Molecular and Genetic Analysis of SHORT INTEGUMENTS1 (SIN1)

植物细胞-细胞信号传导的分子机制尚不清楚。短INTEGUMENTS1 （SIN1）基因对分生组织功能、胚珠中两个细胞层之间的通讯至关重要，在早期胚胎发生中起着重要的母系和后期合子作用。SIN1基因包含20个外显子，它指示一个含有1909个氨基酸的长蛋白，该蛋白具有两部分核定位信号、一个RNA解旋酶C基序、两个RNase III基序和两个类似果蝇Staufen蛋白的羧基端双链RNA结合基序。本研究旨在验证SIN1基因功能机制的模型。将研究SIN1 mRNA的选择性剪接。推测的SIN1启动子增强子区域的鉴定将通过周围区域与报告基因的融合和报告基因在体内的表达进行。SIN1蛋白将通过表达myc标记（c端和n端分别标记）的蛋白版本，然后进行免疫定位，从而在细胞中定位。假设的SIN1靶点将通过相互作用的RNA与针对SIN1- myc的抗myc抗体的共免疫沉淀来鉴定，然后通过RT-PCR构建cDNA文库（在三杂交载体中）。该文库的成员将通过酵母的三杂交试验来确认，并对选定的候选物进行测序。提出的实验将启发植物形态发生的一个研究不足的方面的一个模型，即基因表达的转录后调控跨越细胞边界。

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.