柿果实类胡萝卜素裂解双加氧酶基因家族成员克隆与功能鉴定研究

Aroma is the critical part of fruit flavor quality, and is also one of the important areas of fruit quality research. At present, the studies on fruit aroma biosynthesis are mainly focused on fatty acid and amino acid pathway, while rarely involved in carotenoid pathway based on CCD (carotenoid cleavage dioxygenase) gene. In this study, learned from the research results of the model plants such as Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum), persimmon fruits (Diospyros kaki) are rich in carotenoids are used as materials, aroma composition in mature fruit of different cultivars are analyzed and identified, CCD gene family members are cloned, the change patterns of apocarotenoids aroma, carotenoids and CCDs gene expressions in different organs and tissues and in the fruits of different developmental stages are investigated, then the specific CCD gene family member of aroma formation is determined. Moreover, the function of this specific gene family member is studied and verified by using prokaryotic expression, enzyme activity analysis, subcellular localization, RNAi and overexpressing transgenic tomato. Finally, the molecular mechanism of fruit aroma formation based on carotenoid pathway is clarified. All the results can not only enrich the biological theories of fruit quality, but also provide foundation for exploration of measures to manipulate persimmon fruit aroma formation and breeding new persimmon cultivars with rich aromas.

果实香气是果实风味品质的重要组成部分，针对目前果实香气成分鉴定及生物合成研究主要集中在脂肪酸和氨基酸途径，很少涉及基于CCDs（类胡萝卜素裂解双加氧酶）基因的类胡萝卜素途径的问题，本项目拟借鉴拟南芥、番茄等模式植物的研究成果，以富含类胡萝卜素的柿果实为研究对象，采用色谱化学、生理生化和分子生物学等技术，分析和鉴定不同品种成熟果实主要脱辅基类胡萝卜素香气组分，克隆CCDs基因，研究不同器官组织和果实不同发育阶段类胡萝卜素和脱辅基类胡萝卜素香气组分的变化规律以及CCDs的表达模式，确定可能参与柿果实香气形成的CCDs基因家族成员，并通过原核表达、酶活性分析、亚细胞定位、RNAi及超量表达转基因番茄进行CCDs基因功能研究和鉴定，阐明基于类胡萝卜素途径的柿果实香气形成的分子机制。研究结果不仅可丰富果实品质形成的生物学理论，还将为探索调控香气形成的措施和培育具有浓郁香气的柿品种提供科学依据。

果实香气是果实风味品质的重要组成部分，针对目前果实香气成分鉴定及生物合成研究主要集中在脂肪酸和氨基酸途径，很少涉及基于CCDs（类胡萝卜素裂解双加氧酶）基因的类胡萝卜素途径的问题，本项目以柿果实为研究对象，采用色谱化学、生理生化和分子生物学等技术，分析和鉴定不同品种成熟柿果实主要脱辅基类胡萝卜素香气组分，克隆了CCD1和CCD4基因，研究不同发育阶段、不同采后处理柿果实脱辅基类胡萝卜素香气组分的变化规律以及CCDs的表达模式，并通过原核表达、酶活性分析、亚细胞定位、RNAi及超量表达转基因番茄进行CCDs基因功能研究和鉴定，初步阐明基于类胡萝卜素途径的柿果实香气形成的分子机制。研究结果不仅可丰富果实品质形成的生物学理论，还将为探索调控香气形成的措施和培育具有浓郁香气的柿品种提供科学依据。