LARSON, An Arabidopsis Homeobox Gene in Flower Development

长期目标是揭示决定多细胞生物中细胞如何承担其发育命运的机制。本研究以拟南芥花为研究对象，探讨雄蕊和心皮鉴定规范基因AGAMOUS （AG）的时空特异性表达调控机制。AG mRNA通常在花的内两个轮中表达，而在花的外两个轮中不表达。LEUNIG （LUG）是一种推定的转录共抑制因子，其序列与酵母共抑制因子TUP相似，先前已被证明在抑制外两轮AG表达中起关键作用。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。新发现的LARSON （LSN）基因是LUG dna结合伴侣的极好候选基因。lsn-1突变增强了lug突变在AG调控中的缺陷。此外，初步数据表明，LSN编码一个同源盒蛋白，可以结合AG顺式调控元件，并可以与酵母中的LUG相互作用。此外，由于LUG mRNA在所有花轮中都有表达，因此LUG的外轮特异性抑制活性可能通过与外轮特异性因子的相互作用而获得。LSN能否在提供LUG共抑制因子的时空特异性方面发挥关键作用？提出的实验旨在验证这些假设。首先，通过对LSN -1突变型的转化拯救来确认LSN的分子身份。将采用几种反向遗传方法来鉴定lsn的其他等位基因。其次，通过原位杂交和免疫定位或LSN- gfp测定LSN的RNA和蛋白质表达谱。第三，通过在异位螺旋（LUG表达的地方）中表达LSN cDNA，将测试异位共抑制因子的活性。第四，为了确定LSN直接调控AG，将使用电泳迁移率转移法确定AG顺式调控元件中LSN的结合位点。此外，将利用转基因植物中表达的LSN- vp16嵌合蛋白，在体内建立LSN与AG结合的功能相关性。最后，LSN和LUG蛋白之间的物理相互作用将使用共免疫沉淀法进行测试。其他与lsn相互作用的蛋白将通过酵母双杂交筛选来鉴定。该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.