Study On The Mechanism Of MITF Pathway In The Immune Defence Of The Clam Meretrix Petechialis

The clam Meretrix petechialis is one of the most commercial species of marine bivalves.However,in recent years,clam deseases caused by bacteria and virus are becoming more and more common in the cultivation of Meretrix petechialis.Hence,it will provide molecular basis for cultivating resistant strains of M.petechialis by exploring the signaling pathway response during microbial infection and analyse the innate immune mechanism in the clam M.petechialis.The microphthalmia-associated transcription factor(MITF),a member of the MYC family of transcription factors,is a basic helix-loop-helix-leucine zipper(bHLH-LZ)protein which could regulate downstream genes depending on its bHLH-LZ domain.MITF has been reported to be the key regulator of signalling pathways that control cell proliferation,survival and immune defence.In this research,we identified and characterised the MITF gene family and analysed the functions of MITF in the immune defense and color formation in clams.In addition,we explored the upstream and downstream pathways of MpMITF,which revealed the function of MITF signalling pathway in the immune system.The main results were as follows.1.In this study,two isoforms of MITF were identified from M.petechialis,named MpMITF-1 and MpMITF-2.The full length cDNA of MpMITF-1 is 3564 bp with an ORF of 1365 bp.The tissue distribution analysis showed that MpMITF-1 mRNA was widely expressed in all tissues.The expression level of MpMITF-1 was significantly up-regulated 6 h and 12 h post-Vibrio parahaemolyticus injection(P<0.05).The mRNA expression of MpMITF-1 also increased post-V.parahaemolyticus immersion,which suggested that MpMITF-1 is involved in the immune response in clams.Genotyping in two clam groups with different resistant levels to V.parahaemolyticus(i.e.,11-R and 11-S),five SNPs and two haplotypes were associated with Vibrio resistance.Four SNPs(SNP2,5,6 and 13)and one haplotype(Hap1)were further confirmed to be associated with Vibrio resistance in M.petechialis by association analysis in different clam families.In addition,the full-length cDNA of MpMITF-2 is 2026 bp with an ORF of 1116 bp.The mRNA of MpMITF-2 was more highly expressed in the mantle compared to the other four tissues.Furthermore,there was a significant difference in the expression of MpMITF-2 among three clam strains with different shell colors.These results implied that MpMITF-2 was associated with shell color formation in the clam M.petechialis.When the mRNA expression of MpMITF-2 was knocked down,the new shell showed discontinuous pigment distribution,suggesting that the reduced expression of MpMITF-2 influenced pigment synthesis.A gene encoding phenoloxidase(MpPO)was identified as related to the shell color of the clam and was also a putative downstream gene of MITF.The expression of MpPO decreased significantly when the expression of MpMITF-2 was knocked down(P<0.05).The results indicate the close relationships among MpMITF-2,MpPO and shell color.2.When the expression of MpMITF-1 was knocked down by RNAi,the mRNA expression of phenoloxidase(PO),cathepsin K(CTSK)and BCL-2 was significantly decreased(P<0.05),indicating that PO,CTSK and BCL-2 were the target genes of MpMITF in clams.The ORF of MpCTSK is 1041 bp,encoding a protein of 346 amino acids.The ORF of MpBCL-2 is 705 bp with a predicted protein of 234 amino acids.And the ORF of MpPO is 2019 bp,encoding a protein of 672 amino acids.The tissue distribution analysis showed that MpCTSK,MpBCL-2 and MpPO were widely expressed in all tissues.Meanwhile,the mRNA expressions of MpCTSK,MpBCL-2 and MpPO were all significantly up-regulated post-V.parahaemolyticus challenge(P<0.05),indicating that they take part in the immune response against Vibrio challenge in clams.3.Based on the downstream genes of MpMITF-1 selected by RNAi,we cloned the promoter sequences of MpCTSK,MpBCL-2 and MpPO and detected an E-box sequence in all three of these genes,which is an essential element for the MITF protein to bind to.Our EMSA results showed that MpMITF-1 directly binds to the promoter regions of MpCTSK,MpPO and MpBCL-2.Our yeast one-hybrid assay result supports the conclusion that MpMITF-1 can directly regulate the transcription of MpCTSK,MpPO and MpBCL-2.The purified recombinant proteins,MpPO and MpCTSK,inhibited the growth of Vibrio.Additionally,the apoptosis rate of clam haemocytes rose significantly when the activity of MpBCL-2 was suppressed.These results revealed that MpPO,MpCTSK and MpBCL-2 are involved in the immune defence against V.parahaemolyticus.4.The putative upstream genes p38(MpP38)and p300(MpP300)were detected to directly interact with MpMITF-1 by yeast two-hybrid assay.The full-length cDNA of MpP38 is 1720 bp consisting of a 1095 bp ORF,encoding a polypeptide of 365 amino-acid residues.Meanwhile,the full-length cDNA of MpP300 is 2507 bp consisting of a 1767 bp ORF,encoding a polypeptide of 588 amino-acid residues.The expression levels of MpP38 and MpP300 were significantly up-regulated post-V.parahaemolyticus immersion(P<0.05),suggesting that they respond to Vibrio stimulation.To explore the regulation of MpP38 and MpP300,we found that the MpP38 phosphorylation level was increased in response to V.parahaemolyticus stimulation.And there was a correlation between the MpP38 phosphorylation level and PO expression level.Our results imply that MpP38 participates in the clam immune defence via activating MpMITF-1 and ultimately regulating the expression of an immune-related gene PO.In addition,the yeast two-hybrid assay was adopted to detect the combination between MpMITF-1 and the N-/C-terminal of MpP300.Our result found that the N-terminal of MpP300 could directly bind to MpMITF-1.This result revealed the regulation on MpMITF-1 when it regulates downtream genes.