Cloning And Function Study Of DNA Methyltransferase Gene Family In White Prawn,Exopalaemon Carinicauda

DNA methylation is one of the important modification methods of epigenetics.DNA methyltransferase(DNMT)is an important factor that catalyzes DNA methylation modification in organisms.In order to explore the DNA methylation regulation network of Exopalaemon carinicauda,the full length c DNA sequences of DNMT 1 gene and DNMT 3b gene and the promoter sequences of DNMT 2 gene in the DNA methyltransferase gene family of E.carinicauda were cloned,and the bioinformatics functions of each gene were studied in order to provide a theoretical basis for the molecular regulation network of DNA methylation modification of E.carinicauda.The following are the specific studies:1.Cloning of DNMT 1 gene and analysis of its mitochondrial methylation regulation function in Exopalaemon carinicaudaDNA methylation modification can make organisms produce heritable epigenetic traits without changing the DNA sequence.Research shows that the gene mainly responsible for maintaining the existing DNA methylation modification in organisms is DNA methyltransferase1(DNMT 1).In this study,the full length c DNA sequence of DNMT 1 gene of E.carinicauda was cloned and obtained,including 81 bp in 5’non coding region,934 bp in 3’ non coding region and 4 704 bp in open reading frame.A total of 1 567 amino acids(aa)were translated.The predicted protein molecular weight was 176.47 k Da and the theoretical isoelectric point was 5.83.DNMT 1 contains four special domains: DNMT 1-RFD domain,zf-CXXC zinc finger domain,BAH domain and Dcm domain.The amino acid sequence homology comparison results showed that the amino acid sequence of DNMT 1 gene of E.carinicauda had the highest homology with that of Procambarus virginalis DNMT 1,which was 67.72%.The q RT-PCR detection of DNMT1 gene of E.carinicauda in different tissues found that the expression of DNMT 1 was the highest in gonad tissue and the lowest in heart tissue.The results showed that DNMT 1 gene did not significantly regulate the DNA methylation of COX 3 gene 3’ end sequence,ND 3 gene 5’end sequence and ND 5 gene 3’end sequence in mitochondria.2.Cloning and transcriptional regulation of DNMT 2 gene promoter in Exopalaemon carinicaudaThe 1 315 bp promoter sequence upstream of the 5’ end of DNA methyltransferase 2(DNMT 2)gene was cloned by genome walking technique.The transcription initiation site was236 bp base "A" located upstream of ATG.TATA box transcription binding site was found at 26 bp upstream of the sequence.Analysis of the promoter sequence found that the DNMT 2 gene promoter sequence contained multiple transcription factor binding sites,including STAT 3 and NF-κB,E2 F,GATA-1 and SOX constructed a series of deletion expression vectors and detected the promoter activity of DNMT 2 gene by using double luciferase reporter gene detection technology.The results showed that the sequence of-196 bp ～ + 81 bp could maintain the basic transcriptional activity of DNMT 2 gene promoter,which was the core promoter region of the gene,and there were transcriptional regulatory elements promoting the expression of the gene in the region of-640 bp ～ + 452 bp,There are transcriptional regulatory elements that inhibit gene expression in the region of-940 bp-640 bp.3.Cloning of DNMT 3b gene and analysis of its mitochondrial methylation regulation function in Exopalaemon carinicaudaDNA methyltransferase 3(DNMT 3),also known as de novo methyltransferase,includes many other isomers such as DNMT 3a,DNMT 3b and DNMT 3l.Its main function in organism is related to the newly generated methylation sites in DNA sequence and participates in the de novo methylation process.In this study,the full-length c DNA sequence of DNMT 3b gene of E.carinicauda was 3 865 bp,including 124 bp of 5’ non coding region,147 bp of 3’ non coding region and 3 593 bp of open reading frame.A total of 197 amino acids(aa)were translated.The predicted protein molecular weight was 134.68 k Da and the theoretical isoelectric point was 5.65.DNMT 3b contains two special domains,PWWP domain and addz domain.The expression of DNMT 3b gene in different tissues of white shrimp was detected by q RT-PCR.The results showed that the expression of DNMT 3b gene was the highest in gonadal tissue of E.carinicauda,but not in ventral cord nerve.Through RNA interference under starvation stress and bisulfite sequencing technology,the samples on day 5 and day 10 were detected.It was found that the methylation degree of COX 3 gene 3’ end sequence and ND 3 gene 5’ end sequence in mitochondria was significantly higher than that in the control group,and the methylation degree of ND 5 gene 3’ end sequence was significantly lower.The results showed that,DNMT 3b gene may be involved in the DNA methylation modification of mitochondrial genome of E.carinicauda.