Discovering mechanisms of gene regulation in plant cell specific transcriptomes using bioinformatics and microgenomics

功能细胞和器官是数以万计的基因的惊人集合的产物，这些基因受到不同水平的调控。在最基本的层面上，每个基因表达的可能性是由基因开关控制的，也就是被称为转录因子的蛋白质与之结合的调控序列基序。这些调控基序及其组合可能存在数千个，然而，确切的数字尚不清楚。很少有人熟知或研究它们，但它们对功能细胞、器官和生物体的发育至关重要。 我的NSERC发现资助的研究项目专注于调控基序和植物转录基因调控，使用一种将生物信息学(计算方法)与基因组学(分子方法)相结合的方法。长期目标是了解基因表达的不同基因调节层和机制，以及它们如何对植物功能解剖学做出贡献。我的方法是1)测量目标细胞中的转录组(使用激光捕获显微切割和RNA测序)以确定共调控基因，2)使用新的基序发现算法(例如我实验室开发的Seeder)在基因侧翼区域发现调控基序，3)通过瞬时和稳定的遗传转化和有针对性的基因组编辑(CRISPR/Cas9)验证植物基序在植物中的功能。我们的模式系统是大豆，这是一种在加拿大越来越受欢迎的农作物，长期以来一直处于功能基因组研究的前沿，拥有已测序的基因组和大量其他遗传和基因组资源。我们处于一个很好的位置，可以开始真正的细胞特异性基因表达研究，在那里我们将探索共同调控基因组中的启动子和其他调控基序。这项拟议的研究在大豆分生组织中的主要调控因子(转录因子)和连锁基序以及大豆寄主和大豆相关细菌(病原体和共生体)和大豆胞囊线虫(大豆作物最大的经济损失原因)界面上的细胞方面取得了令人兴奋的发现。这项研究通过增加我们对植物发育和功能的基本知识，通过在生物技术和作物改良战略方面取得进展，通过继续开发生物信息学工具来更好地进行序列分析，通过在这个令人兴奋和迅速扩展的领域对高素质研究生进行关键培训，产生了宝贵的影响。在拟议的研究期间，HQP的培训将继续以同样的高标准为本科生、研究生和博士后提供学习现代分子、基因组和生物信息学技术和科学思维的机会，使他们能够在高影响力的期刊上发表文章，并与学术界和工业界非常需要的技能和知识竞争。

Functional cells and organs are the products of an amazing collection of tens of thousands of genes, orchestrated by different levels of regulation. At the most basic level, each gene’s possibility of expression is controlled by gene switches, regulatory sequence motifs, to which proteins called transcription factors bind. These regulatory motifs and combinations of them probably exists in the thousands, however, the exact number is unknown. Few of them are well-known or well-studied, yet they are crucial for development of functional cells, organs and organisms. My NSERC Discovery funded research program focuses on regulatory motifs and plant transcriptional gene regulation using an approach that integrates bioinformatics (computational methods) with genomics (molecular methods). The long-term objective is to understand different gene regulating layers and mechanisms of gene expression and how they contribute to plant functional anatomy. My approach is to 1) measure the transcriptome in cells of interest (using laser capture microdissection and RNA-Sequencing) to identify co-regulated genes, 2) discover regulatory motifs using de novo motif discovery algorithms (e.g. Seeder, developed in my lab) in the gene flanking regions, and 3) validate the function of the motifs in planta through transient and stable genetic transformation and targeted genome editing (CRISPR/Cas9). Our model system is soybean, an increasingly popular crop plant in Canada that has long been at the forefront of functional genomics research, with a sequenced genome and a multitude of other genetic and genomic resources available. We are in an excellent position to embark on truly cell specific gene expression studies, where we will explore promoters and other regulatory motifs in groups of co-regulated genes. The proposed research pursues exciting findings on master regulators (transcription factors) and linked motifs in soybean meristems, as well as cells at the interface of soybean host and soybean associated bacteria (pathogens and symbionts) and soybean cyst nematode (the cause of the most economical losses to soybean crops). This research has invaluable impact by increasing our basic knowledge of plant development and function, by making progress for biotechnology and crop improvement strategies, by continuing to develop bioinformatics tools for better sequence analysis and by crucial training of highly qualified graduate students in this exciting and rapidly expanding field. The training of HQP to take place during the proposed research will continue with the same high standards to provide opportunities for undergraduate and graduate students and postdocs to learn modern molecular, genomics and bioinformatics techniques and scientific thinking, allowing them to publish in high impact journals and to be competitive with skills and knowledge that are in great demand in academia as well as in industry.