Identification And Functional Characterization Of JNK/Bcl-2 Genes In Swimming Crab(Portunus Trituberculatus)

The swimming crab Portunus trituberculatus is an economically important crustacean in China,but diseases caused by Vibrio parahaemolyticus and WSSV cause serious economic losses in the crab culture industry.As a defense mechanism of host cells,apoptosis plays an important role in the immune process of the organism.However,the mechanism of apoptosis in crab during pathogenic infection is still unclear.This paper mainly discusses the roles of apoptosis related gene in crab during pathogenic infection.The results of this study not only provide a new research idea for the prevention and treatment of the disease,but also provide experimental data and reference for further enriching the role of apoptosis in the immune defense of crustaceans.Three full length cDNA sequences including Pt JNK,PtBax and PtBcl-2 were first obtained by Rapid Amplification of cDNA Ends(RACE)technique.The tissue distribution and the temporal expression of the m RNA levels and protein levels of these genes with the treatment of Vibrio parahaemolyticus and WSSV were measured by Real-time RT-PCR and western blot.In addition,the functional mechanisms of PtBax and PtBcl-2 were analyzed by ds RNA interference and Yeast two-hybrid.The results of this paper are as follows:1.The full-length cDNA of the JNK gene(Pt JNK)was 3240 bp and the open reading frame(ORF)is 1380 bp and encodes a 459 aa polypeptide,which includes a serine/threonine protein kinase(S-TKc)domain with a conserved Thr-Pro-Tyr(TPY)motif,it is a typical feature of JNK gene family.The expression patterns of Pt JNK gene in different tissues were detected by q RT-PCR.The results of tissue expression and distribution show that Pt JNK gene is expressed in all tissues.Hepatopancreas was used as the control in the experiment,the lowest in hepatopancreas,and the highest in stomach and muscle(3.98 times and 3.78 times as compared with the expression levels in hepatopancreas,respectively).Therefore,it is speculated that Pt JNK may play different roles in the Portunus trituberculatus.The transcriptional levels of Pt JNK gene is significantly down-regulated after injection of Vibrio parahaemolyticus(p<0.05),while the expression of Pt JNK gene is significantly up-regulated when WSSV is injected(p<0.05).To sum up,Pt JNK gene is a widely expressed gene,and its expression pattern is different in different pathogen infections,which plays an important role in immune defense.2.The full-length cDNA sequences of PtBax and PtBcl-2 genes were cloned,the full-length cDNA sequence of PtBax is 2571 bp and encodes a polypeptide of 503 amino acids(aa)the full-length cDNA sequence of PtBcl-2 is 2015 bp and encodes a 282 aa polypeptide.Sequences analysis showed that their deduced amino acid sequences contained a typical Bcl domain with three BH motifs.The results of tissue expression and distribution show that PtBax and PtBcl-2 genes are expressed in all tissues.The highest expression levels were found in hepatopancreas.After infection with vibrio parahaemolyticus and WSSV,the m RNA and protein levels of PtBax and PtBcl-2 in hepatopancreas were significantly up-regulated.Fluorescence in situ hybridization was used to detect the main expression sites of PtBax and PtBcl-2 in cells.The results showed that PtBax and PtBcl-2 were mainly expressed in cytoplasm,and were mainly overexpressed in cell membrane after pathogen infection.PtBax and PtBcl-2 genes were silenced by double-stranded RNA injection showed a higher cumulative mortality rate after vibrio parahaemolyticus and WSSV infection compared with control group without RNA interference.But the WSSV copy number has obvious difference,After PtBax gene was silenced,WSSV copy number significantly decreased,while WSSV copy number significantly increased after PtBcl-2 gene knockout.These results indicated that PtBax and PtBcl-2 genes play an important role in pathogen infection.3.In order to understand the interaction between PtBax and PtBcl-2 in Portunus trituberculatus,we detected the interactions between PtBax and PtBcl-2 at m RNA and protein level.After the PtBax gene was silenced,the expression of PtBcl-2 was up-regulated and the expression of PtBax was up-regulated after PtBcl-2 gene was silenced,which indicated that PtBax and PtBcl-2 were antagonistic apoptotic regulatory genes.The interaction between PtBax and PtBcl-2 was further verified by yeast two-hybrid experiment,and the results showed that PtBax could interact with PtBcl-2.These results suggested that PtBax can be involved in the process of pathogen infection by binding to PtBcl-2.