The Mechanism Of Carboxypeptidase B-like Gene And MicroRNA-125 In The Innate Immunity Of Scylla Paramamosain

This article first explored the role of the Carboxypeptidase B-like gene?Cpb?in the infection of the white spot syndrome virus?WSSV?or V.alginolyticus in S.paramamosain.Then explored the role of microRNA-125 of S.paramamosain in the process of WSSV infection 1.The role of Carboxypeptidase B-like gene in WSSV or V.alginolyticus infection in S.paramamosainPrevious studies have found that the Carboxypeptidase B-like may be involved in the innate immune response of S.paramamosain.In this paper,we obtained a 2316 bp gene sequence from S.paramomosain by RACE,with an open reading frame size of 1302 bp,encoding a protein containing 433 amino acid residues,5'UTR and 3'UTR?untranslated regions,UTR?is 161 bp and 853 bp respectively.The molecular weight of Carboxyeptidase B-like was 48.514 kDa and PI was 8.494.The results of amino acid sequence alignment and phylogenetic tree analysis showed that the similarity between the S.paramomosain's Carboxypeptidase B-like gene and Hyalella azteca?XP₀18024120.1?was the highest,and the homology was 50%The expression of Carboxypeptidase B-like gene was detected by real-time quantitative PCR?RT-qPCR?.The results showed that the expression of Carboxypeptidase B-like gene was the highest in hepatopancreas,the second in gills,the moderate in hemolymph and the lowest in heart.Inhibition of Carboxypeptidase B-like gene expression was achieved by RNAi.After the inhibition of Carboxypeptidase B-like gene,the expression of typical innate immune molecules and signal transduction factors in S.paramamosain was detected.The results showed that the expressions of seven important immune genes were significantly down-regulated.After inhibiting the expression of Carboxypeptidase B-like gene,the total hemocyte counts?THC?and the activity of superoxide dismutase?SOD?were significantly up-regulated,while the activity of phenoloxidase?PO?was significantly down-regulated.These results suggested that Carboxypeptidase B-like gene affected part of the immune signal pathway and enzyme activityThe expression of Carboxypeptidase B-like gene in S.paramamosain was significantly down-regulated after infection with V.alginolyticus.The results of mortality showed that the cumulative mortality of S.paramamosain infected with V.alginolyticus was significantly increased after the inhibition of Carboxypeptidase B-like gene.The results of hemocyte phagocytosis showed that after the inhibition of Carboxypeptidase B-like gene,the ability of hemocyte to phagocytize V.alginolyticus was significantly up-regulated,the apoptosis rate of hemocyte was also significantly up-regulated,while the total hemocyte counts decreased significantly.These results suggested that Carboxypeptidase B-like gene could participate in the immune response to V.alginolyticus by affecting phagocytosis,apoptosis,and the total hemocyte counts.The expression of Carboxypeptidase B-like gene in S.paramamosain was significantly down-regulated at 24 h after infection with WSSV,and increased gradually with the passage of time,and the expression at 72 h was significantly higher than that at 0 h.After Cpb-dsRNA treatment,the ability of WSSV phagocytosis of hemocyte of S.paramamosain was significantly increased,the apoptosis rate of hemocyte was also significantly up-regulated,while the total hemocyte counts decreased significantly.It is suggested that in the early stage of WSSV infection,inhibition of Carboxypeptidase B-like gene expression promoted hemocyte phagocytosis and apoptosis,thus inhibited WSSV replication.Survival analysis and WSSV copy number showed that the cumulative mortality and WSSV copy number of S.paramamosain were significantly increased after inhibition of Carboxypeptidase B-like gene.These results suggested that Carboxypeptidase B-like gene of S.paramamosain could participate in the immune response of S.paramamosain to WSSV by affecting immune responses such as phagocytosis and apoptosis.However,due to the complexity of WSSV infection mechanism,Carboxypeptidase B-like gene may be used by WSSV to promote WSSV replication in the later stage of infection2.The role of microRNA-125 in the process of WSSV infection in S.paramamosainmicroRNA-125?abbreviated as miR-125?was involved in the occurrence of many diseases?especially tumors?and host immune response.The purpose of this study was to explore the innate immune function regulated by miR-125 when S.paramamosain is infected with WSSV.AMO-miR-125 was used to interfere with the expression of miR-125,and the interference effect of AMO-miR-125 was detected by RT-qPCR and Northern Blot.After the successful interference of miR-125,the expression of 10 known innate immune molecules and signal transduction factors in S.paramamosain was detected,among which only the expression of Astakine gene was significantly up-regulated,while the expression of other genes such as Carboxypeptidase B-like gene was significantly down-regulated.In this paper,the expression of Astakine gene in various tissues of S.paramamosain was detected,and it was found that it was the highest in hemocyte.Using RNAi,after the expression of Astakine gene was inhibited by Ast-dsRNA,the total hemocyte counts of S.paramamosain was significantly down-regulated,indicating that Astakine gene could affect the total hemocyte counts.After AMO-miR-125 treatment,the apoptosis rate of hemocyte was significantly down-regulated.It is suggested that miR-125 may affect the total hemocyte counts and apoptosis by regulating immune gene expression.According to the results of immune gene expression detected at present,miR-125 may regulate Astakine gene,but it still needs further studyAfter WSSV infection,miR-125 expression was significantly up-regulated,indicating that miR-125 was involved in WSSV infection.The results of WSSV copy number and survival analysis showed that AMO-miR-125 significantly increased the WSSC copy number and the cumulative mortality of S.paramomosain infected with WSSV,indicated that AMO-miR-125 promoted the replication of WSSV and shortened the survival time of S.paramomosain.Phagocytosis showed that the phagocytosis rate of hemocyte decreased significantly,indicated that miR-125 could affect the phagocytosis rate of hemocyte when WSSV was infected.The results of apoptosis showed that the apoptosis rate decreased significantly,indicating that miR-125 could affect the apoptosis rate when WSSV was infected.The above results indicateed that miR-125 plays an important role in WSSV infectionIn conclusion,the full-length sequence of Carboxyeptidase B-like gene was obtained for the first time,and its function in innate immunity was explored.It was proved that Carboxyeptidase B-like gene has the ability to affect the host infected by pathogen?WSSV or V.alginolyticus?,which provided a theoretical basis for improving the innate immune function of S.paramomosain in the future.The function of miR-125 in the innate immunity of S.paramomosain was studied.It was found that miR-125 affected the infection process of WSSV,which provided a theoretical basis for further study of miR-125 function of S.paramomosain.