LARSON, An Arabidopsis Homeobox Gene in Flower Development

0212847 Liu长期目标是揭示决定多细胞生物体中细胞如何承担其发育命运的机制。 以拟南芥花为模型，研究雄蕊和心皮身份特化基因AGAMOUS（AG）的时空特异性表达调控机制。 AG mRNA通常在花的内部两个轮中表达，并且在外部两个轮中不存在。 LEUNIG（LUG），一个假定的转录共阻遏物与酵母共阻遏物TUP的序列相似，以前被证明在抑制AG表达的外两个螺旋中发挥至关重要的作用。 由于LUG不编码DNA结合结构域，因此LUG如何被募集到AG顺式调节元件尚不清楚。 新发现的LARSON（LSN）基因是LUG DNA结合伴侣的极好候选者。 lsn-1突变增强了lug突变在AG调节中的缺陷。 此外，初步的数据表明，LSN编码的同源异型盒蛋白，可以结合到AG顺式调控元件，并可以在酵母中与LUG相互作用。 此外，由于LUG mRNA在所有的花轮中表达，LUG的外轮特异性抑制活性可以通过与外轮特异性因子的相互作用来赋予。 LSN能否在为LUG辅阻遏物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，将通过lsn-1突变体表型的转化拯救来确认LSN的分子身份。 将采用几种反向遗传学方法来鉴定lsn的其他等位基因。 其次，通过原位杂交和免疫定位或LSN-GFP来确定LSN的RNA和蛋白质表达谱。 第三，通过在异位螺旋（其中表达LUG）中表达LSN cDNA，将测试异位共阻遏物活性。 第四，为了确定LSN直接调节AG，电泳迁移率变动分析将用于鉴定AG顺式调节元件内的LSN结合位点。 此外，在转基因植物中表达的LSN-VP 16嵌合蛋白将用于建立LSN与AG结合的体内功能相关性。 最后，将使用免疫共沉淀试验测试LSN和LUG蛋白之间的物理相互作用。 其他的LSN相互作用蛋白将通过酵母双杂交筛选来鉴定。 该研究将阐明转录抑制在高等植物发育中的基本机制，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.