转录因子PuMYBs对冷藏南果梨香气变淡的转录调控机制研究

After harvesting, when the fruit is appropriate ripening, the aroma of Nanguo pears is rich and attractive. Fruit senescence of Nanguo pears is fast at room temperature. Refrigeration can effectively retard the fruit senescence. But the aroma of Nanguo pears weakened after refrigeration. In the previous study, it was found that three MYB genes were related to the fade aroma of refrigerated Nanguo pears. During the shelf-life at room temperature after refrigeration, gene expression levels of the PuMYBs were positively correlated with the expression of PuAAT1. It is speculated that PuMYBs may affect the ester aroma synthesis of Nanguo pears by regulating PuAAT1 after refrigeration. In this study, dual-luciferase assay will be used to analyze the effect of PuMYBs on transcription regulation of PuAAT1 promoter. Then the yeast one-hybrid system and ChIP-qPCR assays will be used to verify the interaction of PuMYBs and PuAAT1. Low temperature conditioning treatment will be used to analyze the gene expression patterns of PuMYBs and PuAAT1 during the shelf-life at room temperature after refrigeration. This study is to explore the transcriptional regulation of PuAAT1 by transcription factor PuMYBs after refrigeration and clear the effect of PuMYBs on aroma fading in refrigerated Nanguo pears. This study will investigate the molecular mechnism of aroma fading in refrigerated Nanguo pears at the transcriptional level. The findings will be valuable for regulating the aroma quality of Nanguo pears after refrigeration.

南果梨采后经适度后熟，果实香气浓郁诱人。果实采后常温下易衰老，冷藏可有效延长其贮藏期，但冷藏后果实香气明显变淡。申请者前期研究发现，三个MYB基因家族成员与冷藏南果梨香气变淡相关，并在冷藏后常温货架期间与酯类香气合成途径关键酶基因PuAAT1的表达呈显著正相关，因此推测冷藏后PuMYBs通过调控PuAAT1影响南果梨酯类香气合成。本项目拟利用双荧光素酶实验，解析PuMYBs对PuAAT1启动子活性的激活特性；同时，利用酵母单杂交和染色体免疫共沉淀技术，分别通过体外和体内验证，鉴定PuMYBs与PuAAT1的基因互作。通过程序降温处理，分析冷藏后PuMYBs与PuAAT1的基因表达模式，探讨冷藏后PuMYBs对PuAAT1的转录调控机理，明确转录因子PuMYBs在冷藏南果梨香气变淡中的作用，从转录调控水平分析冷藏南果梨香气变淡的分子机制，为探索适宜的冷藏南果梨香气品质调控技术奠定理论基础。

南果梨采后经适度后熟，果实香气浓郁诱人。果实采后常温下易衰老，冷藏可有效延长其贮藏期，但冷藏后果实香气明显变淡。AAT是南果梨酯类香气合成的关键限速酶，本项目前期研究表明冷藏后常温货架期间南果梨果实中PuAAT表达量降低，与酯类香气物质生成量变化呈正比。本项目克隆获得了南果梨PuAAT启动子序列，分析其启动子上顺式作用元件后发现，该启动子区域具有MYB、WRKY等转录因子的结合位点。进一步通过酵母单杂交和凝胶阻滞试验验证，发现转录因子PuMYB6和PuWRKY61体内体外均可结合PuAAT基因的启动子；通过亚细胞定位，确定了转录因子PuMYB6和PuWRKY61在细胞中的表达位置为细胞核；通过GUS报告基因和双荧光素酶试验，明确了转录因子PuMYB6和PuWRKY61均能转录激活PuAAT基因的启动子。最后，通过程序降温（LTC）处理冷藏南果梨发现，LTC处理促进了酯类香气物质的生成，并促进了PuMYB6、PuWRKY61和PuAAT的表达水平，三者表达模式基本一致。上述结果综合表明，冷藏后南果梨果实中转录因子PuMYB6和PuWRKY61可通过调控结构基因PuAAT的表达影响冷藏南果梨的香气品质。本项目研究结果为不断完善南果梨贮藏保鲜技术提供了理论指导。