Studyon Salt-Tolerant Gene HOG1of Yeast Added In Soy Sauce

The most aroma compounds in soy sauce are produced by yeast strains. During brewing soy-sauce processes, the metabolic activity of salt-tolerant yeasts is low because the content of salt is17%. The production of flavor components was thus effected badly, resulting in the decrease of density of soy sauce. In our lab, T3-5with higher salt tolerance was constructed from T yeast(Torulopsis versatilis) using genome shuffling. At present, the whole genome and salt-tolerant mechanism of T yeast is unclear. Therefore, the gene HOG1of T yeast and T3-5was studied in order for the further analysis of salt-tolerant mechanism of T yeast. Here, DNA sequence, transcription level and over-expression of gene and protein structure was systematically analyzed.In this study, the RNA expression of HOG1in T yeast and T3-5under different salt contents conditions was analyzed by semi-quantitative RNA expression analysis (RT-PCR). The results indicated that the RNA expression of HOG1in T3-5was higher that of T yeast. The gene HOG1in T yeast and T3-5was cloned and sequenced. Their PCR products were inserted into the vector pUC19, respectively, creating the plasmid pUC19-T and pUC19-T5. The result suggested that the mutant base pairs occurred. Subsequently, the gene sequence was translated into amino acids and the secondary and tertiary structure prediction of protein was carried out by sorting and stitching via NCBI. The data revealed that there were distinct difference of two amino acids and the isoelectric point of T3-5increased compared with T yeast. Finally, the PCR products of HOG1were inserted into the vector YEp195, respectively. The resulting plasmids YEpl95-T and YEp195-T3-5were transformed into W303-1A. Then, the salt tolerance and other indicators of the engineered strains were investigated. The results showed that the salt tolerance of W-Y-T5was higher than that of W-Y-T. Meanwhile, they were obviously stronger compared with the control strain W-Y.At present work, it was found that HOG] was also the key gene in osmo-adaption mechanism of T yeast. Furthermore, the increase of transcription level of HOG1in T3-5was a crucial reason of the improvement of salt tolerance in T3-5.