新疆酿酒葡萄紫檀茋防御酸腐病的机制研究

Grape quality may be affected by a wide range of sour rots. Among them, sour rot is an emergent grapevine disease affecting late ripening cultivars, causing heavy crop losses and being detrimental to wine quality. Our previous work showed that the pterostilbene defensed significantly in both dose-dependent and time-dependent manners to G.citri-aurantii inoculated grape berries, and it was very effective antifungal activities at pharmacological concentrations in vitro. However, it was not well know that the mechanism of pterostilbene defence to G.citri-aurantii in grape. On the base of our previous study, first, the mechanism action of pterostilbene in Xinjiang wine grape against the growth of G.citri-aurantii will be studied by transmission electron, scanning electron microscopy, flow cytometry and qPCR in vitro. Secondly, Xinjiang wine grape is used to study experimental material, which was inoculated with G.citri-aurantii, the structures of metabolic intermediate were elucidated during the course of pterostilbene defence by spectroscopic analysis, including HPLC/ESI-MSn and NMR experiments. Thirdly, differentially expressed gene were identified by using deep sequencing and qPCR. And which was combined with metabolic intermediate of pterostilbene defence to infer possible key gene involved in the process. We will verify the results by expressing these genes using yeast in vitro and grape berries in vivo, respectively. The relationship of metabolic intermediate and key genes of pterostilbene defence will be established. Finally, base on all these research results, the mechanism of pterostilbene defence to G.citri-aurantii in wine grape will be expounded. The results of this project will help us understand the mechanism of stilbenes defensing to G.citri-aurantii in grape.

酸腐病不但引起酿酒葡萄采后期烂果发生，而且还严重影响酿制葡萄酒品质。申请者项目组前期研究葡萄中紫檀茋对酸腐病防御呈显著性，且在药理浓度下紫檀茋体外高效抑制酸腐菌活性，但葡萄中紫檀茋对酸腐病防御机制尚不清楚，故本项目拟在前期研究工作基础上，首先使用扫描电镜、透射电镜、流式细胞、qPCR等技术进行酸腐菌体外抑菌机制研究；其次，以"新疆酿酒葡萄"为试验材料，酸腐菌侵染葡萄，分离紫檀茋防御形成中间产物，使用NMR / HPLC/ESI-MSn技术鉴定中间产物结构；然后使用深度测序（Deep sequence）/qPCR技术获得差异表达基因, 挖掘可能参与紫檀茋防御酸腐病关键基因，并分别在体外酵母、体内葡萄果实真核表达进行验证；最后在上述的研究基础上，根据体外抑菌机制，结合防御中间产物及其防御关键基因阐明酿酒葡萄紫檀茋防御酸腐病的机制。本项目研究结果有助于明确酿酒葡萄茋类化合物防御酸腐病机制。

酸腐病是危害葡萄的重要病害，严重影响葡萄的品质。前期研究发现葡萄中紫檀芪对酸腐菌胁迫呈显著性，且在药理浓度下紫檀芪能高效抑制酸腐菌活性，然而葡萄中紫檀芪对酸腐菌胁迫的防御机制尚不清楚，本实验在以前的工作基础上，围绕葡萄紫檀芪防御酸腐菌的机制进行研究，研究包括四个方面：①以葡萄为材料，采用直接接触的方法，从生理水平检测酸腐菌侵染后葡萄腐烂水平，研究发现侵染48 h时，葡萄开始腐烂，并有腐烂酸臭气味发出；侵染72 h 后，葡萄严重腐烂。为抵御病害侵染，葡萄自身会产生次级代谢产物，通过UHPLC-QQQ-MS2分离得到白藜芦醇和紫檀芪两种防御代谢产物并对其进行定量检测，结果表明酸腐病菌侵染后显著提高葡萄中白藜芦醇和紫檀茋含量。②使用测序技术并结合已有的葡萄基因组数据库获得了紫檀芪合成的关键基因葡萄氧-甲基转移酶基因（VvREOM），并对其进行序列分析，该基因的ORF为1116 bp，编码305个氨基酸，分子量为34.4 kDa，等电点6.52。进化关系分析表明VvREOM基因与欧洲葡萄VvROMT基因属于同一亚家族，并与月季RcOOMT基因的亲缘关系较近。氨基酸序列同源性比对结果显示VvREOM与欧洲葡萄赤霞珠的VvROMT氨基酸序列具有95.86 % 的同源性，与中国月季中RcOOMT1基因预测的氨基酸序列、蓖麻中RsOMT基因预测的氨基酸序列和荷兰鸢尾咖啡酸中IhOMT基因预测的氨基酸序列分别拥有65.61 %、62.10 %和30.57 %的同源性。③利用荧光定量PCR技术对目的基因VvREOM的功能在葡萄体内进行验证。利用酸腐菌侵染葡萄，通过qPCR检测侵染过程中VvREOM基因的表达量，结果表明VvROEM基因的表达随侵染时间先上升后下降。当侵染24 h时，VvREOM表达量达到最高，48 h后表达量迅速降低，侵染72 h时几乎不表达。④利用真核表达系统和HPLC技术对REOM蛋白进行“体外”验证。首先构建真核表达载体pPic9k-REOM，并把含有VvREOM基因的载体转进毕赤酵母表达系统，对其进行诱导表达，使用SDS-PAGE和Western Blot技术检测到相应蛋白，大小约为34.4 kDa。之后向含有重组酵母的BMGY培养基中饲喂底物，利用HPLC技术检测到了紫檀芪，在体外证明了VvREOM具有氧甲基化功能催化产生紫檀芪。