得了重要进展， 但是其脱磷酸和脱敏机制还很不清楚。申请人以BRI1为诱饵，在我们建立的拟南芥磷酸酶的酵母文库中筛选到一个新的磷酸酶BIPP1，并发现它能在短时间内使BRI1完全脱磷酸化。丧失BIPP1基因导致BR信号输出放大。因此我们推测BIPP1可能在BRI1的脱磷酸化起关键作用，是BR信号转导途径负调节因子。本课题将深入研究BIPP1调控BRI1脱磷酸化及其活性的生化机制；遗传分析BIPP1通过BRI1调控信号通路；研究BR信号对BIPP1的影响；以及鉴定新的BIPP1互作蛋白。该项目的完成不仅对于全面揭示BIPP1对BR信号转导途径的调控机制，而且对于其它受体激酶的研究具有重要的理论意义。

在油菜素甾醇信号转导途径的研究中，油菜素甾醇受体激酶BRI1的磷酸化及其激活机制取得了重要进展，但是其脱磷酸和脱敏机制还很不清楚。申请人通过筛选油菜素甾醇受体激酶BRI1相互作用蛋白,获得了一个磷酸酶BIPP1，通过分子生物学和遗传学方法研究了BIPP1与BRI1的相互关系，探讨了BIPP1在油菜素甾醇信号通路中的功能。首先通过广谱和特异的磷酸化抗体证实了BIPP 对于BRI1-JKC具有丝氨酸和苏氨酸磷酸酶活性，但是不具有酪氨酸磷酸酶活性。通过bipp1突变体与油菜素甾醇相关突变体进行遗传杂交分析，并检测双突变体中BR信号的改变，发现bipp1功能失活植株能够恢复BR合成突变体det2-1的矮化表型,却无法挽救BR受体突变体bri1-301的矮化表型,表明BIPP1确实是在BR信号传导中发挥负调节因子的作用，这一结果也进一步被BIPP1过表达导致植株变小，反映油菜素甾醇信号变小所验证。外源eBL能够在转录和翻译水平同时诱导BIPP1的表达，反映了BIPP1的表达受到油菜素甾醇信号通路的负反馈调控。此外，我们通过免疫共沉淀和质谱相结合的方法获得了多个与BIPP1相互作用的蛋白，并对其中的HSP70蛋白进行了深入研究，结果表明通过BIPP1相互作用获得的HSP70蛋白同样展现影响油菜素甾醇信号输出的特征，并也会受到油菜素甾醇信号通路的负反馈调控。这些研究既增加了对油菜素甾醇信号负反馈调节的认识，又确定了BIPP1是一个新的通路负调控元件。同时，还为以后深入研究油菜素甾醇信号通路提供了很多可能的研究对象。这对于在植物中建立油菜素甾醇信号调节模型，以及探索磷酸酶研究相关技术具有非常重要的意义。