Characterization of novel repressors of the embryonic maturation program in Arabidopsis

描述（由申请人提供）：人类消耗的大部分卡路里直接或间接来自种子，种子的营养特性是人类健康的主要贡献者。种子成熟包括导致可食用贮藏产物积累和干燥的所有过程。对于导致种子成熟的发育转换的时间是如何控制的，还没有明确的理解。该项目的长期目标是了解调节种子成熟的遗传机制，特别是防止成熟基因在种子发育早期和萌发后表达的途径。了解成熟是如何调节的将增加我们对发育转录开关的理解。它还可以帮助操纵作物种子的营养成分。该建议集中在两个转录因子，ASIL1和ASIL2（ASIL基因），这是很好的候选人在模式植物拟南芥成熟的阻遏物。由于种子成熟程序似乎在开花植物中是保守的，因此这些发现可以应用于作物植物。该提案旨在研究ASIL基因生物学的两个独立方面。第一个目标是研究这些基因在发育过程中的作用，通过分析它们调节哪些基因和途径。通过比较它们在种子的两个隔室（胚和胚乳）和幼苗中的作用，可以评估它们功能的冗余（或缺乏）。这将有助于确定在种子发育期间和萌发后是否有相同的基因组调节成熟。第二个目的是了解AISL基因的转录调控，看看它们的启动子是否以及哪些离散区域在不同时间和组织类型指导表达。这些信息将使未来的上游监管机构的搜索。第一个目标将通过在适当的阶段收集组织（在胚胎和胚乳的情况下使用激光捕获显微解剖），分离RNA，使用RNAseq定量转录本的丰度，并使用适当的生物信息学分析工具比较不同的组织类型和阶段来实现。第二个目标将通过产生启动子的选定片段与报告基因GUS和GFP的转录和翻译融合，并研究它们在转基因植物中的表达来实现。

DESCRIPTION (provided by applicant): Most of the calories humans consume come directly or indirectly from seeds, and the nutritional properties of seeds are major contributors to human health. Seed maturation includes all the processes that lead to the accumulation of edible storage products and desiccation. There is no clear understanding on how the timing of the developmental switch that leads to seed maturation is controlled. The long-term goal of this project is to understand the genetic mechanisms that regulate seed maturation, in particular the pathways that prevent the maturation genes to be expressed early in seed development and after germination. Understanding how maturation is regulated will increase our understanding of developmental transcriptional switches in general. It may also help manipulate the nutritional content of crop seeds. This proposal focuses on two transcription factors, ASIL1 and ASIL2 (the ASIL genes), which are good candidates for repressors of maturation in the model plant Arabidopsis thaliana. Because the seed maturation programs appear to be conserved among flowering plants, the findings could be applied to crop plants. This proposal aims to investigate two separate aspect of the biology of the ASIL genes. The first aim is to study the roles of these genes during development, by analyzing which genes and pathways they regulate. By comparing their actions in two compartments of the seed (embryo and endosperm) and in the young seedling, the redundancy (or lack thereof) of their functions can be evaluated. This will help establish whether the same set of genes regulate maturation during seed development and after germination. The second aim is to understand the transcriptional regulation of the AISL genes, to see whether and which discreet regions of their promoters direct expression at different times and tissue types. This information will enable the future search for upstream regulators. The first aim will be accomplished by collecting tissue at the appropriate stages (using Laser Capture MicroDissection in the case of the embryo and endosperm), isolating RNA, quantifying the abundance of transcripts using RNAseq, and comparing the different tissue types and stages using the appropriate bioinformatic analysis tools. The second aim will be addressed by generating transcriptional and translational fusions of selected segments of the promoters to the reporter genes GUS and GFP, and studying their expression in transgenic plants.