铜对葡萄果实发育期多酚代谢及果实发酵影响的机制研究

Copper-containing pesticides, a type of chemical drug used for pest control and prevention, have been applied in vineyard extensively for a long time. Long-time application of this kind of pesticides could result to the accumulation of copper in the soil and grape berries, and then the excessive copper, disturbing physiological metabolism of grape cells, would transfer from grapes to fermentation process and lead to slow or stock fermentation. The grape polyphenols, determining the wine color, clarity, taste flavor and structure, is also an important quality index of fruit. Grapes cell suspension system will be used to study physiological feature and respond mechanism under copper stress in this project. The gene transcription and protein expression level of the key enzyme (PAL, CHS, CHI, F3H, DFR, ANS, etc.) in the grape phenylpropanoid pathway and flavonoids synthesis metabolic pathway will be detected under copper stress, which aim to clarify molecular mechanism of the effect of copper on the polyphenol metabolism in grape berries. At the same time, the grape juice contained different concentrations of copper will be used to research the effect of Gu2+ on the viability and metabolism of the yeasts based on the results of changes in glycolysis metabolism flux and molecule level. Flow cytometry will be used to study the effect of copper on the death mechanism of yeast during fermentation. It is expected that from grape development to wine fermentation, we can elaborate the impact mechanism of copper on the wine quality, which can be a guide to control the viticulture and wine making.

长期施用铜制剂农药，会使葡萄果实的铜含量升高，过量的铜在影响果实正常生理代谢的同时，又可进入发酵液，继续影响酵母的发酵，最终导致葡萄酒品质的降低。本项目利用不同发育时期葡萄果实圆片或细胞悬浮体系进行铜逆境生理及胁迫机制研究，测定在铜逆境下葡萄果实多酚苯丙烷类和类黄酮类合成代谢途径中的关键酶的基因转录及蛋白表达差异及应变机制研究，旨在揭示铜影响葡萄果实多酚代谢的分子机制；同时，从糖酵解代谢流量及发酵关键酶基因转录及蛋白表达水平揭示铜对酵母发酵性能影响的机制；并利用流式细胞术分析发酵期间铜对酵母的致死机制。最终阐明铜制剂农药对果实发育和发酵影响的分子生物学机制，为葡萄栽培和葡萄酒酿造的实际生产提供必要的理论依据。

长期施用铜制剂农药，会使葡萄果实的铜含量升高，过量的铜在影响果实正常生理代谢的同时，又可进入发酵液，继续影响酵母的发酵，最终导致葡萄酒品质的降低。本项目利用葡萄果实细胞悬浮体系进行铜逆境生理及胁迫机制研究，测定在铜逆境下葡萄果实多酚苯丙烷类和类黄酮类合成代谢途径中的关键酶的基因转录及蛋白表达差异及应变机制研究，旨在揭示铜影响葡萄果实多酚代谢的分子机制；同时，从糖酵解代谢流量及发酵关键酶基因转录及蛋白表达水平揭示铜对酵母发酵性能影响的机制；并利用流式细胞术分析发酵期间铜对酵母的致死机制。最终阐明铜制剂农药对果实发育和发酵影响的分子生物学机制，为葡萄栽培和葡萄酒酿造的实际生产提供必要的理论依据。研究结果表明：（1）葡萄多酚代谢途径的PAL , C4H , 4CL , CHS , CHI 和 STS 基因都参与了铜胁迫响应，其中VvSTS在响应铜胁迫时上调最为显著。T1代转基因（VvSTS）烟草表型观察表明VvSTS能增强烟草的铜胁迫耐性。（2）铜诱导酵母细胞发生死亡的方式并不是单一的凋亡，还存在其他的死亡形式。(3)我国葡萄酒生产过程中铜的污染情况，包括葡萄园土壤、酿酒葡萄和葡萄酒中的铜的污染情况整体较轻微，主要原因可能在于和传统葡萄酒生产国相比，我国葡萄园的种植时间较短，铜累积较少；(4) 低于国家标准（20 mg/L）的铜含量条件下，铜对于葡萄酒生产，例如甘油、乙酸、琥珀酸等发酵副产物合成，酒精发酵均有显著影响；并且当葡萄酒中含有国标浓度 (1 mg/L)的铜时，已经开始导致小鼠肝脏损伤。上述研究结果为进一步阐明葡萄和酵母在铜胁迫下的响应机制奠定了基础，同时为我国葡萄和葡萄酒生产中铜的限量标准的制定提供了数据基础。