Application of real-time RT-PCR to study gene expression in active dry yeast (ADY) during the rehydration phase.
During the industrial production of active dry yeast (ADY) and its subsequent use in winemaking, the yeast cell is subjected to drastic environmental changes that force it to undergo extensive metabolic modifications and changes in gene expression. In this study, we describe the use of real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor gene expression in ADY Saccharomyces cerevisiae during rehydration in different media. We used three statistical approaches to investigate the expression stability of eight potential reference genes during the rehydration process. The reference system thus obtained was used to normalize the expression values of three genes codifying for the ammonium transporters -- MEP1, MEP2, and MEP3 -- and two genes involved in the osmotic response-SIP18 and GPD1. The results suggested that for the target genes tested, the yeast reacted immediately to rehydration only when a fermentable carbon source was present in the medium. Furthermore, MEP2 expression was modulated by the ammonium concentration, indicating that nitrogen catabolite repression (NCR) is active during the rehydration phase.
Vaudano
Enrico
E
CRA-Centro di Ricerca per l'Enologia, Asti, Italy. biologia.molecolare@isenologia.it
Costantini
Antonella
A
Cersosimo
Manuela
M
Del Prete
Vincenzo
V
Garcia-Moruno
Emilia
E
D003470:Culture Media / Q000737:chemistry; D004734:Energy Metabolism / Q000235:genetics*; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics* / Q000502:physiology*; D007218:Industrial Microbiology; D009997:Osmotic Pressure; D000644:Quaternary Ammonium Compounds / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000494:pharmacology*
D003470:Culture Media; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; D014867:Water