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）通过沉默鳞状启动子结合蛋白（如（SPL）转录因子家族）来调节许多植物特征。 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影响多种植物特征的倾向是由于其调节大型下游基因网络的能力。到目前为止，我们已经分析了从模型植物到主要农作物（包括Medicago Sativa（苜蓿））的MiR156功能，在那里我们发现MiR156通过下调至少七个Spl Genes来影响饲料产量和质量，植物建筑，开花时间，根源发育，结节，结节和非生物胁迫。 我们建议通过研究miR156在苜蓿中发挥的多种作用的分子基础来扩展这项研究。我们的目的是将miR156脱毛的SPL基因和靶基因与通常受苜蓿中miR156影响的特定特征联系起来。分析将涉及miR156和SPL错误表达苜蓿基因型的分子，遗传，表型和生理表征。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）鉴定出SPL调节的下游基因，并结合了苜蓿植物的表型和生理分析，其基因表达改变。目的是产生有关MiR156-SPL遗传网络组成部分的知识，将功能归因于至少某些SPL基因和靶基因的苜蓿中的功能。我们的目标是： 1。表征miR156和靶标在非生物应激中的作用，特别是干旱，铝和盐度。 拟议的研究将使用现有的SPL沉默和过表达苜蓿基因型来测试非生物胁迫耐受性，并确定涉及哪些SPL（SPL）。 我们将进行一系列生理，表型和分子分析，以评估胁迫耐受性和表征基因。 2。评估MiR156/SPL模块在苜蓿中确定根结构，结节和氮固定的作用。 通过以沉默或过表达的miR156靶向SPL基因表征植物，拟议的研究将确定负责上述特征的SPL基因。 3。确定涉及上述特征的EMP调节的靶基因。 NGS将分析显示出最明显的形态或生理变化的SPL RNAi植物，以鉴定差异表达的基因，该基因将通过CHIP-QPCR进一步评估SPL结合，并用于使用沉默和过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中MIR156-SPL基因调节网络的理解，并可能是其他植物。可以利用这些发现来通过基因编辑或标记辅助育种来改善各种苜蓿特征。