杨梅果实杨梅素生物合成及其转录调控机制研究

With the improvement of people's living standard, the nutritional quality of horticultural products attracts more attention and enrichment of health-promoting bioactive compounds in fruit is becoming a new hot topic in the research. As an important dietary bioactive compounds, myricetin is biosynthesized in Chinese bayberry (Myrica rubra) fruit and its content is significantly affected by cultivars and developmental stages. The present study will investigate the biosynthesis of myricetin and its transcriptional regulation in Chinese bayberry by using fruit samples from different cultivars and different developmental stages. Based on RNA-Seq data, the study will focus on the correlation of the gene expression of F3'5'H, FLS and the myricetin content in different fruit. Recombinant proteins of F3'5'H and FLS will be exogenously expressed and their substrate specificity and enzyme kinetic study will be carried out as well. In addition, transcription factors involved in the myricetin biosynthesis such as MYB will be screened and its interaction with the target gene will be studied by various heterologous / homologous transient expression systems or by heterologous transgenic function verification method. The objective of the present study is to elucidate the biosynthesis and transcriptional regulation mechanism of myricetin in bayberry fruit and results will provide important basis for future genetic improvement of the nutritional quality of bayberry fruit as well as providing basis for mass production of myricetin through biotechnology in the future.

随着人们生活水平的提高,园艺产品的营养保健品质日益受到重视，在果实中富集与健康有关的活性物质已成为现今研究的一个新热点。作为膳食来源的重要活性物质，杨梅素在杨梅果实中大量积累，且品种和发育阶段对其含量影响显著。本项目拟以不同品种、不同发育阶段的杨梅果实为试材，在转录组测序基础上，重点研究F3'5'H和FLS基因在果实中的表达模式与杨梅素含量关系；分析F3'5'H和FLS等重组蛋白的底物偏好性和酶动力特征；筛选、鉴别介导杨梅素积累的MYB等转录因子家族成员及靶标基因，通过异源/同源瞬时表达或异源转基因功能验证，探明杨梅素生物合成的关键基因和酶催化特性，阐明杨梅果实杨梅素生物合成的转录调控机制。研究成果可为改良杨梅果实营养品质提供依据，也可为将来利用生物工程富集杨梅素奠定基础。

项目开展了杨梅中杨梅素积累规律的研究，明确了杨梅素生物合成关键结构基因和转录因子的基因表达模式，研究了MrF3'5'H和MrFLS等重组蛋白的酶活性及底物偏好性，探索了MYB12和MYB5/5L等转录因子调控杨梅素积累的分子机制。结果表明： MrF3'5'H 和MrFLS1是杨梅素生物合成关键基因，其中，MrF3'5'H可通过羟基化山奈酚驱动代谢流走向三羟基化黄酮醇杨梅素；MrMYB12是杨梅中调控黄酮醇合成的特异转录因子，但MrMYB12不能直接结合MrF3'5'H启动子；通过MrF3'5'H启动子酵母单杂交筛库获得参与杨梅素积累的重要转录因子MrMYB5和MrMYB5L，酵母双杂和烟草瞬时过表达等分析表明MrMYB12与MrMYB5-MrbHLH1和MrMYB5L-MrbHLH1复合体协同调控杨梅素的生物合成。研究结果丰富了植物杨梅素积累的分子机制，也为将来改良果实营养品质提供科学依据和工作基础。