番茄果实成熟相关外显子环化RNA（circRNA）的鉴定和初步功能分析

Tomato is an economic important vegetable crop worldwide. As the model system for studying fruit ripening, tomato fruit ripening has been characterized in detail at the molecular and genetic levels. Ethylene regulates tomato fruit ripening, but how ethylene-dependent and -independent genes interact to control specific physiological aspects of ripening remains to be further characterized. Exonic circular RNAs (circRNAs) are a class of newly characterized non-coding RNAs that regulate gene expression in humans and animals, but their function in growth and development of plants are poorly characterized. By using high-throughput circular RNA sequencing and bioinformatic analysis, we have identified thousands of putative circRNA sites from tomato fruits at different ripening stages. In addition, a number of circRNAs were confirmed by sequencing and RNase-R protection assay, including six circRNAs originated from ripening regulator ACO2 and ETR4. In this proposal, molecular and functional characterization of circRNAs involved in ethylene-dependent and –independent tomato fruit ripening will be performed: (1) further identification of putative circRNA sites involved in tomato fruit ripening and/or regulated by Never-ripening mutant (Nr); (2) molecular characterization of a large number of circRNAs associated with fruit ripening and/or Nr mutant; (3) functional analysis of two to four circRNAs originated from ethylene and ripening associated genes by over-expression and suppress-expression in tomato. This study will further our understanding of fruit ripening and ethylene physiology at the molecular levels.

番茄是重要的蔬菜作物。作为研究果实成熟的模式植物，其果实成熟机理在分子和遗传水平已有深入研究。乙烯调控番茄果实成熟，但乙烯依赖和非依赖途径中，相关基因如何互作并调控成熟的某一具体生理现象仍需进一步阐述。外显子环化RNA（circRNA）是近年来被证明参与基因表达调控的重要非编码RNA成员，其在植物中的研究极少。通过环形RNA高通量测序，我们从番茄果实中检测出数千个潜在circRNA位点，并用分子手段验证了部分位点，包括来源于成熟调控基因（ACO2和ETR4）的6个circRNA。本项目拟开展番茄成熟（乙烯依赖和非依赖途径）相关circRNA的功能研究：（1）进一步挖掘成熟调控和/或乙烯敏感突变体Nr相关的circRNA；（2）分子鉴定一批所发现circRNA；（3）利用过表达和抑制表达，初步解析2-4个乙烯和成熟相关circRNA的功能。本研究将进一步丰富果实成熟和乙烯调控的分子机理。

circRNA，是一类由前体mRNA反向剪接形成的闭合环状RNA。近年来的研究表明，真核生物circRNA具有调控基因表达的重要生物学功能，然而其在植物中的存在及功能研究却非常少。本研究首先通过对番茄不同成熟期果实中环形RNA的高通量测序鉴定及部分位点的分子生物学验证，证实了circRNA在番茄中的普遍存在，然后通过对部分circRNA的表达模式分析及其在番茄中的过表达分析，揭示了circRNA的重要生物学功能及其可能的作用方式。通过深度测序我们鉴定到了大量circRNA位点信息（Segemehl v0.1.7 软件鉴定到环形RNA共9598个，CIRI v2.0.5软件鉴定到circRNA共1018个，二者共同鉴定到的位点为796个）。利用RNase R耐性实验和Sanger测序我们完成了53个circRNA的分子鉴定工作。通过对8个circRNA在番茄AC和Nr突变体中的表达模式分析，我们发现circRNA的表达与果实成熟有相关性。我们构建了circRNA在植物中的过表达体系，并成功地完成了两个circRNA（ PSY1-circ1和PDS-circ1）在番茄中的高水平、精准表达。进一步的表型分析表明，部分转基因株系中目的circRNA持续高水平表达，其来源基因mRNA、番茄红素及β-胡萝卜素含量均显著下降，果实不能正常变红；另一部分株系中目的circRNA在果实成熟后期表达量下降，其来源基因mRNA、番茄红素和β-胡萝卜素等轻微下降或无显著变化，果实正常变红。因此，circRNA可能通过抑制其来源基因mRNA表达的方式来实现其生物学功能，而这种作用方式可能依赖于circRNA的持续高水平表达。我们还发现，转基因番茄中存在微量的载体来源线性RNA，因此也不排除这些RNA产生的共抑制现象导致了表型的产生。此外，我们构建了以靶向back-splice位点的人工mircoRNA介导的circRNA抑制表达体系，并完成了两个circRNA抑制表达载体的构建和番茄遗传转化工作，相关检测和表型分析正在进行中。本研究揭示了植物circRNA调控基因表达的可能作用方式，给出了植物circRNA具有重要生物学功能的证据，具有重要研究意义。