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

MicroRNAs （miRNAs）是一种短的非编码rna，通过退火和抑制靶信使rna （mrna）的表达，为调控回路提供鲁棒性，并稳定发育决策。成熟MIRNA是通过加工MIRNA （MIR）基因编码的较长的pri- MIRNA而产生的。了解成熟miRNA的调控和表达模式是复杂的，因为每个miRNA都由许多miRNA编码，这往往妨碍了在调控途径中精确定位miRNA。在植物中，miR156/7负调控SQUAMOSA启动子结合蛋白样（SPL）转录因子的表达，SPL转录因子在不同植物物种的发育过程中起着重要作用，包括控制开花起始和花发育。成熟的mir156/7在模式植物拟南芥中由12个基因编码，但mir156/7基因在开花发生的茎尖的功能或表达模式几乎未知。我们和我们的中国合作伙伴（中国科学院王佳伟教授，上海）合作分析了成熟miR156在控制开花时间中的作用，我们提议合作系统地分析拟南芥miR156 /7基因的功能。通过分享所涉及的工作，我们的目标是确定哪些MIR基因在调节开花时间和花分生组织身份方面是重要的。每个实验室都在使用CRISPR-cas9基因组编辑技术，以有效地在每个MIR156/7基因中产生突变，并且每个基因中至少会分离出两个功能缺失的等位基因。同时，每个MIR基因在茎分生组织和发育中的花中的时间和空间模式将通过荧光蛋白的基因融合来评估。基于它们的表达模式，将产生mir156/7突变组合，并对其对开花的影响进行表型评估。同样，基因融合将被用于跟踪茎分生组织中每个MIR156/7基因的转录调控，通过转录因子、环境线索、激素和代谢产物参与成熟MIR156/7水平的调控。最后，拟南芥多年生亲缘植物对冬季低温的开花反应取决于miRNA156/7的水平，根据拟南芥的研究结果，我们将评估MIR156/7基因中的哪一个对这种效应负责。总的来说，这些系统的分析将确定哪些MIR156/7基因参与调控花诱导和花分生组织的身份，并为在控制植物生殖发育早期阶段的调控回路中定位这些基因提供必要的基础。

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.