冷胁迫下番茄果实苯丙氨酸衍生风味物质丧失机制的研究

Tomato fruits are sensitive to chilling injury. Fruit will suffer loss of flavor-associated volatile compounds while stored at chilling temperature. Although the content of aroma compounds derived from Phenylalanine is very low in tomato fruit, they play an important role in flavor production according to the threshold analysis. In the previous study, we found tomato fruit totally lacked the aroma compounds derived from Phenylalanine after cold storage. .Based on the different Phenylalanine derivatives between chilling fruit and un-chilling fruit, the current program will study the metabolism of aroma compounds derived from Phenylalanine in tomato fruit. Metabolomics analysis will be used in this project to focus on the related compounds in Phenylalanine pathway. C13-Phenylalanine will be added to detect the metabolism rule of Phenylalanine and its derivatives in fruit and Phenylalanine metabolism stream will be obtained. In order to demonstrate the molecular mechanism of Phenylalanine derivatives loss under chilling stress, the expression of key genes in Phenylalanine pathway will be analyzed. The homologous MYB transcription factors will also be cloned and verified their functions using chromatin immunoprecipitation assay..The study will help indicate the mechanism of Phenylalanine derivatives loss in tomato fruit under chilling stress and provide a theoretical foundation for further research in seeking control strategies.

番茄是冷敏性果实，低温贮藏会造成果实风味物质减少。虽然苯丙氨酸衍生风味物质在番茄果实中含量不高，但是根据阈值分析，其在果实风味形成中具有重要的作用。我们前期研究发现，番茄经低温贮藏后，苯丙氨酸衍生风味物质完全丧失。本项目以正常成熟和低温贮藏番茄中苯丙氨酸衍生风味物质的差异为出发点，利用天然变异体，研究果实中苯丙氨酸衍生风味物质的代谢规律。使用靶向代谢组学技术，锁定苯丙氨酸代谢上下游途径中相关化合物，对其进行精准定量分析；利用添加外源C13标记苯丙氨酸，检测苯丙氨酸及其衍生物在果实中的代谢规律，绘制苯丙氨酸代谢流；分析代谢途径中关键基因的表达谱，阐释冷胁迫下苯丙氨酸衍生风味物质丧失的分子机制；克隆番茄MYB类转录因子，筛选差异表达基因，通过染色质免疫共沉淀技术，验证其对代谢途径中关键基因的调控。研究有助于揭示番茄果实冷胁迫下苯丙氨酸衍生风味物质的丧失机制，为以后寻求相关调控手段提供理论基础。

香气是决定果实品质的重要指标之一。芳香物质决定了消费者对果实的偏爱程度和购买欲。通过阈值分析，其中苯丙氨酸途径的风味物质，对番茄风味的形成具有重要作用。本项目基于分子感官技术，利用动态顶空（DHS)、搅拌棒吸附萃取（SBSE)、吹扫捕集（P&T)、溶剂辅助蒸发提取（SAFE)、固相微萃取（SPME)等方法，精准分析了番茄果实中风味物质的变化规律，进一步证实苯丙氨酸衍生风味物质在番茄果实风味组成中的重要作用，同时对苯乙醛、苯乙醇等风味物质的时空分布进行了分析，分析了代谢过程中关键基因和转录因子表达情况，阐明了低温胁迫对番茄不同成熟期的风味物质合成中关键基因的表达机制的影响。通过对比正常成熟和低温贮藏番茄的转录组差异，结合KEGG分析、GO分析等多种生物信息学分析筛选出番茄中苯丙氨酸代谢途径的重要差异代谢酶基因：芳香氨基酸转氨酶基因SlAAAT1和SlAAAT2。利用大肠杆菌表达系统，对SlAAAT1和SlAAAT2进行蛋白表达和纯化，进而分析了芳香氨基酸转氨酶的相关酶学性质。利用基因沉默技术研究沉默SlAAAT1和SlAAAT2后苯丙氨酸代谢途径中的苯乙醛和苯乙醇含量的变化情况，发现基因沉默后，苯丙氨酸代谢途径中两种重要的风味化合物苯乙醇和苯乙醛的含量也均显著降低，证明了芳香氨基酸转移酶途径是苯丙氨酸衍生香气物质的合成的重要途径。本项目有助于进一步揭示番茄中苯丙氨酸衍生代谢物质的调控机制，也为利用分子生物学技术能大规模的改善番茄风味，从而提升品质，提高经济效益提供了参考。