| Porphyra, the vital economical algae, widely scatter in the inter-tidal zone along the coast in our country. They live in the so mutable environment that they have the idiosyncratic adaptability to the adversity stresses, especially the fast and efficient regulate mechanism for the changes of the temperature, the osmotic pressure, and the high radiation. Therefore, in-depth study of the stress tolerance-related genes and their functions in Porphyra is very helpful to understand the biotic mechanism of the stress tolerance.In this study, the hsp70 gene of Porphyra haitanensis was chosen as the target gene, and the plasmid p181AINE as the vector, to construct the the recombinant eukaryotic expression vector p181-hsp70, which would be transformed into the yeast in order to analyze the function of this exogenous gene. The vector of p181-hsp70 was proved to be constructed successfully by the evidences of the individual bacterial colonies PCR, double enzymes digestion and sequencing. The p181-hsp70 plasmids were transformed into the wild-type yeast strain BY4741 and SSA3(a reduced gene hsp70) deletion yeast strain YBL075c by the electro-transformation. The empty plasmids p181AINE were transformed into the yeast strains repectively as the controls. The growth curves of the recombinant yeasts were determined. At the same time, the expression of the exogenous gene hsp70 in the recombinant wild-type yeast (overexpression, BY4741+p181-hsp70) and the compensated deletion-type yeast (YBL075c+p181-hsp70) was carried out using the real-time fluorescence quantitative PCR technology after the two yeasts were treated with a short-time heat shock. The NMR technology was also employed in the preliminary studies of the metabonomics in the recombinant yeasts. The results showed as follows: 1,At 30℃and 40℃, there was no obvious difference between the growth rate of recombinant wild-type yeast strains and their controls, the same result as the compensated deletion-type yeast strains. When the yeasts cultivated at 45℃, the compensated deletion-type yeasts and their controls didn′t grow at all;while the growth of the recombinant wild-type yeasts (BY4741+p181-hsp70), were obviously better than the controls(BY4741+p181AINE), but both of them grew worse than those yeasts cultured at 30℃and 40℃.2,Employing the yeast 18S rDNA as the internal reference, the relative expression of hsp70 in the recombinant wild-type yeast (overexpression, BY4741+p181-hsp70) and the compensated deletion-type yeast (YBL075c+p181-hsp70) after the short period of the heat-treatment were detected by the technique of real-time fluorescence quantitative PCR technology. The results showed: the expression of exogenous gene hsp70 in the two recombinant yeasts both declined after heat shock for 5min, increased for 15min, and was decreased slightly for 30min heat-treatment, so the hsp70 expression trend was identical in two yeasts. However, the expression of hsp70 in recombinant wild-type yeasts was lowest after heat shock for 5min, and only the 0.75 times as the control without any heat-treatment, while the one in the compensated deletion-type yeasts increased obviously, with 1.5times as much as their controls. What caused this happened was speculated to be related to the types of the constructive promoter of ADH I in the recombinant plasmid and the yeast SSA3 gene's regulatory elements (inducible element), which could strongly influence on the exogenous gene expression efficiency at the transcription level.3,The metabonomics analysis of the recombinant yeasts indicated that there was no obviously difference between the compensated deletion-type yeasts and its controls. While the significant differences of the metabolites, were found between the recombinant wild-type yeasts and its controls. However, the components and their functions of these metabolites demand for a further analysis. |
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