MADS5调控苹果脂肪酸途径香气物质合成的分子机理

The compositions and contents of volatile aroma were not only an important component of apple flavor quality, but also the critical evaluation indicator of fruit storage quality. On the base of the biosynthesis of aroma in ‘Taishanzaoxia’ apple was regulated by ethylene and the isolation of MADS5 from SSH library, which was closely associated with the biosynthesis of ethylene and aroma, qRT-PCR was used to screen the key genes involved in the biosynthesis of aroma derived from fatty acid pathway and regulated by ethylene. Then, yeast one-hybrid assays, luciferase reporter assay, EMSA, CHIP-PCR and other molecular biology technologies were used to study the protein-DNA interaction of MADS5 with promoters of genes involved in the biosynthesis of ethylene and aroma derived from fatty acid pathway in vitro and in vivo. The gene function of MADS5 will be verified in transgenic plants. These results will elucidate the molecular mechanism of MADS5 involved in the regulation on the biosynthesis of apple aroma derived from fatty acid pathway, further enrich the development and formation mechanism of fruit quality, and provide scientific basis for fruit quality breeding.

香气物质组成及含量不仅是苹果果实风味品质的重要构成因素，同时也是果实贮藏品质评价的重要指标。本申请在明确了‘泰山早霞’苹果香气物质合成受乙烯调控，并从差减文库中分离到与乙烯和香气合成密切相关的转录因子MADS5的基础上，进一步通过qRT-PCR 筛选响应乙烯调控的脂肪酸途径香气物质合成关键基因，并综合利用酵母单杂交、双荧光素酶瞬时表达、凝胶阻滞电泳（EMSA）和染色质免疫共沉淀（ChIP-PCR）等分子生物学技术手段分别在离体和活体状态下研究MADS5与乙烯生物合成和脂肪酸途径香气物质合成基因启动子在蛋白-DNA水平上的互作特点及其调控作用，并进行转基因功能验证，解析转录因子MADS5参与调控苹果脂肪酸途径香气物质合成的分子机制，进一步丰富果实品质发育与形成机理，为果树品质育种提供科学的理论依据。

香气物质组成及含量不仅是苹果果实风味品质的重要构成因素，同时也是果实贮藏品质评价的重要指标。因此，研究探讨果实香气物质的合成与调控机理，对提高果实品质与推动果树品质育种具有重要意义。本项目通过过量表达MADS5的转基因苹果愈伤和番茄，明确了MADS5是位于乙烯信号途径上游的转录因子，直接调控或通过乙烯信号途径间接调控果实香气物质合成与质地品质；通过荧光定量PCR筛选确定了苹果脂肪酸途径香气物质合成的关键基因（LOX1a、AAT1和CXE16等）；通过苹果转基因愈伤进一步明确了LOX1a在香气物质合成中的关键作用；克隆获得了苹果LOX1a的启动子序列，酵母单杂交等实验明确了MADS5/24通过结合LOX1a启动子调控其表达；酵母双杂交等实验明确了MADS24分别与MADS5、ERF1B蛋白互作，ERF1B通过与MADS24蛋白互作促进香气物质的合成。本项目研究明确了MADS5调控苹果脂肪酸途径香气物质合成的分子机理，首次揭示了乙烯间接参与调控果实香气物质合成的分子机制，为乙烯调控果实香气品质形成提供了理论基础。