Study On The Role Of LEPR In Anti-infection Immunity Of Rana Dybowskii And Rana Amurensis

Leptin receptor(LEPR)is a transmembrane protein belonging to the class I cytokine receptor family.The combination of leptin receptor and leptin(LEP)can play many important biological functions such as energy metabolism,reproduction and immunity.In order to explore the role of LEPR in amphibian inflammatory response,this study took Rana dybowskii and Rana amurensis as experimental objects,cloned LEPR gene by RTPCR technology,and analyzed its bioinformatics.Then an inflammatory model of Aeromonas hydrophila(Ah)infection was constructed and the pathological changes of the inflammatory model were observed with hematoxylin-eosin(HE)staining.qRT-PCR was used to detect the dynamic expression of LEPR gene in the two species of Rana dybowskii and Rana amurensis infected by Ah.Immunohistochemical staining and Western blotting were used to investigate the expression of LEPR protein under Ah infection.The results are as follows:The encoding regions of the LEPR gene both Rana dybowskii and Rana amurensis were obtained by RT-PCR,which were 3 405 bp and 3 408 bp,respectively,encoding 1134 and 1 135 amino acid residues,respectively.The LPER protein of Rana amurensis had an additional lysine at 1 111.Both LEPR are hydrophilic and unstable proteins,which are cell membrane proteins with primary transmembrane structure.Nucleotide homology analysis showed that the homology of the two species was 97.1%.The phylogenetic analysis showed that the LEPR of the two species of Rana dybowskii and Rana amurensis converged with amphibians in the genetic relationship,indicating that the LEPR of the two species of Rana was highly conserved.With the extension of Ah infection time,different degrees of pathological changes occurr in liver,spleen,skin,muscle and stomach tissues.The gap of liver tissue is enlarged,the boundary of hepatocyte is blurred,and the nucleus is pyknotic.Hemolysis occurs in spleen cells and an increase in melanin macrophages.The epidermal layer of the skin is damaged,the dermis is dissolved and thinned,and the melanocytes increase.The space between muscle fibers in muscle tissue becomes wider,disintegrates and breaks.The mucosal epithelial tissue of the stomach is damaged and dissolved.The blood vessels in the submucosa are congested,and the blood cells are filled in the lamina propria and submucosa.The above shows that the inflammatory infection model has been successfully established.The m RNA levels of LEPR in the heart,liver,spleen,lung,kidney,skin,muscle and stomach tissues between the two species of Rana were determined with qRT-PCR,it showed that the expression of LEPR m RNA increased at different time points of Ah infection.The expression of LEPR m RNA in lung,kidney,muscle and stomach of Rana dybowskii reached its peak at 16 h,while that in spleen and skin reached its peak at 72 h,and that in heart and liver reached its peak at 24 h and 8 h respectively.The expression of LEPR m RNA in the lung tissue of Rana amurensis reached its peak at 8 h after infection,and the expression of other detected tissues reached its peak at 72 h after infection.Immunohistochemical staining and Western blotting were used to analyse the protein expression of LEPR in the liver,spleen,kidney,skin and stomach of Rana dybowskii and Rana amurensis infected by Ah.It found that they were similar to the qRT-PCR results,and LEPR was mainly located on the cell membrane.To sum up,this study obtained the LEPR gene of Rana dybowskii and Rana amurensis,and analyzed their basic biological characteristics.It was confirmed that the gene was involved in the whole process of Aeromonas hydrophila infection.This study provided a foundation for further analysis of the biological function of LEPR gene in amphibians.