云南弥勒葡萄酒产区云中舞葡萄酵母菌多样性研究

Study on the Biodiversity of Yeasts Isolated from the Variety of Cloud Dancer Grape in Mile of Yunnan Province

【目的】云南弥勒是中国西南地区传统的葡萄酒产区,以该产区云中舞葡萄为试验材料,对其果皮以及自然发酵过程中酵母菌的多样性进行研究。【方法】利用WL培养基的菌落培养形态,酵母细胞微观形态和分子生物学相结合的分类鉴定方法对弥勒云中舞葡萄果皮以及自然发酵过程中酵母菌进行分离鉴定和多样性分析。【结果】从云中舞葡萄分离得到的80株酵母菌共分为5个培养类型,采用26S r DNA D1/D2区序列分析对选出的具有典型特征的15株酵母菌进行了分子生物学鉴定,序列比对分析和WL培养基聚类表明80株被测菌株鉴定为5属5种,分别为葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、陆生伊萨酵母(Issatchenkia terricola)、库德毕赤酵母(Pichia kudriavzevii)、粘红酵母(Rhodotorula glutinis)和桔假丝酵母(Candida quercitrusa),显示了酵母菌种类多样性;其中葡萄汁有孢汉逊酵母占了所分离酵母总数的43.18%,为弥勒云中舞葡萄相关酵母菌的优势种。通过构建15株被测菌株与相关模式菌株的系统发育树,分析酵母菌的遗传多样性和菌株间的亲缘关系,结果表明:各供试菌株均与各自相关模式菌株聚为一枝,和序列比对鉴定结果相一致。另外,鉴定为同种酵母的不同菌株间以及和模式菌株间的亲缘关系具有一定的差异,显示了一定的遗传多样性。【结论】云南弥勒云中舞葡萄相关酵母菌具有多样性的特点,本研究为进一步选育优良的本土葡萄酒酵母提供了材料和参考。

[Purposes]Cloud Dancer grape was selected as experimental material in Mile County of Yunnan, where is a traditional wine producing region in Southwest China.[Methods]Biodiversity of yeasts in the surface of grape and the spontaneous fermentation process were studied by the colony morphology of WL nutrient culture medium, yeast cell morphology, and molecular biology.[Results]80 yeast strains of Cloud Dancer grape isolated from Mile region were clustering analysed by WL nutrient culture medium, and 5 culture types were obtained. 15 wild yeasts selected from the 5 culture types were identified by using 26 S r DNA D1/D2 domain sequences analysis. WL culture media identification and the sequence analysis showed that the selected yeast stains belonged to 5 species of 5 genera. They were Hanseniaspora uvarum, Issatchenkia terricola, Pichia kudriavzevii,Rhodotorula glutinis, Candida quercitrusa. The result revealed the diversity of yeasts related to Cloud Dancer grape in Mile County. H. uvarum was the dominate strain in Mile, which was occupied43.18%. The genetic diversity of yeast and the relationship between strains were analyzed by constructing the phylogenetic tree of 15 strains and type strains. The results showed that all the strains and their type strains clustered into one branch, and the results were similar to the sequence alignment. In addition, the relationship between different strains of the same yeast and the type strains was different,which showed certain genetic diversity.[Conclusions]The result revealed the diversity of yeasts related to Cloud Dancer grape in Mile, Yunnan. This study provides a material and reference for further breeding of indigenous wine yeast.