Control gene em B2M /em served as a non template unfavorable control for each plate. factors were also suggested . Msn2p has been confirmed for its positive regulatory function of em HSP12 /em and most warmth shock protein genes for increased ethanol tolerance [67-69]. A double gene deletion DLEU2 em msn2msn4 /em -mutant showed hypersensitivity to environmental stress including higher ethanol concentrations . We exhibited that the increased expressions patterns of em MSN4 /em overtime were distinct from other transcription factor genes. Our results suggest a potential important role of Msn4p in the dynamic response to the ethanol tolerance. However, limited information is usually available for Msn4p and further studies on its regulatory functions for tolerance are needed. Conclusion The qRT-PCR array assay equipped with the strong mRNA reference and the grasp equation is an efficient means for quantitative gene expression analysis which unifies a large amount of expression data generated under different experimental conditions. The Cannabichromene comparative characterizations of adaptive transcription dynamics for the two Cannabichromene closely related strains are more informative and provide insight into dissection of mechanisms of ethanol tolerance. Analysis Cannabichromene of the expression dynamics and association of other phenotypes allowed identification of candidate and important genes for the ethanol-tolerance and ethanol production under the stress. Enriched background of mRNA large quantity of many genes appeared to be inheritable for the ethanol-tolerant yeast. Most ethanol-tolerance candidate genes were found sharing protein binding motifs of Cannabichromene transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p. The unique expression pattern of em MSN4 /em in the ethanol-tolerant Y-50316 suggested a potential important regulatory role of Msn4p during the adaptive expression in yeast. Unlike repressed in the parental strain, genes able to maintain normal expressions under the ethanol-stress were necessary for the tolerant Y-50316 to function. Ethanol-tolerance candidate genes recognized in this study are primarily associated with functional categories of cytoplasm, membrane, cell wall, response to stress, transportot, protein folding, oxidoreductase activity, protein binding and unknowns classified by gene ontology (GO). However, multiple functions and functions at multiple loci of many candidate genes are common. Ethanol induced genes are involved in at least 79 GO groups and every gene was found to have more than one function . It’s the time to revisit the traditional “one gene-one function” concept when evaluating gene regulatory networks. The complicated gene interactions cannot be overlooked in dissection of mechanisms of ethanol-tolerance in yeast. Methods Yeast strains, medium, and culture conditions Ethanol-tolerant yeast em S. cerevisiae /em NRRL Y-50316 and its inhibitor-tolerant parental strain NRRL Y-50049 (Agricultural Research Service Culture Collection, Peoria, IL, USA) were used in this study. Cultures were maintained and produced on a YM medium (3 g yeast extract, 3 g malt extract, and 5 g peptone, in 1 L distilled water) supplemented with 2 or 10% (w/v) glucose. Cultures were incubated on 300 ml medium in a fleaker system with agitation at 30C as previously explained . A solid YM plate made up of 2% agar was used to examine cell growth and viability. All experiments were carried out with two replications. Yeast adaptation and mutation selection Adaptation procedures were developed based on procedures by Wei et al.  and Dinh et al.  with modifications. Briefly, inhibitor-tolerant strain NRRL Y-50049 was cultured on a YM with 10% glucose made up of ethanol in designated concentrations. Cannabichromene Cultures were treated with a quick freeze at -80C at the mid-log phase and thawed at 30C in a water-bath. The treatment procedures were repeated. Incubations were continued at 30C until a stationary phase was reached. Surviving cultures were sequentially transferred to new medium made up of higher ethanol concentrations. These procedures were repetitively carried out until a target tolerance level reached. Tolerant mutants were selected from at least 40 total cycles using a medium containing no less than 8% ethanol. Culture characteristics were confirmed by.