Molecular and Genetic Analysis of SHORT INTEGUMENTS1 (SIN1)

植物细胞间信号传递的分子机制还知之甚少。短整合基因SIN1在胚珠分生组织功能和两层细胞层之间的通讯中起重要作用，在早期胚胎发育中起着母体和晚期合子后的重要作用。SIN1基因由20个外显子组成，编码一个1909个氨基酸的长蛋白质，具有一个两段核定位信号、一个RNA解旋酶C基序、两个RNaseIII基序和两个与果蝇Staufen蛋白类似的羧基末端双链RNA结合基序。这一建议测试了SIN1基因作用机制的模型。我们将研究SIN1基因的选择性剪接。可能的SIN1启动子-增强子区域的鉴定将通过将周围区域与报告基因融合并通过分析报告基因在体内的表达来进行。SIN1蛋白将通过表达MYC标记的(分别为C-末端和N-末端标记)的蛋白质版本并随后进行免疫定位来定位在细胞中。SIN1的潜在靶点将通过相互作用的RNA与抗SIN1-MYC抗体的免疫共沉淀，然后通过RT-PCR构建(在三杂交载体中)cDNA库。这个文库的成员将通过酵母中的三杂交试验进行确认，并将对选定的候选成员进行测序。拟议的实验将启发植物形态发生中一个研究较少的方面的模型，即跨细胞边界的基因表达的转录后调控。

Molecular mechanisms of cell-cell signaling in plants are poorly understood. The SHORT INTEGUMENTS1 (SIN1) gene is important for meristem function, communication between two cell layers in the ovule, and plays essential maternal and late post-zygotic roles in early embryogenesis. The SIN1 gene, containing twenty exons, instructs a 1909 amino acid long protein with a bipartite nuclear localization signal, an RNA helicase C motif, two RNase III motifs and two carboxy-terminal double stranded RNA binding motifs similar to that of the Staufen protein of Drosophila. This proposal tests models of the mechanism of function of SIN1 gene. Alternative splicing of the SIN1 mRNA will be investigated. Identification of the putative SIN1 promoter-enhancer region will be carried out by fusion of the surrounding region to reporter genes and by assaying expression of the reporters in vivo. The SIN1 protein will be localized in cells by expressing MYC-tagged (C-terminal and N-terminal tags, separately) versions of the protein followed by immunolocalization. Putative targets of SIN1 will be identified by co-immunoprecipitation of interacting RNA with anti-MYC-antibody against SIN1-MYC, followed by RT-PCR to construct a library of cDNA (in a three-hybrid vector). Members of this library will be confirmed by three-hybrid assays in yeast, and selected candidates will be sequenced. The proposed experiments will enlighten one model of a poorly investigated aspect of morphogenesis in plant, that of post-transcriptional regulation of gene expression across cellular boundaries.