番茄果实成熟转录因子和microRNAs的协同调控网络解析

Tomato fruit ripening involves a series of complex physiological and biochemical metabolism process, which was implemented by accurate regulation of multiple functional genes at transcriptional and post-transcriptional level. This project is proposed to construct synergistic regulatory network of tomato fruit ripening-related transcription factors and microRNAs through interaction relationship and coexpression relationship among microRNAs, transcription factors and ripening-related functional genes, which was regulated by the known key transcription factors (RIN, CNR, NOR,TAGL1, AP2a, FUL1, FUL2, MADS1) in the process of tomato fruit ripening. The purpose of this study is to clearly definite the synergistic regulatory pathway of controlling the fruit ripening-related genes expression by key transcription factors and microRNAs with the methods of RNA-seq, small RNA-seq, degradome analysis, EMSA, ChIP-PCR, WGCNA analysis, etc, which help to comprehensively and systematically clarify the regulation mechanism of functional gene expression in tomato fruit ripening, and provide new ideas of develop more storable and high quality tomato fruits.

番茄果实成熟涉及一系列复杂的生理生化代谢过程，并且是通过多个基因的精细调控所实现。这些基因表达调控主要有转录水平调控和转录后水平调控两种形式，并且转录因子和microRNAs分别是它们研究的热点。本项目拟通过研究番茄果实成熟过程中已知关键转录因子（RIN、CNR、NOR、TAGL1、AP2a、FUL1、FUL2、MADS1）所调控的microRNAs、其他转录因子、成熟相关功能基因，采用RNA-seq、small RNA-seq、降解组分析、EMSA、ChIP-PCR、WGCNA分析等技术建立番茄果实成熟的转录因子和microRNAs协同调控网络，明确重要转录因子和microRNAs调控果实成熟功能基因表达的协同调控途径，为全面、系统阐明果实成熟过程中功能基因表达调控机制奠定基础，并为培育耐贮藏、高品质的番茄果实提供新的思路。

番茄果实成熟过程在转录水平调控和转录后水平调控两种形式，其中转录因子和microRNAs是非常重要的两个方面。本研究以番茄为实验材料，通过分子生物学的方法对于成熟过程中的关键转录因子（RIN、CNR、NOR、TAGL1、AP2a、FUL1、FUL2、MADS1）进行沉默。利用深度测序技术，采用生物信息学和分子生物学实验相结合的方法，建立了所有基因包括所有转录因子和功能基因的共表达网络，得到了12个共表达网络模块，并将代谢物进行关联分析。最终筛选了对于抗坏血酸合成途径可能具有调控作用的4个转录因子，同时发现了miR396对于抗坏血酸合成途径的多个基因可能具有调控作用。通过烟草瞬时超表达及VIGS技术初步验证Solyc02g069720对于抗坏血酸的合成具有负调控作用。通过超表达STTM片段抑制miR396的功能证实miR396对于抗坏血酸的合成具有正调控作用。本研究初步建立了番茄果实成熟的转录因子和microRNAs协同调控网络，为阐明番茄果实成熟转录和转录后水平的调控网络提供了一定的理论基础。