| Histones and histone acetylation modification could affect gene expression by controlling the state of chromosomes and involved in the regulation of various biological functions such as cell division,apoptosis and immune response.Histone acetylation and deacetylation are catalyzed by histone acetyltransferase(HAT)and histone deacetylase(HDAC),respectively.HDAC inhibitor(HDI)can inhibit the deacetylation process of histones and effectively reduce the inflammatory response when the body is injured.In this study,histone,HAT and HDAC of P.f.martensii were identified by various bioinformatics methods,their changes in hemocyte transcription after nucleation were analyzed,and the effect of HDI on the transplantation immunity of P.f.martensii was analyzed by RNA-seq and other techniques.The main findings are as follows:1.Identification of histones and their expression changes after transplantation: 22 Histone H1 were identified in the genome of P.f.martensii and present a species-specific expansion pattern.Multiple sequence alignment analysis revealed that the sequence of histone H1 showed lower conservation,and there were some H1 genes with more sequence variation in P.f.martensii and C.gigas,which may be histone H1 variants;A total of 11 histone H1 were expressed in the hemocytes transcription of P.f.martensii,and their expression levels increased significantly from 3 d to 6 d and gradually recovered to the normal level at 12 d after transplantation,indicating that histone H1 is involved in the regulation of transplantation immunity.There were two H2 A,one H2 B,one H3 and two H4 genes in the genome of P.f.martensii,showing contraction phenomenon;sequence analysis revealed one H2 A variant and one H4 variant;except for H2 A variant,other H2 A,the expression of H2 B,H3 and H4 changed after transplantation,indicating that they were involved in the regulation of transplantation immunity.2.Identification of HAT and HDAC and their expression changes after transplantation: the P.f.martensii genome contains eight genes of two HAT gene families(GNAT and MYST),including one KAT2,one KAT1,one KAT5,three KAT8,one KAT7,and one KAT6B;phylogenetic tree analysis shows that these genes cluster together in the same gene families from H.sapiens and C.gigas,respectively;sequence analysis results show that the HAT family includes 18 conserved amino acid sequences,of which sequences 1,2,and 3 are present in all MYST family genes analyzed;sequences 5,8,and 14 are contained in all GNAT family genes;domain analysis results show all genes of the MYST family contain MOZ?SAS domain,genes of the GNAT family contain Acetyltransf?1 domain or Hat1?N domain,indicating the conservation of HAT gene sequences.There were 18 HDAC genes in the genome of P.f.martensii,including 3 genes in class ?,3 in class ?,10 in class ?,and 2 in class ?.The motif analysis of class ?,?,and ? showed that this family contained 21 conserved domains,and their composition and distribution positions were diverse,indicating that there was structural diversity in this family member.Class ? family members contain 7 conserved amino acid sequences,and sequence 2,4,and 5 were contained in all of the members in class III famliy.Domain prediction of the four classes of HDAC protein sequences showed that classes ?,? and ? all contained one or more typical Hist-deacety1 domain,while HDAC ? contains 1-2 SIR2 domains.All HAT and HDAC can be detected in the hemocyte transcription of P.f.martensii,and the expression of most HAT don not change after transplantation,and some HDAC are up-regulated and some are down-regulated.Histone acetylation content analysis showed that at 6 h,12 h and 24 h after nuclear implantation,the proportion of histone H3 and H4 acetylation modification in hemocytes was significantly increased compared with the control group(untransplanted pearl oysters),indicating that histone acetylation modification is involved in the regulation of pearl shell transplantation immunity;3.The effect of HDI on the transplantation immunity of P.f.martensii: After HDI or PBS(control group)injection into the posterior nucleus,hemocytes were obtained at different time points(6 h,12 h,24 h and 48 h)after transplantation for transcriptome analysis.The results showed that there was a total of 3160 DEGs between the HDI control group at 6 h after transplantation,and these DEGs were significantly enriched in the signaling pathways of lysosome,adherens junction,regulation of actin cytoskeleton and local adhesion.Gene expression level analysis showed that lysosome-related enzyme genes were significantly highly expressed in the HDI group,while cell migration-related genes were lowly expressed in the HDI group,indicating that HDI treatment activated the lysosomal signaling pathway and inhibited cell migration;at 12 h after transplantation,there were 2671 DEGs between the HDI and control group,and the functional enrichment pathways were DNA replication,cell cycle,nucleotide excision repair,basal excision repair and mismatch repair,and most of the genes in the pathway were down-regulated,indicating that HDI treatment inhibited the proliferation ability after transplantation;there were a total of 1912 DEGs at 24 h,and these DEGs are mainly enriched in cytoplasmic DNA sensing pathway,NOD-like receptor signaling pathway,NF-k B signaling pathway,apoptosis,TNF signaling pathway,etc.BIRC2/3 was highly expressed in the HDI group,while negative regulation-related genes in the NF-k B signaling pathway(NFKBIA and TRAF3)were highly expressed in the HDI group,indicating that HDI treatment activated the intracellular immune response,but inhibited NF-k B and apoptosis pathways,and there was no significant difference in the number of apoptosis cess between the HDI and control group by apoptosis detection;there were a total of 3113 differential genes at 48 h,and the functional enrichment pathway was consistent with 24 h.Intracellular pattern receptor-related genes were still highly expressed in the HDI group,but the differences in apoptosis and NF-kb signaling pathway between the two groups were lower,indicating the function of HDI was reducing.4.The effect of HDI treatment on serum immunity after transplantation: Compared with PBS(control group),HDI treatment resulted in significantly high enzyme activities of leucine aminopeptidase(LAP)and catalase(CAT)at 6 h,12 h and 24 h after transplantation;the enzyme activities of phenol oxidase(PO)and superoxide dismutase(SOD)were significantly induced at 6 h,12 h,24 h and 48 h after transplantation;the enzyme activities of glutathione peroxidase(GSH-Px)were significantly increased at 24 h and 48 h after transplantation;the enzyme activities of lysozyme(LYS)were significantly increased at 24 h after transplantation;the enzyme activities of alkaline phosphatase(ALP)and malondialdehyde(MDA)were significantly increased at 12 h and 48 h after transplantation;while there was no significantly differen in the enzyme activities of acid phosphatase(ACP)between the two groups(P < 0.05). |
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