Study On The Function Of DNA Methylation In The Salinity Adaptation Of Portunus Tritubereulatus

The Portunus trituberculatus is one of the main crab species in our country and It is an important economic ways.It is one of the most important environmental factors in the breeding of Portunus trituberculatus,which directly affects the growth,development and survival of the Portunus trituberculatus.DNA methylation modification is the most common epigenetic phenomenon,It can directly affect gene activity,It become a hot topic of epigenetics research field in recent years,the aquatic animal growth,development,sexual reversal and play an important role of adaptation.However,whether DNA methylation plays a certain role in the salinity adaptation of crustaceans has not been reported.For analysis of DNA methylation in Portunus trituberculatus salinity to adapt to the function,this study mainly in two aspects:1,the first cloned with the function of maintaining genome methylation status of Portunus trituberculatus methyltransferase gene(PtDNMT1),through the q RT-PCR analyses the expression level in the three different Portunus trituberculatus the organization,and in the expression change rules were studied under salinity stress,preliminary discussion on DNA methylation in three wart swimming crab in low salt to play a function.2,through the use of advanced technology of Methyl-RAD,from the level of whole genome reveals Portunus trituberculatus gills before and after salt stress change rule of DNA methylation in the organization,and from the perspectives of epigenetics methylation modification in the salinity in the process of adaptation function,the main results were as follows:1.Expression analysis of PtDNMT1 gene cloning and salinity stress.Methyltransferase gene(DNMT1)is one of the important genes,maintain genomic methylation status of the gene has a specific structure domain and conservative at the end of the N C at the end of the catalytic domain structure,DNA methylation catalyzed by DNA methyltransferase family,what for the study of DNMT1 gene functions Portunus trituberculatus salinity to adapt to,this experiment by using SMART-RACE cloned from Portunus trituberculatus methyltransferase(PtDNMT1)gene.The c DNA sequence of PtDNMT1 gene is 5919 bp,including the open reading frame of 4833 bp,encoding 1610 amino acids,and the predicted molecular weight is 148.15 kd,and the theoretical equivalence point is 4.68.Structure prediction found that there are two special PtDNMT1 domain structure,respectively,the structure of zf-CXXC zinc finger domains and methyltransferase familial Dcm structure domain,the evolutionary tree analysis showed that PtDNMT1 gene and insects DNMT into one.Tissue expression analysis revealed that PtDNMT1 gene was expressed in hepatic pancreas,gill,ovary,muscle,stomach,heart and blood,among which the expression was highest in hepatic pancreas,followed by ovary and gill.After further research the low salt stress PtDNMT1 gene expression in gill,liver,pancreas and muscle tissue change rule: 6 h gill tissue found stress PtDNMT1 gene expression in the peak(5.3 times),and continuing to 12 h(4 times),then decline gradually,at 72 h is still significantly higher than the control group(2.3 times);The expression of PtDNMT1 gene in hepatic pancreas is similar to that of gills,whereas the peak time is slightly later than the gill(24h),and the increase is relatively higher than the gills(8 times).After low salt stress,the expression of PtDNMT1 gene in muscle was initially down-regulated,and then(24h)was up-regulated to peak(2.2 times),and the expression was up-regulated to 72 h.This part of the research results show that the PtDNMT1 genes in gill,liver,pancreas tissue after salinity stress in a large number of expression,and gill,liver,pancreas and muscles are Portunus trituberculatus body to adapt to the major organs of salinity.It is speculated that PtDNMT1 gene is involved in the regulation mechanism of the salinity adjustment of the three warthos,while the PtDNMT1 gene is an important gene to maintain the DNA methylation state.The results suggest that DNA methylation may play a role in the salinity adaptation of the Portunus trituberculatus.This paper provides data support for the mechanism of salinity adjustment mechanism of the Portunus trituberculatus and other crustaceans.2.Analysis and validation of DNA methylation in the gills of the Portunus trituberculatus under salinity stress.DNA methylation is a kind of regulation of epigenetics,That is the most important way,In order to study the DNA methylation in Portunus trituberculatus.The salinity to adapt to the function,the experiment using advanced Methyl RAD technology,the salinity stress 0 h,12 h and 72 h after Portunus trituberculatus gills tissue material for DNA methylation detection,found CCGG pattern of methylation site 93418,CCWGG pattern of methylation site 44218.Five methylation sites were selected and verified by sulfite method,and the accuracy rate was 100%.By comparing salinity stress before and after the sequencing of the gill tissue DNA methylation site depth,were screened CCGG mode methylation site altogether 7575 differentially expressed,CCWGG mode methylation site 923 differentially expressed.According to the laws of the expression will be differentially expressed methylation site expression pattern types classified into four categories,the main type of the expression pattern of salinity stress after the first hike after cut,accounting for 67% of the total variance loci.The results showed that the m RNA expression of the sites with high methylation levels was significantly reduced by the detection of 7 different methylation sites by q PCR.Further analysis revealed that 1,086 differential DNA methylation sites had clear gene annotation information.The results of GO enrichment analysis showed that the different methylation sites were mainly concentrated in the process of growth reproduction,metabolic process,cell process,biological regulation and immunity.The results of this study showed that: 1.Using Methyl RAD technique,it can accurately and efficiently analyze the methylation change rule in the gill tissues of the three warty swimming crab.2.DNA methylation plays an important role in the process of salinity adaptation of the gill tissue of the Portunus trituberculatus.