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Cloning Glycerol-3-Phosphate Dehydrogenase Gene of Yeast and Analysis of Its Various Transcriptional Levels of mRNA in the Hyper-Osmotic Conditions

Author: ZhangFan
Tutor: YanChunTai；LiangYunXiang
School: Huazhong Agricultural University
Course: Microbiology
Keywords: gpd1 Saccharomyces cerevisiae Osmolarity pressure
CLC: Q78
Type: Master's thesis
Year: 2005
Downloads: 97
Quote: 0
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Abstract


The environmental osmotic pressure is a key factor that would affect yeast cell survival. When the environmental osmotic pressure increases, the yeast Saccharomyces cerevisiae ’s survival will be threatened greatly. The cell would accumulate intracellular glycerol which would help yeast adapt hyper-osmotic stress. GPD1 is a protein with 391 amino acids. It would catalyze the reaction from dihydroxyacetone phosphate to glycerol-3-phosphate, and it play an important role in the hyper-osmotic stress adaption. Hyper-osmotic stresswill increase the expression and transcription of GPD1 protein , and mutation on the locus of gpd1 gene will lead to lethal effect in hyper-osmotic environment.Three yeast strains of Saccharomyces cerevisiae named YY、 WFB、 FHS are studied in this article. To reveal how the gpd1 gene affects the yeast adaptability of hyper osmotic stress, different concertration of Sodium Chloride is added into the medium. When the yeast strains YY、 WFB、 FHS were disposed with 1%、 3% and 5% concentration of NaCl, different growth rates in each disposal were observed and analyzed. The results show that the influence of growth rate caused by NaCl on YY and WFB is more magnificant than FHS when disposed with concentration of 1% and 3%. Further more, when Yeast strains were disposed with a concentration of 1% NaCl, it indicated that the influence of growth rate caused by NaCl on strain WFB is more dramatically than YY. And when Yeast strains were cultured in NaCl solutions with a concentration of 5%, it indicated that the influence of growth rate caused by NaCl on strain WFB is more magnificant than FHS, furthermore FHS is more magnificant than YY. But the difference between the test stains isn’t so obvious. Then it could be inferred that FHS strain is best, YY is better, WFB is worse concerning the adaptability of hyper-osmotic stress.In order to find out the difference adapability of hyper-osmotic stress in the three yeast strains, gpd1 gene was Cloned and sequenced of YY、 FHS and WFB strains respectively. By use of biological software Vector NTI, compared the gpd1 gene sequence in YY、 FHS and WFB strains with the gpd1 gene sequence (X76859) published in Gene bank, the nucleotide homology of gpd1 gene fragments between those originated from strains WFB、 YY、 FHS and the one presented on the Gene Bank(X76859) are 99.32%、 99.83%、 99.83% respectively. Translating all these nucleotide sequence into amino acid sequence, then align these amino acid sequence, the results indicate that the amino acid sequence of gpd1 gene fragments between those originated from strains YY、 FHS and the one presented on the Gene Bank(X76859) is the same. The homology between WFB and the one presented on the Gene Bank(X76859) is 98.5% only.Differential mRNA transcription Levels on hyper-osmotic stress of gpd1 gene was also analyzed in this study. . RNA Dot blotting showed that in YY、 FHS strains the mRNA transcriptional level of gpd1 gene increased corelated to the concentration of NaCl, butit’s contrary to the strain WFB. The result show that increase the transcriptional mRNA Levels of gpdl gene could improve the yeast adaptability of hyper-osmotic environment.

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