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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712787
• 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启动子结合蛋白样(SPL)转录因子家族来调节许多植物性状。Spls进而调节包含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它有能力调节一个庞大的下游基因网络。到目前为止，我们已经分析了miR156在从模式植物到主要作物(包括紫花苜蓿)中的功能，我们发现miR156通过下调至少7个SPL基因来影响牧草产量和品质、植株结构、开花时间、根发育、结瘤和非生物胁迫耐受性。 我们建议通过研究miR156在紫花苜蓿中扮演的多种角色的分子基础来扩展这项研究。我们的目标是将miR156沉默的SPL基因和目标基因与紫花苜蓿中通常受miR156影响的特定性状相关联。分析将涉及miR156和spl错误表达的紫花苜蓿基因型的分子、遗传、表型和生理特性。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)，结合对基因表达改变的紫花苜蓿植株的表型和生理分析，寻找SPL调控的下游基因。其目标是产生关于miR156-SPL遗传网络组件的知识，将功能归因于紫花苜蓿中至少一些SPL基因和目标基因。我们的目标是： 1.研究miR156和靶标Spl在非生物胁迫，特别是干旱、铝和盐分胁迫中的作用。这项拟议的研究将利用现有的SPL沉默和过表达的紫花苜蓿基因型来测试非生物胁迫的耐受性，并确定哪些SPL(SPL)参与其中。我们将进行一系列的生理、表型和分子分析，以评估抗逆性并确定基因的特征。 2.评价miR156/SPL模块在确定紫花苜蓿根构型、结瘤和固氮中的作用。通过表征沉默或过度表达miR156靶向SPL基因的植物，拟议的研究将确定与上述特征有关的SPL基因。 3.筛选与上述性状相关的SPL调控靶基因。表现出最明显的形态或生理变化的SPL RNAi植株将被NGS分析以确定差异表达的基因，这些基因将通过芯片-qPCR进一步评估SPL结合，并用于进一步的分子和表型鉴定使用沉默和过度表达的植物。 本研究将加深我们对紫花苜蓿及其他植物中miR156-SPL基因调控网络的理解。这些发现可能被利用来通过基因编辑或标记辅助育种来改良紫花苜蓿的各种性状。