拟南芥ATOFP家族蛋白在BR调节的生长发育中的作用

While the brassinosteroid signal transduction pathway has been well studied, future research will be more focused on transcription activity regulation of BR specific transcription factors BZR1/BES1, and the mechanism study of BR directly regulated genes. ATOFPs is a family of proteins specific in plant species and has not been well studied. Previous study showed that some of the ATOFP genes are BZR1 directly regulated genes. Plant overexpress ATOFP genes showed phenotypes similar to BR deficent mutants, such as short petiole and hypocotyl, rounder leaves, and are hyper-sensitive to BR and hypo-sensitive to BR biosynthesis inhibitor BRZ treatment. These data suggested ATOFP might be involved in BR regulated growth. In this project, we propose to use ChIP-seq coupled with Digital Gene Expression Profiling (DGE) to analyze the genes affected by ATOFP over-expression at genome level. The results will illustrate the mechanism of ATOFP in regulating plant physiological responses. Meanwhile, our preliminary data showed that ATOFP2 and ATOFP5 interact with BZR1/BES1 in yeast two-hybrid system. We will confirm this interaction by methods such as gel overlay, immunoprecipitation etc, and investigate if the interaction would affect the transcription activity of BZR1 and BES1, in a way similar to the activity regulation of TALE family transcription factors by ATOFP1 and ATOFP4. Our study of the role of ATOFP proteins in BR regulated plant growth as well as BZR1/BES1 related transcriptional regulation will add more insight into our understanding of BR regulated plant growth and development.

在BR信号转导通路研究的较为清楚的情况下，BR特异的转录因子BZR1/BES1的转录调节方式及其下游基因的作用机理将成为BR研究领域的热点之一。拟南芥ATOFP蛋白是植物中特异存在并尚未被深入研究的一个蛋白家族，编码这一家族的部分基因是BZR1的靶基因。初步的研究结果表明，部分ATOFPs高表达植株的表型与BR缺失突变体相似。对高表达ATOFP植株的生理学分析显示ATOFPs可能参与BR调节的生理反应；而ATOFP2和ATOFP5与BZR1/BES1的直接结合则暗示ATOFP2和ATOFP5可能参与对BZR1/BES1的转录活性的调节。我们将结合遗传学及基因组测序的方法分析ATOFP在BR调节的生长发育中的作用，同时通过蛋白相互作用结合分子生物学手段探索ATOFP在BZR1/BES1的转录活性调节中的作用。对ATOFP蛋白的双重作用机理的研究将进一步展示BR对植物生长发育调节的具体方式。

在BR信号转导通路研究的较为清楚的情况下，BR如何通过下游基因调节细胞的生长发育过程将成为BR研究领域的热点之一。拟南芥ATOFP蛋白是植物中特异存在并尚未被深入研究的一个蛋白家族，部分成员的高表达植株的表型与BR缺失突变体相似。对ATOFP高表达植株和多重缺失突变体的生理学分析显示突变体对BR合成抑制剂PCZ的敏感程度降低，而高表达植物对PCZ的敏感程度增加，并且ATOFP2和ATOFP5可以与BZR1/BES1在多种体系中直接结合，说明ATOFPs可能参与BR调节的生理反应， 但是ATOFP2的ChIP和数字基因表达谱的结果显示ATOFP2可能并不通过调节BR转录因子BZR1的活性来影响细胞的伸长过程。 我们通过对酵母双杂交文库的筛选，得到了ATOFP2的结合蛋白TON2。后续的实验证明TON2确实可以与ATOFP2相互作用。烟草瞬时转化实验显示ATOFP2和ATOFP5在烟草表皮细胞中呈纤维状排列，并且纤维的分布与ATOFP的表达量相关。高表达ATOFP2和ATOFP5的拟南芥下胚轴细胞中微管的重排明显慢于野生型，而其幼嫩的子叶中，在细胞伸长之前，微管的排列方向已经与野生型不同，说明ATOFP高表达子叶的表皮细胞沿横轴伸长的表型是由于ATOFP影响微管的排布造成的。我们的结果证明ATOFP通过与可以影响微管骨架的蛋白TON2的相互作用，直接影响了细胞的伸长过程，为BR调节的细胞伸长的机理提供了新的思路。