Comparision And Application Of The Promoters Osmoregulated By HOG In Candida Glycerinogenes

Promoter is a cis-acting element regulating gene expression. Its activity has great influence on the gene expression. Therefore screening for excellent promoters has great significance to heterologous gene expression and strains transformation. Although a number of promoters are cloned and identified, the constructive promoters which used commonly in yeasts are uncontrollable and the inducible promoters need inducers. The Candida glycerinogene isolated from nature has good performance in terms of osmotolerant and glycerol production. It contains Cg Hog1, the core element of the high osmolarity glycerol mitogen-activated protein kinase(HOG-MAPK) pathway, which is the homologous protein of Hog1 from Saccharomyces cerevisiae.In this study, we did the following works aimed at selecting the osmo-regulated promters possessing excellent performance for further research of the industrial yeast C. glycerinogenes:At first, in order to obtain promoters regulated by Cg Hog1, we used Phosphorylation p38 as the specific antibody for Chromatin Immunoprecipitation(Ch IP) and constructed the gene library from the Cg Hog1-promoters compound. PCg STL1, PCg STL3, PCg ZWF were selected in this study. Analysing the genetic sequences of the three promoters by bioinformatics database presumed that PCg STL1, PCg STL3, PCg ZWF were regulated by Cg Hog1 under hyperosmotic pressure. In addition, PCg PGI, PCg TPI and PCg STL2 which were regulated by stress in prediction weren’t gotten in this study.Then, we constructed integration vectors containing PCg STL1, PCg STL2, PCg STL3, PCg ZWF, PCg PGI, PCg TPI to check the intensity of the promoters under stress. Using green fluorescent protein as a reporter showed the performance of the candidate promoters under hyperosmotic pressure. The transcription levels of gfp in the recombinant strains under hyperosmotic stress were checked by q RT-PCR. This experiment explained the changes of corresponding promoters under stress. The results showed that the transcription level of gfp driven by PCg STL1, PCg STL3 and PCg ZWF increased with the osmotic pressure. But PCg PGI, PCg TPI and PCg STL2 had little effect on the gfp transcription.At last gfp expression of the recombinant strains were checked in order to compare the performance of the candidate promoters under different glucose pressure. The results indicated that the fluorescence of gfp induced by PCg STL1, PCg STL3, PCg ZWF enhanced with hyperosmotic stress. Among the tested stains, the expression of gfp reached by PCg STL3 had the largest increase and the fluorescence intensity reached the maximum value in 30% glucose solution. It means that PCg STL1, PCg STL3 and PCg ZWF are new promoters having good effect on the expression of heterologous gene and their strength are adjustable by osmotic stress. As PCg STL3 showed the most sensitivity to osmotic pressure, it could be used as an adjustably strong promoter for overexpression of heterologous gene.