番茄果实中乙烯相关circRNAs的分离及其与miRNAs的协同调控功能研究

Tomato is a model system for study fruit ripening and ethylene, and tomato fruit ripening is a highly harmonious coordination genetic regulation process. Ethylene is the initiation factor of fruit ripening and the ethylene pathway is regulated by many factors. CircRNAs and miRNAs are newly discovered post-transcriptional regulators which play important regulation roles in fruit ripening and plant hormone pathway and all kinds of physiological and biochemical processes. In order to explore the coordinated regulations of circRNAs and miRNAs on ethylene pathway in tomato, circRNAs and transcriptome and miRNAs are chosen for deep sequencing, bioinformatics and molecular biology methods are combined to analysis the targets and the coordinated regulations of circRNAs, mRNA and miRNAs. Identify the circRNAs involved in ethylene pathway and analysis and verify the coordinated regulation functions of circRNAs and miRNAs in ethylene biosynthesis and signal transduction, all of which would lay a theoretical foundation for illuminating the mechanisms of the participation of non-coding RNAs in ethylene signal pathway.

番茄是研究果实成熟和乙烯的模式材料，其成熟衰老是一个高度协调的遗传调控过程，乙烯是果实成熟衰老的启动因子且乙烯信号途径受很多因子调控。CircRNAs和miRNAs是新发现的转录后调控因子，其功能多种多样，涉及到果实成熟衰老和植物激素调控等许多生理生化过程。为了研究circRNAs和miRNAs协同调控乙烯信号途径的功能，我们采用生物信息学和分子生物学相结合的方法，对番茄果实中circRNAs、转录组、miRNAs进行深度测序，通过对circRNAs和miRNAs靶基因预测和功能分析、circRNAs、mRNA和miRNAs的协同表达关系分析，分离出与乙烯信号途径相关circRNAs，并对circRNAs与miRNAs协同调控乙烯生物合成和信号转导途径的功能进行分析和转基因验证，为最终阐明非编码RNAs参与调控乙烯信号途径的机理奠定理论基础。

番茄是研究果实成熟衰老的模式材料，其成熟衰老过程是一个高度协调的遗传调控过程，乙烯是果实成熟衰老的启动因子且乙烯的信号途径是受很多转录因子调控，MiRNA具有通过直接的碱基互补配对结合靶向mRNA的非编码区，进而影响其翻译的功能。circRNA分子富含miRNA结合位点，在细胞中起到miRNA海绵 (miRNA sponge) 的作用，进而解除miRNA对其靶基因的抑制作用，升高靶基因的表达水平, 这一作用机制被称为竞争性内源RNA(ceRNA)机制。本项目以绿熟期、红熟期和乙烯信号途径突变体Gr的番茄果实为材料，采用深度测技术、生物信息学和分子生物学相结合的方法，在绿熟期和红熟期比较组中，共发现了65个差异表达的circRNAs和100个差异表达的miRNAs，在红熟期和Gr突变体的比较组中，发现了33个差异表达的circRNAs和34个差异表达的miRNAs。并对靶基因进行了预测和功能分析，3个差异表达的circRNAs的靶基因涉及乙烯的生物合成（ACO）和色素合成途径（ZISO）；27个差异表达的miRNAs的靶基因涉及乙烯的生物合成（ACS，ACO）和信号转导途径（ERF，MADS-box，EIN3等），研究建立了番茄果实成熟过程中的ceRNAs调控网络。通过构建circRNAs过表达载体，进行circRNAs的遗传转化与分子鉴定，成功培育circRNAs过表达转基因株系，验证了circRNAs调控乙烯途径的靶基因，为进一步明确非编码RNA参与番茄乙烯途径的机理研究提供理论依据。