Cloning Of Apoptosis-related Genes In Macrobrachium Nipponense: Its Role In Response To Hypoxia

Macrobrachium nipponense is one of the important varieties of freshwater shrimp in China.Especially in recent years,the production of freshwater shrimp has been increasing,economic benefits have been improving,and it plays an important role in the aspect of nutritional value.However,hypoxia is a more serious environmental stress factor in the process of aquaculture,especially for Macrobrachium nipponense with benthic living habits,hypoxia may lead to adverse effects on the growth and reproduction of shrimp in all aspects,and even death.Tissue hypoxia is the main cause of animal morbidity and mortality.Bcl-2 family proteins(Bax,Bnip3,Bok)play an important role in regulating mitochondrial function and the release of cytopigment C,and play their functions in mitochondria-mediated apoptosis pathway.When cells are stressed by external environmental factors(such as hypoxia),Genes produce the instructions for cell apoptosis by promoting the apoptotic pathway,which Caspase9 carries out and ultimately leads to cell apoptosis.The research content includes the following three parts:1.Cloning and expression analysis of Bax genes in Macrobrachium nipponense under hypoxia stress.In order to explore the role of cell apoptosis related gene Bax(B-cell 2 associated X protein)in hypoxia stress of Macrobranchium nipponense.In this study,the full-length c DNA sequence of apoptosis gene Bax of Macrobrachium nipponense was obtained by c DNA terminal rapid amplification(RACE)PCR.Semi-quantitative RT-PCR and realtime fluorescence quantitative PCR(q PCR)were used to analyze the expression of Bax gene in different tissues and hypoxic stages of Macrobrachium nipponense.Western blot and immunohistochemistry were used to analyze the expression and localization of Bax protein in Macrobrachium nipponense under hypoxia.The results showed that the full length of the c DNA of Bax gene of Macrobrachium nipponense was 2,287 bp(NCBI entry number: MZ823353),including the 5 ’non-coding region(UTR)of 42 bp,the 3’ UTR of 814 bp,the open reading frame(ORF)of 1,431 bp,encoding 476 amino acids.The sequences were analyzed by software and bioinformation website,and amino acid similarity comparison showed that the apoptotic gene Bax of Macrobrachium nipponense was rich in highly conserved BH1,BH2 and BH3 domains.Phylogenetic tree analysis showed that Bax gene of Macrobrachium nipponense was closely related to Bax of Penaeus nipponense.RT-PCR results showed that Bax gene expression of Macrobrachium nipponense was the highest in hepatopancreas and the lowest in brain.q PCR results showed that Bax gene expression levels in gill and hepatopancreas tissues of Macrobrachium nipponense were significantly higher than those of the control group at 1-96 h of hypoxia stress.Western Blot analysis also confirmed that the abundance of Bax protein expression was basically similar to the gene transcription level.The recombinant Bax protein was obtained in vitro by constructing prokaryotic expression vector,and the purified recombinant protein was immunized with rabbits to obtain antiserum.Immunohistochemical results showed that with the increase of hypoxia time,the positive signal of Bax protein in gill and hepatopancreas was gradually deepened and mainly localized in gill epithelial cells and hepatocytes.The above studies indicated that Bax gene of Macrobrachium nipponense plays a pro-apoptotic role in the molecular response to hypoxia stress,which provides a theoretical reference for the molecular mechanism of hypoxia intolerance of Macrobrachium nipponense.2.Cloning and functional analysis of Bnip3 gene in Macrobrachium nipponense under hypoxia stressIn order to explore the role of apoptosis-related gene Bnip3 in the process of hypoxia stress in Macrobranchium nipponense,the full-length c DNA sequence of apoptosisrelated gene Bnip3 was obtained by c DNA terminal rapid amplification(RACE)PCR.Semi-quantitative RT-PCR and real-time quantitative fluorescence PCR(q PCR)were used to analyze the expression of Bnip3 in different tissues and hypoxia stages of Macrobrachium nipponens.Western blot and immunohistochemistry were used to analyze the expression and localization of Bnip3 in Macrobrachium nipponens under hypoxia.The results showed that the full-length c DNA of Bnip3 gene of Macrobrachium nipponens was 1,483 bp(NCBI entry number: MW480557),including the 5 UTR of 383 bp,3’ UTR of 500 bp,ORF of 600 bp,encoding 199 amino acids.Through the