LARSON, An Arabidopsis Homeobox Gene in Flower Development

长期目标是揭示决定多细胞有机体中细胞如何承担其发育命运的机制。以拟南芥花为模型，本研究的重点是揭示AGAMOUS(AG)的时空特异性表达的调控机制，AGAMOUS(AG)是一种雄蕊和心皮特征指定基因。银杏核糖核酸通常在花的内两轮中表达，而在外两轮中不表达。Leunig(LUG)是一种与酵母TUP序列相似的转录共抑制因子，在抑制外两轮的AG表达中起着至关重要的作用。由于Lug不编码DNA结合域，因此Lug如何被招募到AG顺式调控元件尚不清楚。新近发现的Larson(LSN)基因是成为LUG DNA结合伙伴的极佳候选基因。LSN-1突变增强了Lug突变在AG调节中的缺陷。此外，初步数据表明，LSN编码一个同源框蛋白，可以与AG顺式调节元件结合，并可以与酵母中的LUG相互作用。此外，由于lug在所有的花轮生中都有表达，因此外轮特异性的抑制活性可以通过与外轮特异性因子的相互作用来实现。LSN能否在为LAG共抑制因子提供空间和时间特异性方面发挥关键作用？拟议的实验旨在验证这些假设。首先，通过对LSN-1突变表型的转化挽救来确认LSN的分子同一性。将使用几种反向遗传方法来识别LSN的其他等位基因。其次，LSN的RNA和蛋白质表达谱将通过原位杂交和免疫定位或LSN-GFP来确定。第三，通过在异位轮生中表达LSN基因(其中表达了lug)，将测试异位共抑制物的活性。第四，为了确定LSN直接调节AG，将使用凝胶迁移率改变分析来确定AG顺式调节元件中LSN的结合位点。此外，在转基因植物中表达的LSN-VP16嵌合蛋白将被用于在体内建立LSN与AG结合的功能相关性。最后，将使用免疫共沉淀试验来测试LSN和Lug蛋白之间的物理相互作用。其他与LSN相互作用的蛋白将通过酵母双杂交筛选来鉴定。这项研究将阐明高等植物发育中转录抑制的基本机制，并为本科生和研究生提供良好的培训机会。

0212847Liu The long-term objective is to reveal the mechanisms determining how cells in a multicellular organism assume their developmental fates. Using Arabidopsis flower as a model, the focus of this proposal is to reveal the regulatory mechanism for the spatial and temporal-specific expression of AGAMOUS (AG), a gene for stamen and carpel identity specification. AG mRNA is normally expressed in the inner two whorls of a flower and is absent from the outer two whorls. LEUNIG (LUG), a putative transcriptional co-repressor with sequence similarity to the yeast co-repressor TUP, was previously shown to play a crucial role in repressing AG expression in the outer two whorls. Since LUG does not encode a DNA binding domain, how LUG is recruited to the AG cis-regulatory element is unknown. The newly identified LARSON (LSN) gene is an excellent candidate for being a DNA-binding partner of LUG. lsn-1 mutation enhances the defects of lug mutation in AG regulation. Further, preliminary data indicated that LSN encodes a homeobox protein, can bind to the AG cis-regulatory elements, and can interact with LUG in yeast. In addition, as LUG mRNA is expressed in all floral whorls, the outer whorl-specific repression activity of LUG could be conferred by interaction with an outer-whorl-specific factor. Could LSN hold the crucial role in providing the spatial and temporal specificity to the LUG co-repressor? The proposed experiments are aimed at testing these hypotheses. First, the molecular identity of LSN will be confirmed by transformation rescue of the lsn-1 mutant phenotype. Several reverse genetic methods will be employed to identify additional alleles of lsn. Second, the RNA and protein expression profile of LSN will be determined by in situ hybridization and immuno-localization or LSN-GFP. Third, by expressing LSN cDNA in ectopic whorls (where LUG is expressed), the ectopic co-repressor activity will be tested. Fourth, to establish that LSN directly regulates AG, the electrophoretic mobility shift assay will be used to identify the binding sites of LSN within AG cis-regulatory elements. Further, LSN-VP16 chimeric protein expressed in transgenic plants will be used to establish the functional relevance of LSN binding to AG in vivo. Finally, physical interactions between LSN and LUG proteins will be tested using a co-immunoprecipitation assay. Other LSN-interacting proteins will be identified by a yeast two-hybrid screen. The proposed study will elucidate the basic mechanism of transcriptional repression in higher plant development and provide excellent training opportunities for undergraduate and graduate students.