| Heat shock proteins(HSPs)are highly conserved stress proteins that are ubiquitous in various organisms.When organisms are exposed to various adverse environmental conditions,these proteins are synthesized in large quantities with the stress response of the body to reduce the damage caused by external stimuli.The special physiological characteristics and living environment of amphibians determine that they are more vulnerable to adverse factors such as pathogenic microorganisms during their survival.As an important member of the heat shock protein family,heat shock protein 70(HSP70)plays an important role in stress resistance.Therefore,it is necessary to study the function of hsp70 gene in amphibians under infection conditions.In this study,the dominant amphibian species in Northeast China,Rana dybowskii and Rana amurensis,were used as the research objects.Based on the method of homologous cloning,the cloning and identification of hsp70 family genes of two species of Rana were completed by RT-PCR and homologous sequence alignment.The sequence characteristics,three-dimensional structure and phylogenetic relationship of Rd-hsp70 and Ra-hsp70 family genes were analyzed by bioinformatics analysis software.The inflammatory infection model of R.dybowskii and R.amurensis under the stress of Aeromonas hydrophila(Ah)was constructed,and the histopathological changes of the two species were observed by hematoxylin-eosin staining(HE).The tissue expression characteristics of hsp70 family genes under bacterial infection were detected by real-time quantitative PCR(qRT-PCR).The expression of HSP70 protein was obtained by immunohistochemistry(IHC)and Western Blot(WB).It lays a theoretical foundation for the functional study of amphibious hsp70 gene in response to bacterial infection.In this study,the full-length coding regions of hsp70 family genes,hspa5,hspa8 and hspa13 genes of R.dybowskii and R.amurensis were successfully cloned.The lengths were 1 920 bp,1 962 bp,1 944 bp and 1 383 bp,encoding 639,653,647 and460 amino acid residues,respectively.The nucleotide and amino acid sequence homology between the corresponding family members of the two Rana species were :hsp70(98.38 % and 98.74 %),hspa5(98.06 % and 98.92 %),hspa8(98.55 % and99.22 %)and hspa13(97.90 % and 98.47 %).Phylogenetic analysis showed that R.dybowskii and R.amurensis were closely related to amphibians such as R.temporaria.By constructing the Ah infection model of R.dybowskii and R.amurensis,HE staining was used to observe that with the prolongation of infection time,different degrees of pathological damage occurred in the three tissues of liver,skin and stomach,and the tissue cells were significantly damaged,indicating that the inflammation model had been successfully established.The results of qRT-PCR showed that the hsp70 family genes were generally expressed in the heart,liver,spleen,lung,kidney,skin,muscle and stomach of R.dybowskii and R.dybowskii under normal physiological conditions.The four members showed the lowest expression level in the heart tissues of the two species of Rana,and the expression levels in the spleen,kidney,skin and stomach were generally high.This suggests that hsp70 family members play a role in maintaining the normal physiological state of the body.The results of mRNA dynamic expression profiles of Rd-hsp70 and Ra-hsp70 family genes in different tissues after Ah stress showed that the relative expression levels of the four members in different tissues of the two Rana species were significantly up-regulated(P < 0.01).However,the expression trends of each gene in different tissues were different,and the peak expression levels were also different,with tissue specificity,indicating that the hsp70 family genes responded to the biological process of bacterial infection to varying degrees.At the same time,it was found that the three members of hspa5,hspa8 and hspa13 had the same expression trend in the two Rana species,indicating that the three members may play a synergistic role in the process of responding to pathogen infection.The results of immunohistochemistry showed that HSP70 protein was expressed in different degrees in the cytoplasm of 8 tissues of R.dybowskii and R.amurensis.The quantitative analysis confirmed that the expression of protein level was basically consistent with the fluorescence quantitative results.At the same time,Western blot experiments confirmed that about 72 kDa HSP70 protein bands were detected in the liver and muscle tissues of the two species of Rana,and the expression of HSP70 protein was consistent with the expression trend of hsp70 mRNA in the correspondin g tissues,that is,the expression was significantly up-regulated after infection.It is speculated that this result is related to the response of HSP70 to inflammatory response in Rana chensinensis.The expression of Rd-hsp70 and Ra-hsp70 family genes is up-regulated to varying degrees,indicating that different family members may play different biological functions in the anti-inflammatory immune response in vivo. |
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