Molecular and Genetic Analysis of SHORT INTEGUMENTS1 (SIN1)

植物细胞间信号传导的分子机制知之甚少。 SIN 1基因对分生组织的功能、胚珠中两层细胞之间的通讯起着重要作用，在早期胚胎发生中起着重要的母性和后期合子后作用。 SIN 1基因含有20个外显子，编码一个1909个氨基酸长的蛋白质，具有一个二分核定位信号、一个RNA解旋酶C基序、两个RNA酶III基序和两个羧基末端双链RNA结合基序，与果蝇的Staufen蛋白相似。 该建议测试SIN 1基因的功能机制的模型。将研究SIN 1 mRNA的选择性剪接。 推定的SIN 1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以及通过测定报告基因的体内表达来进行。 SIN 1蛋白将通过表达MYC标记（C-末端和N-末端标签，分别）形式的蛋白，随后通过免疫定位而定位在细胞中。 SIN 1的推定靶标将通过相互作用RNA与针对SIN 1-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中的三杂交试验进行确认，并对选定的候选人进行测序。 拟议中的实验将启发一个模型的形态发生在植物中，跨细胞边界的基因表达的转录后调控方面的研究不足。

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.