| Cadmium(Cd)is a bioaccumulative heavy metal,which can cause irreversible damage to the aquatic immune system and endanger the health of aquatic organisms.The oriental river prawn,Macrobrachium nipponense is one of the most common prawn species grown in East China,which has high economic and nutritional value.Due to its small size and fast reproduction,it is a better choice for the study of cadmium immunotoxicology and biological monitoring molecular markers.In the present study,the hepatopancreas transcriptome of M nipponense exposed to the toxic metal Cd was compared to that of the unexposed prawn.The goal was to provide new insights into the biological effects of Cd on crustaceans,as well as sensitive innate immune biomarkers that could be used for aquatic Cd risk assessment.Transcriptome analysis,molecular biology was used in the research to find out the tissue distribution of immune-related genes especially Paxillin(PXN)in the hepatopancreas of M.nipponense,as well as the effect of cadmium stress on its expression pattern.And RNA interference was used to reveal the effect of cadmium stress on its immune response function.The specific results are listed as follows:1.Transcriptome analysis on the effects of cadmium stress on the mRNA level of immune-related genes in hepatopancreas of M.nipponenseTranscriptome analysis and molecular biology techniques were used to analyze the immune response mechanism in hepatopancreas of M.nipponense exposed to cadmium and qRT-PCR was used to verify its effect on the mRNA level of immune-related genes.The results showed that 51,740 unigenes was detected from the experimental group and the control group.A total of 580 different expressed genes were identified and annotated into 36 GO terms,including 413 genes whose expression levels were significantly upregulated,and 167 genes whose expression levels were significantly downregulated,11 of which were related to immunity and belonged to 8 pathways.2.The effect of cadmium stress on the characterization of MnPXNRT-PCR and RACE-PCR were used to clone the homologous gene of PXN from M.nipponense,named MnPXN,whose full-length cDNA is 3655 bp,including a coding sequence of 1491 bp,encoding a protein of 496 amino acids,with a molecular weight of 54.02 kDa,an isoelectric point of 5.07,4 low-complex regions and 3 conservative LIM domains.The homology alignment and phylogenetic tree showed that the PXN amino acid sequence encoded by M nipponense and P.vannamei were in a same cluster,indicating that it was evolutionarily conserved and closely related to insects,fish,birds,amphibians and mammals,have the farthest relationship with nematodes.The results of qRT-PCR showed that MnPXN was widely distributed in different tissue and it has a higher expression levels in gills,stomach and hepatopancreas,with the lowest expression in heart.After exposed to cadmium for 6 h,the expression level of MnPXN mRNA in hepatopancreas began to be higher than that of the control group significantly(P<0.05),and reached the highest level at 24 h(P<0.001).Western Blot analysis showed that MnPXN protein was expressed in hepatopancreas.Cadmium stress will increase its expression level and it reached the highest level at 24 h,which is consistent with its mRNA expression level,indicating that cadmium stress can affect the immune response of MnPXN protein in M.nipponense.The results showed that MnPXN protein was present on the cell membrane of hepatopancreas,and was located in the adhesion after analysis.3.Effect of cadmium stress on the immune function of MnPXNRNA interference results showed that double-stranded RNA can significantly reduce the expression level of MnPXN mRNA in hepatopancreas(P<0.05).Cadmium stress after MnPXN silencing can significantly increase the mortality of M.nipponense(P<0.001),indicating that MnPXN participates in the innate immune response of M.nipponense to cadmium stress,and cadmium stress has a significant effect on its immune function.The result showed a feasibility of MnPXN as an innate immune biomarker for cadmium risk assessment of M.nipponense. |
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