The Role Of Na~+/H~+ Exchanger And Carbonic Anhydrase In Fenneropenaeus Chinensis In Response To PH Stress

In recent years,intensive breeding mode has been promoted continuously,aquaculture industry has been developed rapidly,at the same time,it also faces many environmental problems caused by high breeding density,frequent feed feeding,drug abuse and so on.Aquaculture animals also face a variety of environmental factors of stress.As an important influence factor in water environment,p H mutation can cause stress on the acid-base balance and osmotic adjustment of shrimp,reduce the immune ability of shrimp,and then inhibit its growth.To study the role of Na~+ / H~+-exchanger(NHE)and carbonic anhydrase(CA)in the response of Fenneropenaeus chinensis to ph stress.Firstly,that p H value of 72 h acid-base semi-lethal of Fenneropenaeus chinensis was determine by hydrostatic toxicity test.The Na~+ / H~+-exchanger isoform 3,cytoplasmic carbonic anhydrase cytoplasmic carbonic anhydrase and glycosylphosphatidylinositol carbonic anhydrase were cloned by race technique.The expression characteristics and functions of the protein under p H stress were analyzed by fluorescence quantitative PCR and RNA interference technique.The distribution difference of three genes in gill tissue of Penaeus vannamei before and after stress was observed by in situ hybridization.In order to understand the mechanism of NHE3 and CA in the adaptation of Fenneropenaeus chinensis to p H stress,and provide a theoretical basis for the study of the adaptation mechanism of crustacean acid-base water environment.1.Statistical analysis was conducted on the mortality of Fenneropenaeus chinensis under different p H gradients.It was found that when the p H was 4.6 and 9.7,the cumulative mortality reached 100% at 72 h,and there was no dead individual at p H 6.6 and 8.1.The quasi-lethal p H and quasi-lethal p H of Fenneropenaeus chinensis were calculated to be 5.2 and 9.1,respectively.The shrimp fleas under different p H stress were subjected to HE staining.The results showed that the p H value was 9.1.Under stress,the damage of shrimp larvae was severer than that of the control group and the acid half-lethal p H(p H 5.2)stress group,and there was a significant cocoon swelling and blood cell swelling.2.The Na~+/H~+-exchanger isoform 3 gene of Fenneropenaeus chinensis was obtained and named as Fc NHE3.The full-length c DNA sequence was 3508 bp with an open reading frame of 2805 bp and encoded 934 amino acids with signal peptide and 12 genes.Transmembrane domains;homologous protein analysis revealed that Fc NHE3 had the highest homology with Carcinus maenas,reaching 74%;phylogenetic analysis showed that Fc NHE3 was related to Portunus trituberculatus and Carcinus maenas.The relationship is recent.Quantitative real-time PCR analysis showed that the expression of Fc NHE3 gene was significantly higher in iliac tissues than in other tissues(P<0.05);the Fc NHE3 gene was significantly up-regulated during the entire stress period under acid lethal p H(p H 5.2)stress(P<0.05).Under the condition of quasi-lethal p H(p H 9.1),Fc NHE3 gene was down-regulated in the first 48 h(P<0.05).The expression of Fc NHE3 was lowest at 12 h,and only up-regulated at 72 h.The results of in situ hybridization showed that the signal of acid half lethal p H(p H 5.2)stress group was stronger than that of alkaline half lethal p H stress(p H 9.1)group and the control group,indicating that the expression of Fc NHE3 gene was higher under acidic half lethal p H stress.Same as the q PCR experiment results after 24 h of p H stress.The results of RNA interference showed that after injection of si RNA,the expression of Fc NHE3 gene was inhibited,and the survival rate of shrimp under p H 5.2 stress was significantly lower than that of the control group.When Fc NHE3 gene was disturbed for 3 h,the interference efficiency was highest in si-3 group.At this time point,Fc CAc,Fc CAg,V-H~+-ATPase(VHA)and Na~+/HCO3-cotransporter(NBC)were down-regulated compared to the control group.HCO3-Cotransporter(AE2),Na~+/K+/Cl-cotransporter(NKCC)gene expression was relatively up-regulated.Studies have shown that Fc NHE3 may play an important regulatory role in response to p H stress in Fenneropenaeus chinensis,especially in acidic water environment.3.Two kinds of carbonic anhydrase genes from Fenneropenaeus chinensis were obtained by cloning,namely cytoplasmic Carbonic Anydrase,named Fc CAc and glycosyl-phosphatidylinositol-linked Carbonic Anhydrase,named Fc CAg.Phylogenetic analysis showed that both genes were closely related to Litopenaeus vannamei.The results of tissue expression and distribution showed that the expression levels of both genes were highest in iliac crest tissue.Under acidic half-lethal p H(p H 5.2)stress,Fc CAc gene expression showed a wave-like change,increased first and then decreased,and basically recovered to control at 72 h.At the level of the group(P<0.05),the expression was down-regulated for 0-24 h under the alkaline lethal p H(p H 9.1)stress,and the expression was the lowest at 24 h,then the expression level increased,and the expression was up-regulated at 72 h(P< 0.05);For the Fc CAg gene,the gene was significantly upregulated during the whole process at the acid lethal p H(p H 5.2)stress,with the highest expression at 12 h(P<0.05);at the half lethal p H Under p H 9.1 stress,the first 24 h of gene expression was significantly down-regulated,while after 24 h,the expression level increased and reached the level of up-regulation at 72 h.The results of in situ hybridization on the samples treated with acid and alkali stress for 24 hours showed that the Fc CAc was weaker in the acid semi-lethal p H(p H 5.2)and alkaline semi-lethal p H stress(p H 9.1)groups than the control group.The results showed that the expression of Fc CAc gene was decreased in 24 h under p H stress;the Fc CAg was stronger in the acid semi-lethal p H stress group than in the quasi-lethal p H stress group and the control group,while the basal half lethal p H stress group had the highest hybridization signal.Weak,indicating that Fc CAg gene expression is high under low p H stress;it is consistent with the 24 h gene expression level in stress experiments.After RNA interference,both Fc CAc and Fc CAg were inhibited.The survival rate of shrimp under p H 5.2 stress was significantly lower than that of the control group.When Fc CAc was interfered for 3 h,the interference efficiency was highest in the si-3 group.At this time point,the expression of ion transport related genes Fc NHE3,Fc CAg,Fc VHA,and Fc AE2 genes was relatively downregulated,while the expression of Fc NBC and Fc NKCC genes was relatively upregulated;when the Fc CAg gene was disturbed for 3 h After the si-1 group had the highest interference efficiency,the selected ion transport related genes were down-regulated at this time point.This suggests that the two genes may play an important role in response to p H stress in Fenneropenaeus chinensis by interacting with other ion transport related genes.