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

功能性细胞和器官是由成千上万个基因组成的惊人集合的产物，这些基因由不同水平的调控精心编排。在最基本的水平上，每个基因表达的可能性都是由基因开关控制的，基因开关是一种调控序列基序，被称为转录因子的蛋白质与之结合。这些调控基序和它们的组合可能存在于数千个，然而，确切的数量是未知的。它们中很少有人是众所周知的或经过充分研究的，但它们对功能细胞，器官和生物体的发育至关重要。我的NSERC发现资助的研究计划的重点是调控基序和植物转录基因调控使用的方法，将生物信息学（计算方法）与基因组学（分子方法）。长期目标是了解不同的基因调控层和基因表达的机制，以及它们如何有助于植物功能解剖。我的方法是1）测量感兴趣的细胞中的转录组（使用激光捕获显微切割和RNA测序）鉴定共调节基因，2）使用从头基序发现算法发现调节基序（例如，我实验室开发的Seeder）在基因侧翼区，3）通过瞬时和稳定的遗传转化和靶向基因组编辑（CRISPR/Cas9）验证这些基序在植物中的功能。我们的模型系统是大豆，这是加拿大越来越受欢迎的作物，长期以来一直处于功能基因组学研究的前沿，拥有测序的基因组和大量其他遗传和基因组资源。我们处于一个非常有利的位置，开始真正的细胞特异性基因表达研究，在那里我们将探索启动子和其他调控基序的共调控基因组。拟议的研究追求在大豆分生组织中的主调节因子（转录因子）和连接基序，以及大豆宿主和大豆相关细菌（病原体和共生体）和大豆孢囊线虫（大豆作物最经济损失的原因）界面的细胞上的令人兴奋的发现。这项研究通过增加我们对植物发育和功能的基本知识，通过生物技术和作物改良策略的进展，通过继续开发生物信息学工具以进行更好的序列分析，以及通过在这个令人兴奋和快速扩展的领域中对高素质研究生的重要培训，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和研究生以及博士后提供学习现代分子，基因组学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. **