| The phenotype switching of Candida.albicans is closely related with their infection and diseases, but the mechanism about the phenotype switchin of Candida.albicans is not clear. Some related genes such as VPS11, CaCLA4 , INT1 and CaRSR1, and the signal conducting network including MAPK , cAMP have been reported to be involved in the phenotype switching . However most of the research is confined to a certain gene or the composition of signal conduction, and there is a lack of comprehensive and systematic studying on the gene network that adjusting and controlling the phenotype switching of Candida.albicans. The sequencing work on the genome of Candida.albicans has been completed, but a large number of unknown genes need to be identified, and the key genes participating in the phenotype switching of Candida.albicans have not been demonstreted. In the past years, , the LongSAGE libraries of different phenotype of Candida.albicans have been established using serial analysis of gene expression (SAGE) and thousands of expressed sequence tags (ESTs) have been obtained. Furtherly total mRNA of genes expressed in the cell can be measured and qualitatively analysed in short time, which can not merely be used in the analysis on the difference of gene expression on different fungus, but also can be used in finding the potential unknown genes. Based on the above-mentioned research work, 3 ′RACE technology was used to obtain gene sequence from the label to the end of mRNA-3 ′representing the differently expressed SAGE labels, and amplification of the 3′end of those different genes possibly functioning in the phenotype switching was carried out to obtain the EST of these genes, which finally represented differently expressed genes of Candida.albicans in phenotype switching. Our results will be useful for studying positive capacity of the LongSAGE library and detecting those genes participating in the regulation and control phenotype switching or some new genes of Candida.albicans. Our work were performed as follows : 1. The induction of hypha of Candida.albicans ATCC90028; 2. Preparation and analysis of total RNA of Candida.albicans ATCC90028 in different phases; 3. Establishment of the difference of the differently expressed genes of LongSAGE library labels in different fungi using 3 ' RACE technology. Main results and conclusion are as follows: 1. By shake culture method, the induction ratio of germ of Candida.albicans was greater than 95% in1640 culture medium or DMEM culture medium, with spore density at the 5×106 / L ( pH7.35, 37℃). It proved that pure hypha of Candida.albicans can be harvested under optimized conditions. 2. Using improved fungus rupture method (mechanical glass pearls), total RNA of different cell both in yeast-form and hyphal-form was obtained successfully in high quality, which were verifed by RT-PCR at the same time . It is confirmed that the improved fungus rupture method with mechanical glass pearls is suitable for different forms of Candida.albicans. 3. 3′RACE technology is applied to amplify different EST of Candida.albicans, then the products were purified and connected by T/A, transformed into E coli.DH5 and induced to express by X-gal/IPTG, the plasmids were extracted and positive bacterial strain digested by the edonuclease and sequenced. Two DNA fragments can be found their homologs in Genbank. Their Score value was < 40 and E-value was < 0.5. EFT2, 26S rRNA and SHMI were identified to be that represented by LongSAGE labels of JMD8 ,JSW4 and JSW10 respectively. In summary, our work has successfully proved that three different EST representing genes through gene clone technology of molecular biology. It not only has elementarily proved the establishment of LongSAGE library, but also provided a suitble method for the verification of the positive capacity of LongSAGE library of Candida.albicans . The above work will be helpful for further studying the precise biological function and molecular mechanisms of those genes involved in the phenotype switching of Candida.albicans.
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