HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette
HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette
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DOI：
10.1007/s002530000490
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发表时间：
2001-04
影响因子：
5
通讯作者：
Y. Tamai;Keiko Tanaka;Y. Kaneko;S. Harashima
中科院分区：
文献类型：
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作者：
Y. Tamai;Keiko Tanaka;Y. Kaneko;S. Harashima
工业酵母的 Southern 印迹分析表明，本研究中测试的所有顶部发酵酵母、酿酒酵母和部分葡萄酒酵母均产生杂交信号（约 7 kb），对应于酿酒酵母型 HO 基因 (Sc-HO)。它还表明底部发酵酵母产生 7-kb 和 4-kb 杂交信号，分别对应于 Sc-HO 基因和啤酒酵母 HO 基因 (Lg-HO)。两种葡萄酒酵母产生 4 kb 杂交信号，对应于 Lg-H2O；一种葡萄酒酵母产生 2.5 kb 和 1.5 kb 杂交条带，对应于 aS。 uvarum 型 HO 基因 (Uv-HO)。从这些葡萄酒酵母扩增的HO基因的部分核苷酸序列分别与Lg-HO和Uv-HO基因完全匹配。通过在Lg-HO或Uv-HO基因内插入显性选择标记PGK1p-neo和交配型检测盒MFα1p-PHO5构建HO破坏载体。从携带单破坏基因的转化体中，通过减数分裂很容易获得具有交配能力的后代。此外，以非常低的频率出现的交配活性衍生物可以从无减数分裂的双破坏ho转化体（甚至从缺乏孢子形成能力的葡萄酒酵母）中获得，因为灵敏的磷酸酶染色方法允许通过随机孢子法从汇合菌落中检测Pho+交配活性衍生物。我们的研究描述了一种从工业酵母中分离具有交配能力的克隆的快速便捷的方法。
Southern blot analysis of industrial yeasts showed that all top-fermenting yeasts, distiller's yeasts and a proportion of wine yeasts tested in the present study produced a hybridization signal (approximately 7 kb), corresponding to aSaccharomyces cerevisiae-typeHOgene (Sc-HO). It also showed that bottom-fermenting yeasts gave rise to 7-kb and 4-kb hybridization signals, corresponding to the Sc-HOgene and the lager yeastHOgene (Lg-HO), respectively. Two wine yeasts produced a 4-kb hybridization signal, corresponding to Lg-HO; and one wine yeast produced 2.5-kb and 1.5-kb hybridization bands, corresponding to aS. uvarum-typeHOgene (Uv-HO). Partial nucleotide sequences ofHOgenes amplified from these wine yeasts perfectly matched those of Lg-HOand Uv-HO, respectively.HOdisruption vectors were constructed by inserting a dominant selective markerPGK1p-neoand the mating-type detection cassetteMFα1p-PHO5within the Lg-HOor Uv-HOgene. From transformants carrying a single-disruptedhogene, mating-competent progenies were easily obtained through meiosis. Moreover, mating-competent derivatives appearing at very low frequency could be obtained from a double-disruptedhotransformant without meiosis (even from a wine yeast lacking sporulation ability), because the sensitive phosphatase-staining method allowed detection of the Pho+mating-competent derivatives from confluent colonies by the random spore method. Our study describes a rapid and convenient method for isolating mating-competent clones from industrial yeasts.