Immune Responses Of ShToll1 And ShToll3 From The Freshwater Crab Sinopotamon Henanense To Cadmium Exposure And Aeromonas Hydrophila Infection

Toll signal is the key to express immune genes,which plays an important role in invertebrate resistance to bacterial,fungal and viral infection.Although some Tolls have been identified in crustaceans,they have not been found in the freshwater crab S.henanense.In addition,the effect of Cadmium(Cd)on Toll has not been studied in freshwater crabs that live in sediment and are prone to accumulate heavy metals.Our transcriptome analysis of hepatopancreas from S.henanense showed that Cd exposure reduced the expression of Toll3.Therefore,this project studied the immune responses of Sh Toll1 and Sh Toll3 from the freshwater crab S.henanense to Cd exposure and A.hydrophila infection.The main contants of this study are as follows:1.Sequence analysis and distribution pattern of Shtoll1 and Shtoll3 from the freshwater crab S.henanense: the full-length sequences of Shtoll1 and Shtoll3 were cloned by RACE method;then the protein domains of Sh Toll1 and Sh Toll3 were predicted to analysis characteristics of the functional domains of Sh Toll1 and Sh Toll3;also phylogenetic trees among similar species were constructed to predict the evolutionary status of Sh Toll1 and Sh Toll3,and further to predict the functions of Sh Toll1 and Sh Toll3.At the same time,total RNA of the seven tissues haemolymph,gills,hepatopancreas,midgut,muscle,testis and ovary from S.henanense were extracted and reverse transcribed into c DNA.Specific primers were designed to detect the expression of Shtoll1 and Shtoll3 in the tissues to analysis the target tissues of Shtoll1 and Shtoll3 involved in immune response by q PCR.2.The establishment of A.hydrophila infection model and analysis of the response of Shtoll1 and Shtoll3 from S.henanense to A.hydrophila infection: the virulence genes of A.hydrophila were tested,and the virulent strain was propagated and preserved;the CFU of A.hydrophila was masured and the relationship between CFU and OD600 of A.hydrophila was build;the LD50 of S.henanense infected by A.hydrophila was calculated through counting the number of dead individuals of S.henanense infected by A.hydrophila,in order to establish the infection model of S.henanense infected by A.hydrophila.By q PCR,the expression patterns of Shtoll1 and Shtoll3 in response to A.hydrophila infection were analyzed.3.Analysis of the response of Sh Toll1 and Sh Toll3 from S.henanense to Cd exposure and A.hydrophila infection: the total RNA in four tissues of gills,midgut,heamocytes and hepatopancreas from S.henanense were extracted and reverse transcribed into c DNA after Cd exposure for 7 d and A.hydrophila infection for 24 h;the expressions of Shtoll1 and Shtoll3 from S.henanense to Cd exposure and A.hydrophila infection were detected by q PCR;meanwhile,the expression patterns of Sh Toll1 in the heamocytes and Sh Toll3 in the midgut and gills to Cd exposure and A.hydrophila infection were analysized by immunohistochemical analysis.4.Verifing the responses Sh Toll1 and Sh Toll3 from S.henanense to Cd exposure and A.hydrophila infection by functional deletion methord: synthesis the si RNA for RNAi of Shtoll1 and Shtoll3,respectively,and verify the interference efficiency.Western blotting method was used to compare the response of Sh Toll1 and Sh Toll3 to Cd exposure and A.hydrophila infection before and after RNAi of Shtoll1 and Shtoll3;at the same time,q PCR method was used to analyze the response of downstream AMPs of the Toll signaling pathway from S.henanense to Cd exposure and A.hydrophila infection before and after RNAi of Shtoll1 and Shtoll3.The main conclusions of this study are as follows:Firstly,sequence