Determining the roles and regulation of MIRNA156 genes in reproductive development of annual and perennial Brassicaceae species

确定 MIRNA156 基因在一年生和多年生十字花科植物生殖发育中的作用和调控

• 批准号: 391673110
• 负责人: Professor Dr. George Coupland, Ph.D.
• 金额: --
• 依托单位: National Natural Science Foundation of China
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/391673110?language=en
• 关键词: Determining; roles; regulation; MIRNA156; genes

MicroRNAs (miRNAs) are short non-coding RNAs that provide robustness to regulatory circuits and stabilize developmental decisions by annealing to and repressing expression of target messenger RNAs (mRNAs). Mature miRNAs are generated by processing longer pri-miRNAs that are encoded by MIRNA (MIR) genes. Understanding the regulation and patterns of expression of mature miRNAs is complicated because each miRNA is encoded by many MIRs, which often precludes precisely positioning the miRNA in regulatory pathways. In plants, miR156/7 negatively regulates expression of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors, which have important roles in diverse plant species in controlling different aspects of development, including initiation of flowering and flower development. Mature mir156/7 is encoded by 12 genes in the model plant species Arabidopsis thaliana, but the function or expression patterns of individual MIR156/7 genes in the shoot apex where flowering occurs is almost unknown. We and our Chinese co-operation partner (Professor Jiawei Wang, Chinese Academy of Sciences, Shanghai), with whom we have co-operated in analysing the role of mature miR156 in controlling flowering time, propose to collaborate to systematically analyse the functions of the MIR156/7 genes in A. thaliana. By sharing the work involved, we aim to define which of these MIR genes are important in regulating flowering time and floral meristem identity. CRISPR-cas9 genome editing is being used in each laboratory to efficiently generate mutations in each of the MIR156/7 genes, and at least two loss of function alleles will be isolated in each gene. In parallel the temporal and spatial patterns of each MIR gene in the shoot meristem and developing flower will be assessed using gene fusions to fluorescent proteins. Based on their patterns of expression, combinations of mir156/7 mutations will be generated and their effects on flowering assessed phenotypically. Similarly, the gene fusions will be used to follow transcriptional regulation of each MIR156/7 gene at the shoot meristem by transcription factors, environmental cues, hormones and metabolites implicated in the regulation of mature miR156/7 levels. Finally, the flowering response of perennial relatives of A. thaliana to cold winter temperatures has been shown by the applicant and co-operating partner to be dependent on miRNA156/7 levels and based on the results obtained in A. thaliana, we will assess which of the MIR156/7 genes are responsible for this effect. Overall these systematic analyses will define which MIR156/7 genes are involved in regulating floral induction and floral meristem identity, and form an essential basis for positioning these genes within the regulatory circuits that control the early stages of plant reproductive development.

MicroRNAs(MiRNAs)是一种短小的非编码RNAs，通过与靶信使RNAs(MRNAs)的结合和抑制表达，为调控电路提供稳健性，并稳定发育决策。成熟的miRNAs是通过处理由miRNA(MiR)基因编码的较长的pri-miRNAs而产生的。理解成熟miRNAs的调控和表达模式是复杂的，因为每个miRNA由许多miR编码，这往往排除了miRNA在调控途径中的准确定位。在植物中，miR156/7负调控类鳞状启动子结合蛋白(SPL)转录因子的表达，这些转录因子在不同植物中控制不同发育的不同方面，包括开花和花发育。在模式植物拟南芥中，成熟的miR156/7由12个基因编码，但单个miR156/7基因在开花的顶端的功能或表达模式几乎未知。我们和我们的中国合作伙伴(上海中科院王家伟教授)合作分析了成熟miR156在控制开花时间中的作用，建议合作系统地分析A.thaliana中miR156/7基因的功能。通过分享所涉及的工作，我们的目标是确定这些MIR基因中的哪些在调节开花时间和花分生组织特性方面是重要的。CRISPR-Cas9基因组编辑正在每个实验室使用，以有效地在每个miR156/7基因中产生突变，并在每个基因中分离出至少两个功能缺失的等位基因。同时，每个MIR基因在新梢分生组织和发育中的花中的时间和空间模式将通过与荧光蛋白的基因融合来评估。根据它们的表达模式，将产生miR156/7突变的组合，并评估它们对开花的影响。同样，基因融合将被用来跟踪在茎分生组织中每个miR156/7基因的转录调控，这些转录因子、环境信号、激素和代谢产物与成熟miR156/7水平的调控有关。最后，申请者和合作伙伴已经证明多年生拟南芥的多年生亲缘植物对寒冷冬季温度的开花反应取决于miRNA156/7水平，并根据在拟南芥中获得的结果，我们将评估哪些miR156/7基因对这种影响负责。总体而言，这些系统分析将确定哪些miR156/7基因参与调控花的诱导和花分生组织的鉴定，并形成重要的基础，将这些基因定位在控制植物生殖发育早期阶段的调控回路中。