EST Construction And Global Transcriptional Analysis Of Trichophyton Rubrum Conidial Germination

Compared to bacteria and virus, the research of fungi remained undeveloped. Now, with the increase of the incidence of disease caused by fungi, the important role of fungi in the research on evolution, comparative genomics, pathogenicity, and the influence on industry and agriculture have been gradually understood. Among the numerous pathogenic fungi, dermatophytes are the most common fungus . They are the etiologic pathogens of various dermatophytoses, affecting up to 25% of infections to the world's population,. The percentage of infection has continued to increase in recent years. Thichophyton rubrum is a very important human pathogen which causes at least 60% of infections of various dermatophytoses in the world.In this reseach, T. rubrum BMU01672 cDNA libraries from 8 growth phases were constructed and 10118 unique expression sequence tags (EST), including 3416clusters and 6772 singlets, were obtained by sequencing near 60, 000 cDNA clones. The average length of these clusters and singletons were 720bp and 443bp respectively. The ESTs were then analyzed by comparision with the database and their putative encoded proteins were classified in COGs. The acquired results and information was used to find the functions of some important genes and to elucidate pathways involved in the information storage and processing, cellular processes and metabolism in T. rubrum.In order to get to know the physiological process, pathogebic mechanism and feature of metabolism of the fungus, a cDNA microarray including all 10250 ESTs of T. rubrum were prepared. The array was then used to analyse the gene expression profiling in the process of spore germination.In this research, Total RNA samples were collected at 1 hour intervals starting immediately from spore (0 hour) and ended when 16 pieces of samples , At every time-point, two biological replicates containing 4 hybridized data were used for cDNA microarray hybridizationThe processed slides were scanned with a Genepix 4100B scanner. After spots flag and normalization process, 9470 spotsin a microarray were used for the gene expression profiling analysis. The expression variations during the time-course were analyzed by ANOVA (p < 0.01) for each gene. 2772 genes were obtained that their expression levels were significantly altered during the germination. In order to identify genes whose expression levels were altered dramatically during germination, 1576 out of the 2772 genes fitting expression levels altered exceeding twofold were used for further analysis. These genes were clustered by Tiger TMEV 3.1 software. The genes enrichment within cluster and the statistical significance of gene distribution across the clusters were estimated by post-hoc tests(p <0.05) using SPSS 10.0 For Windows.To identify the correlation between gene expression profile and physiological transitions during germination, ESTs of the selected 1576 genes were matched to the sequences in the Gene Ontology (GO) database by BLASTX searches..ESTs of the selected genes were matched to the sequences in the Gene Ontology. Total 780 out of 1561 genes were assigned to different biological process, and 666 and 960 out of 1561 genes were assigned to GO terms: "Cell Components"and"Molecular Function"respectively. Individual gene can have multiple Gene Ontology assignments. About 454 genes have no hints in the Gene Ontology.Use the GO terms, we analy for expression profiling associated with putative biological and physiological events during T. rubrum conidial germination To better understand the cellular processes and signalling pathways involved in conidial germination, the EST sequences of all 1561 genes were matched to GO terms at the Saccharomyces Genome Database. A total of 37 homologues of genes involved in several signalling pathways or regulation modules, such as the cAMP/PKA signaling pathway, the Ras-GTPase-related signalling pathway, the MAPK signaling pathways and two-component signal transduction systems were found and their expression levels were induced during the process. Comparison of signalling transduction pathways and cellular events involved in conidial germination in T. rubrum and yeastTo analyze the function of pre-stored mRNA in conida of Trichophyton rubrum. The EST sequences of 560 genes pre-stored in conidia were noted by comparison with the database of other filamentous fungi database in NR. Physiological processes that these genes may be involved in were classified by GO database. Some important genes and signaling pathways may be relative to induce conidial germination were foundIn order to verify the microarray result, the relative expression levels of 10 genes at several time points (0, 4, 10, and 15) were estimated by Quantitative real-time RT-PCR. Our results may provide insights into molecular mechanisms of conidial germination at the cell level, and may enhance our understanding of the regulation modulus related to the morphological construction of T. rubrum.