analysis and distribution pattern of Shtoll1 and Shtoll3 from the freshwater crab S.henanense: the full length of Shtoll1 was 4746 bp,including 255 bp of 5'-untranslated region(UTR),1713 bp of 3'-UTR and 3033 bp of ORF,in which ORF could encode a putitave proteinc with 1011 amino acids;the full length of Shtoll3 was 4488 bp,including 414 bp of 5'-UTR,781 bp of 3'-UTR and 3690 bp of ORF,in which ORF could encode a putitave proteinc with 1230 amino acids.Phylogenetic analysis results showed that Sh Toll1 was clustered into the group with most of the crustaceans Toll1 and Toll2,and Sh Toll3 was clustered into the group with all of the crustaceans Toll3.Sh Toll1 possessed the highest identity to Cm Toll of 68.59%,and the lowest to Dm Toll3 of 22.71%;Sh Toll3 possessed the highest identity to Pt Toll3 of 93.22%,and the lowest identity to Dm Toll2 of 39.31%.The results of tissue expression pattern showed that Shtoll1 iiiand Shtoll3 were distributed in different tissues of S.henanense.Shtoll1 was the highest in the heamocytes,and Shtoll3 in the gills.The results provide sequence basis for further study of Sh Toll1 and Sh Toll3 functions.Secondly,the establishment of A.hydrophila infection model and analysis of the response of Shtoll1 and Shtoll3 from S.henanense to A.hydrophila infection: seven virulence genes of aer A,hly A,alt,ast,ahp B and lip were examed in A.hydrophila.The CFU of A.hydrophila was 3.82×106 cfu/m L,and the relationship between CFU and OD600 was y = 0.191 x + 0.045(R2=0.973).The LD50 of S.henanense infected by A.hydrophila was 5.2 × 105 cfu/m L.A.hydrophila infection model was established successfully.The expression of Shtoll1 in heamocytes was increased post 6 h infection of A.hydrophila and the expression of Shtoll1 in midgut was decreased post 12 h.The expression of Shtoll3 in midgut was decreased post 12 h infection of A.hydrophila.Thirdly,analysis of the response of Sh Toll1 and Sh Toll3 from S.henanense to Cd exposure and A.hydrophila infection: the q PCR and immunohistochemical analysis results showed that in heamocytes,the expression of Shtoll1 increased under 29 mg/L Cd exposure and A.hydrophila infection indicating Shtoll1 mainly respond to Cd exposure and A.hydrophila infection in the heamocytes.In the gills and midgut,the expression of Shtoll3 increased under 14.5 mg/L Cd exposure and A.hydrophila infection indicating Shtoll3 mainly respond to Cd exposure and A.hydrophila infection in the gills and midgut epithelial cells.The results of immunohistochemistric analysis results showed that the Sh Toll1 from the freshwater crab S.henanense stained in the heamocytes Cd exposure and A.hydrophila infection.And the Sh Toll3 from the freshwater crab S.henanense stained in the gills Cd exposure and A.hydrophila infection.No stain changes of Sh Toll3 were found in the midgut.Fourthly,verifing the responses Sh Toll1 and Sh Toll3 from S.henanense to Cd exposure and A.hydrophila infection by functional deletion methord: si RNAs for RNAi of Shtoll1 and Shtoll3 from S.henanense were successfully synthesized,and the interference efficiency of Shtoll1 in heamocytes was 90%,and that of Shtoll1 in gills and midgut was 68% and 78%,respectively.After RNAi Shtoll1 and Shtoll3,respectively,the protein expressions of Sh Toll1 and Sh Toll3 were inhibited in response to Cd exposure and A.hydrophila infection.After RNAi Shtoll1,alf5,alf6 and c-lys in the downstream of the Toll signaling pathway were detected to participate in the immune response to Cd exposure and A.hydrophila infection through Shtoll1;After RNAi Shtoll3,crustin and c-lys in the downstream of the Toll signaling pathway were detected to participate in the immune response to Cd exposure and A.hydrophila infection through Shtoll3.