Cloning And Expression Characterizations Of Some Apoptotic Genes In Litopenaeus Vannamei Immune Reponse

Pacific white shrimp,Litopenaeus vannamei was introduced to China in 1988.And so far,diseases have been the major “bottleneck” restriction of shrimp aquaculture,among which the white spot syndrome virus(WSSV)has been resulted in huge economic losses.Therefore,it was necessary to in-depth understand the molecular of the immune response of shrimp in defending pathogenic stimulation.In present study,we obtained the full length cDNA sequences of some apoptotic genes,including Cytochrome C(CYC),Apoptosis-inducing factor(AIF),autophagy protein(ATG5),microtubule associated protein 1 light chain 3(LC3)by RT-PCR and RACE-PCR techniques,and analyzed their bioinformatics characteristics.We detected the tissue expression and the transcripts after WSSV infection in hepatopancreas and hemocytes by semi-quantitate PCR technique.The caspase3 activation,Reactive oxygen species(ROS)production after WSSV infection were also detected.Construction of the PGEX-4T-2-ATG5 recombinant expression vector and using Escherichia coli expression system to obtain the recombinant protein of ATG5 were done.The results are as follows:(1)Apoptotic genesThe full-length cDNA of CYC was 582 bp,including a 5'-terminal untranslated region(UTR)of 50 bp,an open reading frame(ORF)of 315 bp encoding for 104 amino acids(aa),and a 217 bp 3' UTR.The predicted CYC included a conservative cytochrome_C domain.Phylogenetic analysis revealed that CYC showed the closest relationship with its counterparts from shrimp Marsupenaeus japonicus.Tissue expression profiles showed that CYC existed in most tissues,with the highest expression of CYC was detected in muscle,and the weakest expression was in hemocytes.The full-length cDNA of AIF was 1664 bp,including a 154 bp 5'-UTR,a 1323 bp ORF encoding a polypeptide of 440 aa and a 3' UTR of 187 bp.The deduced protein of AIF contained a conserved Pyr_redox and AIF_C domain at the N-ternimal.Phylogenetic analysis revealed that AIF belonged to AIF1 subfamily and showed a close relationship with AIF1 from vertebrates.Tissue expression profiles showed that AIF existed in most tissues,with the most predominant expression of AIF in intestine,then followed muscle and the weakest expression in gill.Additionally,after white spot syndrome virus(WSSV)infection,the transcripts of AIF,CYC and caspase3 were induced significantly in hepatopancrease at most time points(P < 0.05).However,most of them decreased in hemocytes after infection.WSSV proliferation and shrimp mortality showed a time-dependent manner and the production of ROS in hemocytes were significantly increased at 6 and 12 h after infection.Our results showed that the apoptotic genes AIF,CYC and caspase3 could be induced in hepatopancreas after WSSV infection.However,the production of ROS in hemocytes might be important in shrimp defense against WSSV infection.(2)Autophagy related genesThe full-length cDNA of ATG5 was 1481 bp,including a 96 bp 5' UTR,an 810 bp ORF encoding for 269 aa,and a 575 bp 3' UTR.The predicted ATG5 included a conservative APG5 domain.Phylogenetic analysis revealed that ATG5 showed the closest relationship with its counterparts from crab Callinectes sapidus.Tissue expression profiles showed that both ATG5 existed in most tissues,with the highest expression of ATG5 was detected in muscle,and the weakest expression was in gill.The full-length cDNA of LC3 was 945 bp,including a 161 bp 5' UTR,a 447 bp ORF encoding for 148 aa,and a 343 bp 3' UTR.The deduced protein of LC3 contained a conserved APG12 domain.Phylogenetic analysis revealed that LC3 belonged to LC3 C subfamily and showed a close relationship with LC3 C from vertebrates.Tissue expression profiles showed that LC3 existed in most tissues,with the most predominant expression of LC3 in skin,then followed intestine and the weakest expression in hemocyte.Additionally,after WSSV infection,the transcripts of ATG5 were induced significantly in hepatopancreas at 24 h(P < 0.05).However,most of them decreased in hemocytes after infection.Our results showed that the Autophagy related gene ATG5 could be induced in hepatopancreas after WSSV infection.However,the ATG5 and LC3 could be inhibited in hemocyte after WSSV infection.The expression of PGEX-4T-2-ATG5 recombinant protein was mainly expressed in the inclusion body.