番茄YFT1基因在乙烯-类胡萝卜素果色形成途径中的表达和功能分析

In tomato (Solanum lycopersicum), fruit color is a very important quality trait, which decides tomato appearance, and is also related closely to formation of the qualities and nutritional components, hence one of the most important research diction and hot-points for improvement of the tomato qualities is to expose molecular mechanism of development of the fruit color and formation. We found that yft1（yellow fruit tomato 1）, a tomato mutant compared with wild type of the M82 present phenotypes of the ethylene insensitive and decreasing carotenoides contents, in other words turned into yellow fruited phenotype from red-fruited M82. The YFT1 isolated from tomato by map-based cloning is a homologue to the EIN2(ETHYLENE INSENSITIVE 2) in Arabidopsis thaliana, the EIN2 protein is a core component for ethylene signaling pathway in plant kingdom. But, the molecular mechanisms remain unclear for the EIN2 gene expression and regulation yet, as well as how would changes of the EIN2 gene expression influence ethylene signaling pathway in plant. For this, several experiments would be designed to reveal molecular mechanisms of the YFT1 expression and regulation that including construction of the mutational promoter of the YFT1, the functional region analysis of the promoter, screening of the yeast one hybridization library, EMSA (electrophoretic mobility shift assay) and dissection of genetics functions for YFT1, the aim is to define key cis-acting elements and essential transcript factors binding with those elements. And those results would clarify biological mechanisms of that the YFT1 control the tomato fruit color via ethylene regulate carotenoides synthesis pathway in tomato. This research would not only provide a novel insight to ethylene signaling pathway in plant, and would also offer scientific basis for accurate improvement of the tomato fruit color.

番茄（Solanum lycopersicum）果色不但决定和影响商品美观，与品质和营养形成更加密切相关；因而目前番茄品质改良一个主要研究方向和热点是阐明果色发育和形成分子机理。我们发现，番茄突变体yft1（yellow fruit tomato 1）表现出乙烯不敏感和类胡萝卜含量下降特征；图位克隆显示，番茄YFT1是拟南芥EIN2（ETHYLENE INSENSITIVE 2）同源基因。EIN2是植物乙烯信号转导核心分子，但是EIN2表达调控分子机制目前还不清楚。为此，我们拟通过创造YFT1突变启动子、功能区段分析、酵母单杂交筛选、凝胶阻滞（EMSA）和遗传学功能分析途径；明确YFT1表达关键元件和重要调控因子，在揭示YFT1表达调控分子机制基础上，阐明YFT1介导乙烯调控类胡萝卜素途径控制番茄果色形成生物学机制。该研究将为植物乙烯信号调控提供新思路，也将为番茄果色精准改良提供科学依据。

果色是番茄（Solanum lycopersicum）重要商品和品质性状，与番茄品质和营养密切相关；解析和揭示番茄品质特别是果色形成分子机制对番茄品质改良具有重要的科学意义。.番茄黄果突变体yft1（yellow fruited tomato 1）是由野生型番茄M82经快中子辐射获得的黄果突变体；遗传学分析发现，黄果表型由单隐YFT1基因控制。构建yft1突变群体，用图位克隆技术分离获得了位于第9号染色体靶基因的YFT1，候选基因是EIN2（ETHYLENE INSENSITIVE 2）基因。YFT1 allele在yft1番茄中表现出显著下调表达。乙烯信号转导途径核心因子基因EIN2，因在yft1番茄中的下调表达扰乱了乙烯信号转导、并反馈抑制乙烯合成、有色体发育延迟，还抑制了类胡萝卜素在yft1番茄果皮有色体中累积，而使yft1呈现黄果表型。yft1番茄YFT1 allele在启动子与野生型M82番茄的YFT1相比，在ATG上游 -318 bp处发生两个突变事件：IF573(Insertion of 573 bp DNA fragment)和Del 13(Deletion of 13 bp segment)。启动子突变分析确认YFT1 allele在yft1中下调表达与Del 13无关，因IF573事件所致。进一步深入解析发现，YFT1 allele下调表达是IF573使YFT1 allele在转录水平发生可变剪接，打破了5' UTR 的uORF和同时引入负调控DNA序列所致。为了调控乙烯信号途径核心基因YFT1表达，发现并确认一个关键转录因子WRKY32，其通过正向调控YFT1表达。并通过遗传学、生物化学和分子生物学手段明确了WRKY32通过调控乙烯信号转导途径关键核心因子EIN2而影响番茄果色的形成。该项目研究结果不仅将植物乙烯信号转导途径向前推进了一小步，也将对番茄品质改良提供重要理论参考。