Esteban Rosa, Rodríguez-Cousiño Nieves
Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, Universidad de Salamanca, Consejo Superior de Investigaciones Científicas, Salamanca 37007, Spain.
Yeast. 2008 May;25(5):359-69. doi: 10.1002/yea.1594.
In this study we have developed a useful method to identify a particular yeast strain within a mixture of strains during must fermentation, based on the presence or absence of a stable genetic element derived from Saccharomyces cerevisiae 23S RNA autonomous replicon. 23S RNA is a natural virus-like RNA replicon present in some S. cerevisiae strains, which encodes only its own RNA-dependent RNA polymerase named p104. A modified version of 23S RNA (23S-tagged RNA) was generated after transformation of S. cerevisiae wine strains with a launching plasmid, where six nucleotides were changed in the 23S RNA cDNA sequence without modifying the amino acid sequence of p104 RNA polymerase. Once generated, the 23S-tagged RNA can replicate autonomously (without the plasmid), is very stable, is present in high copy number in stationary phase or nitrogen-starved cells and confers no phenotype to the host, like the endogenous 23S RNA replicon. However, it can be distinguished from endogenous 23S RNA by reverse transcription followed by polymerase chain reaction (RT-PCR) with specific oligonucleotide primers. 23S RNA-derived replicon can be used to tag wine yeast strains in order to monitor easily their prevalence over endogenous strains during wine fermentation.
在本研究中，我们开发了一种有用的方法，可在葡萄汁发酵过程中，基于源自酿酒酵母23S RNA自主复制子的稳定遗传元件的有无，从菌株混合物中鉴定出特定的酵母菌株。23S RNA是存在于一些酿酒酵母菌株中的一种天然病毒样RNA复制子，它仅编码其自身名为p104的RNA依赖性RNA聚合酶。在用启动质粒转化酿酒酵母葡萄酒菌株后，产生了23S RNA的修饰版本（23S标记RNA），其中23S RNA cDNA序列中有六个核苷酸发生了变化，而p104 RNA聚合酶的氨基酸序列未改变。一旦产生，23S标记RNA就可以自主复制（无需质粒），非常稳定，在稳定期或氮饥饿细胞中以高拷贝数存在，并且像内源性23S RNA复制子一样，不会赋予宿主任何表型。然而，通过逆转录然后用特异性寡核苷酸引物进行聚合酶链反应（RT-PCR），它可以与内源性23S RNA区分开来。源自23S RNA的复制子可用于标记葡萄酒酵母菌株，以便在葡萄酒发酵过程中轻松监测它们相对于内源性菌株的流行情况。
