采后葡萄果实内选择性淬灭单线态氧Scirpusin A生物合成及其调控的 分子机制研究

Abundant polyphenols are the most important component for grapes and other fruits, it play decisive role in the nutritional quality of postharvest fruits in storage period. Our previous work showed that ε-viniferins is the precursor of biosynthesis of the scirpusin A as selective singlet oxygen quencheris, and its biosynthesis is closely correlated with grape fruits irradiated by UV-C. However, it was not well known that the biosynthesis pathway, on the base of our previous study, ‘postharvest grape’ was used to study the scirpusin A biosynthesis and regulation mechanism of postharvest UV-C. 13C stable-isotope technique was to mark ε-viniferins of precursor. The mark system from ε-viniferins to scirpusin A was established. the structures of intermediate were elucidated by spectroscopic analysis, including NMR and HPLC/ESI-MSn experiments; Differentially expressed gene were identified by using deep sequencing and qPCR. And which was combined with metabolic intermediate to infer possible key gene involved in the scirpusin A biosynthesis. Bosed on verifying results by using yeast and grape fruits expression, the biosynthesis linear model was constructed, which make the mechanism of scirpusin A biosynthesis linear; Finally, the effect of postharvest UV-C on all key steps of scirpusin A biosynthesis was analized systematically to understand mechanism of regulation of different time UV-C to scirpusin A biosynthesis. The results of this project will help us understand the mechanism of the concrete pathway of polyphenols biosynthesis in postharvest fruits.

葡萄等果品内丰富的多酚化合物对采后贮藏期果实营养品质具有决定性作用。申请者研究发现，选择性淬灭单线态氧多酚化合物Scirpusin A生物合成的前体为ε-viniferins，且合成与紫外线(UV-C)照射密切相关，但生物合成途径尚不清楚，故本项目拟在前期研究基础上，以“采后葡萄”为材料，通过稳定同位素13C饲喂技术标记前体物质，建立从前体物质到产物合成的线性标记体系，采用NMR/HPLC/ESI-MSn技术鉴定中间产物结构；利用深度测序/qPCR技术，挖掘参与Scirpusin A合成关键基因，并在酵母/葡萄果实蛋白表达进行验证，在上述基础上，建立葡萄Scirpusin A合成线性模型，明确合成机制；最后通过紫外线照射对Scirpusin A合成线性模型影响，阐明紫外线对葡萄果实Scirpusin A合成的调控机制。本项目研究结果将有助于明确采后果实内多酚合成具体途径。

芪类化合物Scirpusin A 作为葡萄品质功能因子，其药理活性备受关注，但合成路径却鲜有研究报道。本研究以葡萄为材料，探究品质功能因子Scirpusin A 生物合成机制，利用转录组测序技术筛选获得 Scirpusin A 生物合成关键基因（SAH 和 VvCP）并对其进行功能验证，主要研究内容有以下几点：（1）UV-B 辐射对葡萄的品质有影响。UV-B辐射后葡萄SOD、POD酶活性升高，CAT 酶活先升后降。随着处理时间的增长，葡萄中糖含量升高，UV-B 辐射影响单种有机酸在总有机酸的比例。葡萄中芪类化合物的含量与 UV-B 辐射时间呈正相关，白藜芦醇含量对 UV-B 辐射响应幅度最大，其次是 ε-viniferins 和 Scirpusin A，UV-B 辐射对 δ-viniferins 和紫檀芪的影响最小。（2）转录组测序结果表明，UV-B 辐射 30 min 后与未处理的相比，共有 13906 个差异表达基因(DEGs)，根据 GO 注释分类，其中 5560 个DEGs属于生物过程分类，5024 个DEGs被归类到细胞组成分类中，3322 个DEGs归类于分子功能范畴。KEGG 代谢途径分类和功能富集结果共有 4891 个DEGs被归类到 5 个主要代谢途径中，其中1017 个DEGs参与遗传信息加工，2988个DEGs参与新陈代谢，394 个DEGs参与生物体系统。DEGs参与主要富集在核糖体、植物病原相互作用和黄酮类化合物生物合成三大区域。 注释信息与参照基因 HsCYP1B1 比对，筛选获得 5 个潜在Scirpusin A 生物合成基因并对其进行 RT-qPCR 验证。（3）综合基因差异表达倍数和基因同源性分析，选择与目标基因功能高度吻合的 SAH 和 VvCP 构建系统进化树，基因结构分析，染色体定位分析和顺式作用元件分析。（4）对 SAH和 VvCP 蛋白进行体外验证。将含有目的基因的载体转进毕赤酵母构建真核表达载体 pPic9k-SAH/pPic9k-VvCP对其进行诱导表达，SDS-PAGE 和 Western Blot 技术检测目标蛋白。对含有重组酵母的BMGY 培养基饲喂底物，利用 HPLC 技术检测 Scirpusin A。亚细胞定位表明SAH 目的蛋白表达部位是细胞核，少量在细胞膜表达，VvCP 目的蛋白的表达在细胞膜。