analysis of its sequence through software and bioinformation website,amino acid similarity comparison showed that the apoptotic gene Bnip3 of Macrobrachium nipponens had only BH3 domain.According to phylogenetic tree analysis,the Bnip3 gene of Macrobrachium nipponens has the closest genetic relationship with crustaceans such as Penaeus nipponense.RT-PCR was used to analyze the expression of Bnip3 gene in different tissues of Macrobrachium nipponens.The results showed that the expression of Bnip3 was relatively high in the testis and ovarian tissues,but relatively low in the brain and intestinal tissues.Real-time quantitative PCR(q PCR)was used to analyze the expression of Bnip3 gene in different hypoxia stages of Macrobrachium nipponens.The m RNA expression level of Bnip3 gene in the gill tissues of Macrobrachium nipponens was higher than that of the control group at 1-24 h hypoxia,and reached the maximum in the experimental group at 24 h hypoxia.The m RNA expression level of Bnip3 gene in hepatopancreas of Macrobrachium nipponens was also higher than that of control group(P <0.05)at 1-96 h of hypoxia,and showed a gradually increasing trend with the increase of time,with the maximum up-regulation rate at 96 h of hypoxia,Finally,Western blot and immunohistochemistry were used to analyze the expression and localization of Bnip3 protein in Macrobrachium nipponens under hypoxia.The recombinant protein Bnip3 was obtained by constructing prokaryotic expression vector,and the rabbit was immunized with purified recombinant protein to obtain antiserum.Double luciferase reporter gene assay confirmed that Hif-1a/Bnip3 signaling pathway played a role in the testis of Macrobrachium nipponens.3.Cloning and expression analysis of Bok and Caspase9 genes in Macrobrachium nipponense under hypoxia stress.Bok and Caspase9 cDNA sequences of Macrobrachium nipponense were cloned.The full-length c DNA sequence of Bok gene was 2,391 bp,including four domains BH1,BH2,BH3 and BH4.The full length of Caspase9 gene c DNA is 2,274 bp sequence,open reading frame 1,515 bp,encoding 504 amino acids.Multiple alignment of amino acid sequences of Bok and Caspase9 genes revealed that the BH domain was conserved between crustaceans and fish species.The evolutionary tree showed that Bok and Caspase9 genes were clustered together with other shrimp and crab genes.Bok gene was the most closely related to Exopalaemon carinicauda and Macrobrachium rosenbergii,and Caspase9 gene was the most closely related to Chionoecetes opilio.Semiquantitative detection showed that Bok and Caspase9 genes were expressed in different tissues of Macrobrachium nipponense,Bok gene was relatively highest expressed in hepatopancreas,but relatively low expressed in heart,intestine and muscle,Caspase9 gene was relatively highest expressed in hepatopancreas,but relatively low expressed in brain and heart tissues.Quantitative PCR showed that Bok gene m RNA expression level in gill tissues of Macrobrachium nipponense was significantly higher than that of control group at 1-48 h of hypoxia(P<0.01),and reached its maximum level at 24 h of hypoxia.Bok m RNA expression in hepatopancreas of Macrobrachium nipponense was significantly higher than that of control group at 3 h,12 h,24 h and 48 h of hypoxia,and the up-regulation rate was the largest at 3 h of hypoxia.At 3 h,12 h,24 h,48 h and 96 h of hypoxia,the m RNA expression of Caspase9 gene in the gill tissue of Macrobrachium nipponense was significantly higher than control group(P<0.01),and reached the maximum at 48 h of hypoxia.At 3h,12 h,24 h and 96 h hypoxia,the m RNA expression of Caspase9 gene in hepatopancreas of Macrobrachium nipponense was significantly higher than control group,and the up-regulation amplitude was the largest at 24 h hypoxia.The apoptosis rate of hemocytes in Macrobranchium nipponense was significantly higher than that in the control Flow cytometry at 96 hours of hypoxia stress.TUNEL assay showed that there was no significant apoptosis in the gill tissue of Macrobranchium nipponense after hypoxia stress,and the apoptosis rate in hepatopancreas was more significant,but there was no significant difference in the apoptosis rate under hypoxia.In this study,it was confirmed that Bax,Bok and Caspase9 played a role in promoting apoptosis in apoptotic mitochondrial pathway of Macrobranchium nipponense,so as to explore the molecular pathway of apoptosis induced by hypoxia stress,and provide theoretical reference for high-density and intensive culture of Macrobranchium nipponense.