Cloning And Expression Of MyD88and IRF3Gene Of Schizothorax Prenanti

Schizothorax prenanti, which is one of the most important economical cold water fish, distributed in China uniquely. In recent decades, along with the fish disease outbreak constantly, which caused a lot of economic loss. Therefore, to understand its immune mechanism is very important for Schizothorax prenanti diseases control and healthy farming. Toll-like receptors (TLRs) construct the first anti-infection line in the Schizothorax prenanti, which recognized the conservative pathogen-associated molecular patterns (PAMPs) of pathogen structure.In our study, the parcial cDNA sequence of MyD88and IRF3gene were cloned from Schizothorax prenanti by RT-PCR, and the tissue expression of two genes were also analyzed by RT-PCR. The results are as following:(1) The length of obtained cDNA of MyD88was518bp, encoding172amino acids. By blast analysis in NCBI, the sequence identity of MyD88nucleotide sequence between Schizothorax prenanti and Cyprinus carpio, and Danio rerio was96%and93%, respectivly. Based on the MyD88amino acids in GenBank, the phylogenetic relationship analysis was performed by Neighbor-Joining method using Mega5.1software. The result found that phylogenetic tree was divided into two branchs. Schizothorax prenanti and other12fish species clustered into a separate branch. Birds and mammals clustered into a sub-family, respectively, and formed anther branch. It same as the traditional classification methods. The relationships between Schizothorax prenanti and Cyprinus carpio, and Danio rerio was most near in all tested species.(2) The length of obtained cDNA of IRF3was586bp, encoding193amino acids. By blast analysis in NCBI, the sequence identity of IRF3nucleotide sequence between Schizothorax prenanti and Cyprinus carpio was77%. The results of phylogenetic relationship analysis suggested that phylogenetic tree was divided into two branchs. Schizothorax prenanti and other8fish species clustered into a separate branch. Birds and mammals clustered into a sub-family, respectively, and formed anther branch. The relationships between Schizothorax prenanti and Cyprinus carpio was most near in all tested species.(3) RT-PCR analysis revealed a broad expression of MyD88in all examined tissues, with highest expression in the spleen and liver respectively. IRF3expressed in all the examined tissues, such as spleen, liver, muscle, gill, intestine, kidney, and blood, with highest expression in the liver and lowest expression in the muscle respectively. The expreesion of MyD88in spleen and IRF3genes in liver of Schizothorax prenanti changed statistically significant after induced with Aeromonas hydrophila. Overall, this study provides theoretical basis for understanding immune mechanism in depth of Schizothorax prenanti and orther economically important fish species in the Yangtse River.