基于iTRAQ技术研究新疆无核白葡萄荫干过程中酚类物质代谢及对酶促褐变的调控机制

The browning of Thompson seedless grape during drying in the barn has been well-acknowledged as the primary factor affecting the development of the raisins industry in Xinjiang. The enzymatic browning is the main reaction in the shade drying of the Thompson seedless grapes. It is closely related to the phenolic compounds in grape. However, the mechanisms of phenolic metabolism and its regulation on browning during shade dry has been insufficiently studied. Besides, the relevant mechanisms are unclear. Thompson seedless grapes are used as experimental materials in this study. The effect of phenolic metabolism related proteins during shade drying was studied using iTRAQ technology. Systems analysis on differential proteins and metabolic pathways before and after browning was performed. Moreover, real-time polymerase chain reaction was applied to analyze the expression of key genes. Based on changes of phenolic metabolism physiological experiment, the effect of phenolic metabolism because of dehydration stress was investigated. Based on the effect of phenolic metabolism, the changes of main types of phenolics content and the research of the role of different phenolics in browning was combined in our study. The regulation mechanism of phenolic metabolism on enzymatic browning could be clearly obtained. Study on its mechanism will lay base to a better understanding on enzymatic browning, and it will also provide guidance to control the quality of Thompson seedless grapes in practice. This study is of important significance to the development of Xinjiang Thompson seedless grapes industry.

新疆无核白葡萄荫干褐变已成为制约新疆葡萄干产业健康发展的关键问题。无核白葡萄晾房荫干褐变主要为酶促褐变，酚类物质与酶促褐变关系密切。但在新疆晾房光照条件下，脱水胁迫对无核白葡萄酚类物质代谢及对酶促褐变的调控机制尚不清楚。项目以新疆无核白葡萄为原料，采用iTRAQ蛋白质组学技术研究荫干对酚类物质代谢中相关蛋白质表达的影响，并对褐变前后的差异蛋白和代谢通路进行系统分析，FQ-PCR对关键酶基因表达进行验证，结合影响酚类物质代谢的生理生化变化，阐明荫干脱水胁迫对无核白葡萄酚类物质代谢的影响。基于酚类物质代谢，以及荫干过程中无核白葡萄主要酚类物质种类和含量的变化和不同酚类物质在褐变中的作用，进一步明确酚类物质代谢对酶促褐变的调控机制。通过对其机制研究，为无核白葡萄干制酶促褐变奠定理论基础，为实践中采取有效控制措施提供指导，对新疆无核白葡萄产业的发展具有重要意义。

本项目以无核白葡萄为原料，对新鲜和荫干褐变的葡萄进行DIA（Data independent acquisition）蛋白质谱分析，并进行了平行反应监视（Parallel Reaction Monitoring，PRM）验证；对酚类物质代谢中关键酶进行了生理活性分析；研究无核白葡萄多酚氧化酶的提取工艺，采用超高效液相-离子淌度飞行时间质谱测定荫干过程中无核白葡萄的酚类物质变化，鉴定了引起无核白葡萄酶促褐变的底物。项目研究为无核白葡萄荫干酶促褐变机理提供了科学依据，为实践中控制无核白葡萄荫干褐变提供了新思路。结果表明：. （1）新鲜葡萄和褐变葡萄中鉴定到5431个蛋白，739个蛋白存在显著差异。进一步对差异蛋白利用Blast2GO进行生物学过程、分子功能和细胞成分功能注释。发现：“有机物代谢过程”、“分子功能调节”、“酶调节活性”和“异构酶活性”相关蛋白在褐变过程中变得非常活跃。与褐变相关的差异蛋白主要参与苯丙烷类生物合成、酪氨酸代谢、苯丙氨酸代谢、氧化磷酸化代谢、谷胱甘肽代谢、过氧化物酶体途径和脂肪酸降解途径。另外，5种差异蛋白被PRM验证。PRM结果与DIA数据一致。. （2）通过研究无核白葡萄荫干过程中相关酶活性的变化，发现苯丙烷代谢的关键酶（4-香豆酸:辅酶A连接酶、肉桂酸-4-羟化酶、苯丙氨酸解氨酶、β-葡聚糖酶、过氧化物酶）在无核白葡萄酚类物质和总黄酮合成过程中起到主要的作用。. （3）采用丙酮提取法、超声波辅助浸提法和缓冲溶液浸提法分别提取无核白葡萄多酚氧化酶，在单因素的基础上，利用响应面法优化了多酚氧化酶的提取工艺。通过检测无核白葡萄荫干过程中的酚类物质变化发现，无核白葡萄中主要的酚类物质有14种，原花青素B2、对羟基肉桂酸、儿茶素、顺式-白藜芦醇、反式-咖啡酸在无核白葡萄干制过程中与褐变度呈现显著的相关性，经验证，这几种物质也是无核白葡萄酶促褐变的底物。另外，实验中检测到的杨梅黄素、香草酸，因其本身颜色淡黄，且随着干制的进行而含量增加，推测这两种酚类物质在无核白葡萄干制褐变中也起到了一定的作用。