Research On Gene Expression And Proteomics Of Chinese Brown Frog (rana Chensinensis)skin

Chinese brown frog skin and its secretions play important roles in fighting against the invasion of microbes and pathogens. At present, the researches on Chinese brown frog skin and its secretions are mainly focused on a certain or several proteins or peptides, and the comprehensive information on the immune proteins and defensive peptides from Chinese brown frog skin and its secretions at molecular level is rare. We studied gene expression of proteins and peptides from Chinese brown frog skin and its secretions on RNA transcription and protein expression levels using EST technology and proteomics. We expect that this work will help us to have a comprehensive understanding on amphibian skin and its secretions physiological function and defence mechanism. It will provide a candidate data set for screening Chinese brown frog skin functional genes in the future.Firstly, we constructed a non-normalized cDNA library from the skin. By sequencing cDNA clones at the 5'end, we obtained 5,976 high-quality EST sequences, which clustered into 512 contigs and 1379 singletons(in all 1,891 clusters). After BLAST searches of the protein and nucleic acid databases in GenBank, we found 46.6% of clusters to have significant similarity to known sequences, and 53.4% of clusters have no hits. They are likely to be new genes. Gene annotationresults indicated that genes related to secretion and defensive function, such as ubiquitin, lectin, proteinase inhibitors and whey acidic-domain proteins, are highly expressed in the skin. To investigate gene function in Chinese brown frog skin, we classified 826 (43.7%) clusters into three different categories (molecular function, biological process and cell component) based on gene ontology. For further annotation, we compared the 1891 clusters with the protein-domain database by using InterProScan. Of these, 638 (33.7%) clusters could be assigned to known domain families. In addition, the KEGG metabolic pathway analysis on Chineses brown frog skin expressed genes help us to have a better understanding on physiological functions and metabolic pathways of Chineses brown frog skin at the molecular level.Notably, we found 518 ESTs (8.67%) related to immunity and defense in the current library. They included whey acid protein family (WAP), non-digestible enzymes and proteinase inhibitors, antimicrobial peptides and lectins. The discovery and analysis of these genes provides important molecular informations on Chinese brown frog skin immunity and defense mechanism. On the other hand, we studied Chinese brown frog skin and its secretions on protein expresstion level using two-dimensional electrophoresis and mass spectrometry technology. We found proline -4 hydroxylase, L-allo-threonine aldolase, galectin, Ranasmurfin, BTB /POZ family proteins, macrophage migration inhibitory factor, antimicrobial peptide chensirin-3 precursor and Phosphoglycerate kinase. They all play an important role in the skin immunity and defense process.