Analysis Of DNA Methylation And Function On Different Regions Of Mantle Tissue From Pinctada Fucata Martensii

DNA methylation is an important epigenetic phenomenon,which was closely related to gene expression regulation,gene mapping and so on.Pinctada fucata martensii is one of China's important marine pearl cultivation of shellfish,its mineralization mechanism,immune defense system has been the concern of researchers.In this study,we conducted a basic study of DNA methylation in the mantle,obtained the gene with methylation and functionally studied.This sthdy provides a basis for further research on DNA methylation.The main findings are as follows:(1)In this research,methylation-sensitive amplification polymorphism(MSAP)technology was used to analyze the methylation levels of mantle tissues from Pinctada fucata martensii,including mantle edge(ME),mantle pallium(MP),mantle central(MC).The results showed that there were no significant difference in the bands of three tissue(P>0.05).The percentages of methylation levels were(17.07 ± 2.19)% in ME,(15.48 ± 2.34)% in MP,(19.61 ± 2.88)% in MC(P<0.05),the methylation levels from high to low was MC > ME > MP.After sequencing the obtained specific bands,we got eight gene sequences which were methylated.Among them,there were three sequences which were homologous with 40 S ribosomal protein SA,interference hedgehog(iHog)and Zinc finger protein castor by gene annotations.Real-time PCR showed that iHog was expressed in ME,MP and MC,with the highest expression level in ME,the lowest expression level in MC(P<0.05).This indicates that the expression of iHog in MC was inhibited by DNA methylation.(2)Using MeDIP-Seq to screen for genes with difference of DNA methylation between ME and MC.The sequencing results showed that both of ME and MC were got 122 M original Reads with 58702,55721 Peaks,respectively.The bioinformatics analysis showed that there were about 311 genes with differences in DNA methylation.GO and KEGG pathway enrichment indicated that the differential genes were enriched in the immunization and mineralization pathway of Pinctada fucata martensii.TRAF2,TRAF3 and MyD88,with differences in DNA methylation,were detected with difference expression in ME and MC by RT-PCR(P<0.05).(3)In this study,by using rapid amplification of cDNA ends(RACE)technology,we have obtained the full length of MyD88 cDNA from Pinctada fucata martensii(PmMyD88),analyzed its sequence characteristic and function.The results reveal that the cDNA full length of Pm-MyD88 is 1591 bp,including 257 bp of 5' UTR,257 bp of 3' UTR,and 1077 bp of open reading frame(ORF)encoding 358 amino acids.Furthermore,quantitative real-time PCR analysis demonstrated that Pm-MyD88 mRNA was widely expressed in all detected tissues with the highest expression level in gill.After LPS stimulation,the expression level of Pm-MyD88 began to increase and reached the highest level at 12 h,then gradually declined to the normal level(P<0.05).In Site Hybridization indicated Pm-MyD88 is mainly present in the immune cells of hemolymph,and the number of immune cells will increase after LPS stimulation.Dual luciferase analysis illustrated that the luciferase activity of pNF?B-Luc was significantly increase about 1.9372 times,after co-transfection pcDNA3.1-Pm-MyD88 recombinant plasmid.The analysis of the software RNAhybrid and microTar showed that miR-4047 was predicted as the candidate miRNAs of Pm-MyD88.The expression of Pm-MyD88,NF-kB and IL-17 were significantly downregulated about 90.16 %(P<0.05),97.09 %(P<0.05)and 99.99 %(P<0.01)by overexpression miR-4047 in vivo.Dual luciferase analysis illustrated that the luciferase activity of Pm-MyD88-3'UTR was significantly downregulated about 36.95 % by miR-4047.In conclusion,the findings point out that Pm-MyD88 plays an important role in the immune response in mollusk by regulating NF-kB signaling pathway.(4)Using rapid amplification of cDNA ends(RACE)technology,we have obtained the full length sequence of Pm-Dnmt1,Pm-Dnmt1 b,Pm-Dnmt3 a,Pm-Dnmt3 b gene,which were 4848 bp,2661 bp,2405 bp,2775 bp,respectively,and encoded 1550,797,705,803 amino acid.Real-time PCR indicated that Pm-Dnmt1,Pm-Dnmt1 b,PmDnmt3a,Pm-Dnmt3 b were easily detected in different tissues,especially that PmDnmt1 b,Pm-Dnmt3 a,Pm-Dnmt3 b gene were highly expressed in the gonads.And PmDnmt1 was highly expressed in the mantle central,which suggest that Pm-Dnmt1 may be involved in the regulation of shell formation by DNA methylation.DAC treatment experiments indicates that the expression of DNA Methyltransferase on ME,MC were significantly decreased after DAC treatment(P<0.05).Meanwhile,the expression of TRAF2,TRAF3 and MyD88,were also significantly up-regulated(P<0.05).