酿酒葡萄果实成熟过程中褪黑素对铜胁迫的响应机制

With the long time using of copper-containing pesticides in grape production, the copper stress has become an important challenge to grape and wine production. In previous study, we found that melatonin could significantly relieve the copper stress of wine grapes. In this project, the grapes discs and the grapes cell suspension system will be used to perform copper stress. Firstly, the response of the physiological and oxidative stress reaction, antioxidant enzyme and non-enzymatic antioxidant protection system of wine grape to the copper stress will be determined, and the relationship between melatonin and the antioxidant defense system of wine grape will be built to investigate the protective way of melatonin to the copper stress. Then the 15N stable isotope tracer method combines with HPLC-MS/MS will be used to detect the changes of melatonin precursor, melatonin, melatonin isomers and its metabolites, so as to analyze the synthesis rule of melatonin under copper stress. The key enzyme gene transcription and protein expression differences will also be analyzed to reveal the molecular mechanism of melatonin response to copper stress during fruit ripening. Finally, the molecular biological mechanism of melatonin in response to copper stress through fruit ripening will be elucidated, which can be as a direction to control the high quality viticulture under copper stress.

随着铜制剂农药在葡萄生产中的长期施用，铜胁迫逐渐成为影响葡萄与葡萄酒生产的重要挑战。本项目基于申请人前期研究发现褪黑素可显著缓解酿酒葡萄铜胁迫的事实，利用不同发育时期葡萄果实圆片/果实悬浮细胞体系进行铜逆境生理胁迫，首先通过测定酿酒葡萄生理与氧化应激反应、抗氧化酶及非酶抗氧化剂保护系统对铜胁迫的响应，建立褪黑素与酿酒葡萄抗氧化防御系统的关系，探究果实发育过程中褪黑素对铜胁迫的保护途径；之后采用15N稳定同位素标记结合HPLC/MS/MS测定褪黑素前体、褪黑素/褪黑素异构体及其代谢物的变化与合成规律，并分析其合成通路关键酶基因转录与蛋白表达差异，揭示果实成熟过程中褪黑素响应铜胁迫的分子机制。最终阐明酿酒葡萄果实成熟过程中褪黑素对铜胁迫响应的分子生物学机制，为铜胁迫下葡萄优质栽培提供必要的理论依据。

随着铜制剂农药在葡萄生产中的长期施用，铜胁迫逐渐成为影响葡萄与葡萄酒生产的重要挑战。课题组前期发现褪黑素可显著缓解葡萄铜胁迫，而褪黑素参与葡萄果实发育过程铜逆境胁迫应答的作用机制尚不清楚。本项目(1)首先通过文献计量学，对葡萄铜胁迫研究现状进行了系统可视化分析。(2)适度的低铜胁迫会促进总酚、总类黄酮及总黄烷醇的生成；高浓度的铜胁迫会抑制它们的生成。而总花色苷在转色期以后的生长发育过程中整体呈现含量上升的趋势，且其对铜胁迫处理较为敏感，较低浓度的铜胁迫对其合成具有一定的促进作用，高度铜胁迫都会对其合成具有抑制作用，且铜胁迫浓度越高，抑制效果越明显。(3)外源喷施铜农药进行胁迫处理会造成葡萄果实中铜含量的激增。低浓度的铜胁迫在一定程度上提高了葡萄果实的抗氧化能力。在高铜浓度范围，葡萄酒的抗氧化能力显著降低。低浓度的铜胁迫处理对褪黑素的合成有较轻微的抑制作用；相反，较高浓度的铜胁迫处理对褪黑素的合成具有明显的促进作用。控制褪黑素合成的关键基因表达量的表现与褪黑素含量的表现基本一致。(4)适度的低浓度铜胁迫会轻微抑制MDA的生成，高浓度的铜农药则会显著激发MDA的生成。低浓度的铜农药处理组轻微抑制了SOD、CAT、POD的活性，但是影响不大，而高浓度的铜农药处理组显著增加了三种抗氧化酶的活性。在发育期内，从整体上来看，适度的低浓度铜农药处理对AsA/DHA及GSH/GSSG比值影响不大，与抗氧化酶活性的变化基本一致，高浓度的铜胁迫则会显著降低该比值，高浓度的铜胁迫会对葡萄果实产生严重的氧化胁迫。(4)铜胁迫诱发的氧化胁迫会导致AsA/DHA及GSH/GSSG比值降低，而通过外源喷施褪黑素可以提高抗坏血酸-谷胱甘肽循环中关键物质的含量和AsA/DHA及GSH/GSSG比值。褪黑素在作为氧化剂直接清除自由基的同时，不仅可以提高抗氧化酶的活性，也提高了非酶抗氧化系统中抗坏血酸-谷胱甘肽循环中关键物质的含量及比值，从而帮助植物抵御逆境胁迫。项目研究结果将为铜胁迫逆境下酿酒葡萄高效栽培提供创新性理论依据。