Discovery Research towards Alfalfa Improvement

苜蓿改良的发现研究

• 批准号: RGPIN-2018-04241
• 负责人: Hannoufa, Abdelali
• 金额: $ 2.62万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686597
• 关键词: Discovery; Research; towards; Alfalfa; Improvement

MicroRNA156 (miR156) regulates numerous plant traits by silencing SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors family. SPLs in turn regulate other downstream genes (target genes) that contain a SPL binding cis acting element. Thus, the propensity of miR156 to affect multiple plant traits is due to its ability to regulate a large network of downstream genes. So far, we have analyzed miR156 function in plants ranging from model plants to major crops, including Medicago sativa (alfalfa), where we found that miR156 affects forage yield and quality, plant architecture, flowering time, root development, nodulation, and abiotic stress tolerance by downregulating at least seven SPL genes. ******We propose to expand this research by investigating the molecular basis for the multitude of roles that miR156 plays in alfalfa. We aim to associate each of miR156-silenced SPL genes and target genes to specific traits normally affected by miR156 in alfalfa. Analysis will involve molecular, genetic, phenotypic and physiological characterization of miR156 and SPL mis-expression alfalfa genotypes. We will identify SPL-regulated downstream genes through Next Generation RNA Sequencing (NGS), promoter analysis, and chromatin immunoprecipitation-qPCR (ChIP-qPCR), combined with phenotypic and physiological analyses of alfalfa plants with altered gene expression. The goal is to generate knowledge about the components of the miR156-SPL genetic network, ascribe functions to at least some of the SPL genes and target genes in alfalfa. Our objectives are:******1. To characterize role of miR156 and target SPL in abiotic stress, specifically drought, aluminum and salinity. The proposed research will use existing SPL silencing and overexpression alfalfa genotypes to test for abiotic stress tolerance, and determine which SPL (SPLs) is involved. We will conduct a range of physiological, phenotypic and molecular analyses to evaluate stress tolerance and characterize genes. ******2. To assess the role of miR156/SPL module in determining root architecture, nodulation and nitrogen fixation in alfalfa. By characterizing plants with silenced or overexpressed miR156-targeted SPL genes, the proposed research will identify SPL genes responsible for above traits. ******3. To identify SPL-regulated target genes involved in above traits. SPL RNAi plants showing the most visible morphological or physiological changes will be analysed by NGS to identify differentially expressed genes, which will be further assessed for SPL binding by ChIP-qPCR, and used for further molecular and phenotypic characterization using silencing and overexpression plants.******This research will deepen our understanding of the miR156-SPL gene regulatory network in alfalfa, and potentially other plants. The findings could be exploited to improve various alfalfa traits by gene editing or marker-assisted breeding.

MicroRNA156 (miR156) 通过沉默 SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) 转录因子家族来调节多种植物性状。 SPL 反过来调节包含 SPL 结合顺式作用元件的其他下游基因（靶基因）。因此，miR156 影响多种植物性状的倾向是由于其调节下游基因的大型网络的能力。到目前为止，我们已经分析了 miR156 在从模式植物到主要作物（包括苜蓿）的植物中的功能，我们发现 miR156 通过下调至少 7 个 SPL 基因来影响饲料产量和质量、植物结构、开花时间、根系发育、结瘤和非生物胁迫耐受性。 ******我们建议通过调查 miR156 在苜蓿中发挥的多种作用的分子基础来扩展这项研究。我们的目标是将每个 miR156 沉默的 SPL 基因和靶基因与苜蓿中通常受 miR156 影响的特定性状相关联。分析将涉及 miR156 和 SPL 错误表达苜蓿基因型的分子、遗传、表型和生理特征。我们将通过下一代 RNA 测序 (NGS)、启动子分析和染色质免疫沉淀 qPCR (ChIP-qPCR) 结合基因表达改变的苜蓿植物的表型和生理分析来识别 SPL 调节的下游基因。目标是生成有关 miR156-SPL 遗传网络组成部分的知识，将功能归因于苜蓿中至少一些 SPL 基因和目标基因。我们的目标是：*****1。表征 miR156 和目标 SPL 在非生物胁迫（特别是干旱、铝和盐度）中的作用。 拟议的研究将使用现有的 SPL 沉默和过度表达苜蓿基因型来测试非生物胁迫耐受性，并确定涉及哪种 SPL (SPL)。 我们将进行一系列生理、表型和分子分析，以评估应激耐受性并表征基因。 ******2.评估 miR156/SPL 模块在确定苜蓿根结构、结瘤和固氮方面的作用。 通过表征具有沉默或过度表达的 miR156 靶向 SPL 基因的植物，拟议的研究将鉴定负责上述性状的 SPL 基因。 ******3.鉴定与上述性状相关的 SPL 调控靶基因。 显示出最明显的形态或生理变化的 SPL RNAi 植物将通过 NGS 进行分析，以确定差异表达的基因，并通过 ChIP-qPCR 进一步评估 SPL 结合，并用于使用沉默和过度表达植物进行进一步的分子和表型表征。******这项研究将加深我们对苜蓿和其他植物中 miR156-SPL 基因调控网络的了解。这些发现可用于通过基因编辑或标记辅助育种来改善苜蓿的各种性状。