转录因子RIN调控番茄果实软化的靶基因确定和分子机制研究

MADS domain protein is a big transcription factor family that has been implicated in the regulation of a number of plant developmental control and signal transduction processes. RIN is a member of the MADS box transcription factor family. The mutation of LeRIN gene inhibits most all measurable tomato fruit ripening phenomena. Therefore, RIN is thought to be one of key factors in regulating fruit ripening and softening, and has been proved to be an important developmental regulator in tomato fruit ripening and softening. However, it is still not yet completely clear about the regulation mechanism of RIN involved in tomato fruit softening. In this study we make use of the tomato fruit genome microarray data obtained in the preliminary work, analyze the genes related to RIN in tomato fruit softening process by bioinformatics, and select the high correlation genes to next research. High-resolution two-dimensional (2D) electrophoresis, mass spectrometry technology and bioinformatics methods are used to analyze the expression differences of proteins in tomato fruit at proteomics aspect. Then the proteins that have large expression differences are selected to further research. In particular, we perform electrophoresis mobility shift assay (EMSA) coupled with chromatin immunoprecipitation (ChIP) assay to identify RIN-regulated direct target genes for control of tomato fruit softening in vitro and in vivo. The purpose of our research is to reveal the molecular regulation mechanism of RIN in tomato fruit softening process.

MADS box 转录因子家族在植物的许多发育过程和信号转导途径中发挥调控作用。RIN是MADS box 转录因子家族成员之一，其基因的突变可抑制番茄果实成熟的大部分表型，因此RIN被认为是调控果实成熟软化的关键因子之一，但是到目前为止转录因子RIN调控果实软化的机制和路径尚不清楚。本项目利用前期工作所获得的番茄果实基因芯片数据，采用生物信息学方法分析番茄果实软化过程中与RIN相关的基因，选取相关度较高的基因进行深入研究。同时采用高分辨率的双向电泳技术、质谱鉴定技术结合生物信息学方法在蛋白质组学方面分析不同番茄果实中蛋白的表达差异，确定与果实软化相关的表达差异较大的蛋白，并选取这些蛋白进行深入研究。最后通过凝胶阻滞实验和染色质免疫共沉淀技术，在番茄果实体外和体内共同确定转录因子RIN直接作用的且与果实软化相关的新的靶基因，为进一步阐明转录因子RIN对番茄果实软化的调控机制奠定基础。

转录因子RIN在果实成熟软化过程中发挥关键的调控作用，LeRIN基因的突变可抑制番茄果实成熟的大部分表型，但是到目前为止转录因子RIN调控果实软化的机制和路径尚不清楚。本项目将破色期AC和rin突变体番茄果实用不同浓度的外源乙烯和1-MCP处理，通过测定一系列相应的生理生化指标，确定100 μL/L 外源乙烯和1.0 μL/L 1-MCP分别有效地促进和延缓了AC番茄果实的软化进程，但是外源乙烯和1-MCP处理对rin突变体番茄果实没有显著影响。然后以突变体rin、野生型AC、100 μL/L外源乙烯和1.0 μL/L 1-MCP处理的AC番茄果实为样本，采用高分辨率的双向电泳技术、质谱鉴定技术和生物信息学方法在蛋白质组学方面分析AC和rin突变体番茄果实中蛋白的表达差异，确定与果实软化相关的表达差异较大的蛋白是蛋白点3（ACO4）和蛋白点26（α-Gal）。同时根据项目负责人前期工作的基因芯片数据，选出表达差异较大的基因是PG、TBG4、LeMAN4和LeEXP1，并且通过实时定量PCR技术精确确定了LeACO4、α-Gal、PG、TBG4、LeMAN4和LeEXP1基因的表达差异。最后应用凝胶阻滞实验和染色质免疫共沉淀技术，从番茄果实体外和体内确定转录因子RIN调控番茄果实软化的新的直接靶基因是LeACO4和α-Gal。这一结果为阐明转录因子RIN调控番茄果实软化的分子机制奠定了理论基础。