新疆葡萄酒酿酒酵母遗传多样性及其发酵特性高通量分析

There must be rich in diverse and unique metabolic indigenous Saccharomyces cerevisiae in Xinjiang because of local ecology environment with much diverse and extremely different climates and ground configurations.Howerver,the diversity and enology characters of indigenous S.cerevisiae have maybe homogenized with planting the wine grape varieties with great large area and inoculating commercial yeasts for long time. In this project, the several molecular markers, radom amplified polymorphic DNA（RAPD）,inter simple sequence repeat （ISSR）,simple sequence repeat (SSR）,COX1 and δ sequence typing are used to research on the genetic diversity of indigenous S.cerevisiae with commercial S.cerevisae strains used for local wine fermentation as references.The representative strains are screened out based on genentic diversity assay of these S.cerevisiae isolates, meanwhile considering their isloated local background. The enology characters of the strains screened out are assayed with high-through methods as micro-plates to culture, plus quickly measures as GC-MS, micro-plates reader and otherwise to check. The S.cerevisiae sexual and asexual offspring populations from the strains with significant different enology characters are used to assay their heritability and hereditary stability. The gene expressions are assayed by the complementary DNA-Amplified Fragment Length Polymorphism（cDNA-AFLP）for the strains with high quality enology characters and good heritability.The aim of the project are to reveal the genetic diversity and enology characters of the indigenous S.cerevisaie in Xinjiang and how much they has been homogenized by commercial S.cerevisae, and then the heritability, hereditary stability of the main enology characters in the indigenous S.cerevisiae population, lastly the highy qulaity functional genes of S.cerevisaie strains with high qualty and typical indigenous enology characters. The work of the project will lead to construct the resource database of local wine S.cerevisaie in Xinjiang. Addition to these, the scientific work will lay on a good foundation to further protect and utilize the resource of wine S.cerevisiae in Xinjiang and afford strains with high quality for wine industry. Besides, the assay system could be a valuable reference for a large scale research on wine yeasts.

新疆特殊生境，蕴含着独特且多样的本土葡萄酒酿酒酵母，但酿酒葡萄大面积种植及商用酵母长期使用可能已导致其多样性受减与发酵特性同质化。本项目提出利用δ 等多重分子标记对新疆本土葡萄酒酿酒酵母（本地商用酿酒酵母做对照）进行遗传多样性分析；筛选代表株，利用微培养技术，GC-MS、酶标仪等快速检测方法，进行发酵特性高通量测试，分析发酵特性多样性及确定主发酵特性；筛选发酵特性差异显著株，构建有性与无性遗传群体，分析主发酵特性遗传性；利用cDNA-AFLP技术对高质量菌株做基因差异表达分析。上述研究旨在探明新疆本土葡萄酒酿酒酵母遗传多样性与发酵特性及其同质化状况，土著发酵特性与主发酵特性及其遗传力、遗传稳定性等，挖掘优质功能基因，建立本土葡萄酒酿酒酵母种质资源库（含信息数据库），为新疆葡萄酒酿酒酵母资源保护与利用、我国葡萄酒行业提供优质菌种奠定基础，为葡萄酒酵母菌多样性及发酵特性大规模探索提供科学参考。

酿酒酵母为葡萄酒发酵过程的优势菌，对葡萄酒高效发酵及保证葡萄酒优秀品质有着重要的意义。本土葡萄酒酿酒酵母具有极其丰富的遗传多样性与发酵特性多样性，且具有赋予葡萄酒典型地域风格的土著发酵特性，并能进行稳定遗传，是工业用葡萄酒酿酒酵母良好候选菌；新疆特殊生境，蕴含着独特且多样的本土葡萄酒酿酒酵母，但大面积酿酒葡萄种植及进口商用菌长期使用，可能已导致其多样性消减与发酵特性同质化。本项目完成新疆南北疆60个县市、13个师的565份葡萄、104土样、33份葡萄酒厂设备采样、169份白酒酒糟、马奶酒和传统发面酵头共计871份样品的采集， 走访葡萄生产基地15个，调研6个白酒厂，6个红酒厂，分离获得上万株酵母菌，其中酿酒酵母菌株6000余株，构建了新疆葡萄与葡萄酒酵母种质资源库1个，获得降酸、产香、高起发性等优良酿酒酵母共计12株，基于2株优良菌研发的慕萨莱思现代发酵工艺专利2项。通过本项目的研究，获得重要学术发现有：新疆葡萄与葡萄酒相关酵母种类丰富多样，同一地区酵母菌群结构相似，地貌、水纹等自然特征、葡萄品种等均会影响酵母菌的种群结构，但随着酿酒酵母的大规模引入，现代发酵工艺的扩大化，土著酵母菌的多样性受到侵扰。新疆酿酒酵母菌具有丰富的表型多样性与遗传多样性，且具有明显地域特征，其进化中南疆地域受到人类酿酒干扰小，表现出丰富的种内和种间多样性，在世界酿酒酵母群体中，南疆酿酒酵母单独形成2支和北疆1支。可能受到白酒和葡萄酒的酿制工业影响，北疆地区酿酒酵母表现出人为干扰的痕迹。WL在可分离的优势酵母菌菌群结构判断上和葡萄酒自然发酵过程控制上具有潜在的优势。在自然发酵中酵母的最大遗传力发生在第四代，而纯种酿酒酵母的最大遗传力发生在第三代。基于定性指标相关性，有助于低成本低劳动量的筛选优质酵母菌的筛子设计。新疆葡萄与葡萄酒相关的酿酒酵母其演化历程可能与新疆内的丝路文明传播耦合，但需要大样本量的测试分析。