The Role Of Danio Rerio Dnmt1 Gene Under Low Temperature Pressure

As ectotherms,environment temperature can regulate the growth and development of fish through influencing the body temperature because water temperature makes influence on the temperature of fish.Low temperature will have a great impact on fish growth,development,metabolism,immunity and sometimes it causes the death of fish.Despite the current global warming,there is still a cold wave every year that has caused huge amounts of fish die,which makes great influence to the environment and economy.Therefore,studying the physiological effects of low temperature pressure on fish and its molecular regulation mechanism is of great significance.Epigenetic modification is of great importance during the cold adaption of animals and plants,especially DNA methylation,which participates the regulation and expression of cold adaption of a huge number of plants.However,the role of DNA methylation in the process of cold adaptation in fish is not much studied.DNA methylation refers to the transfer of a methyl group onto the position of the genomic Cp G dinucleotide cytosine through DNA methyltransferase which affects the accessibility of DNA and transcription factors and promotes or inhibits transcription.The level of DNA methylation is primarily regulated by the DNA methyltransferase(DNMT)protein family.DNMT protein family includes DNMT1,DNMT2,DNMT3 A,DNMT3B and DNMT3 L,and DNMT1 plays a leading role in the regulation of DNA methylation process.DNMT1 can suppress or activate the translation of the gene that determines the gene to express normally or not through regulating the level of DNA methylation.Zebrafish(Danio rerio)is a widely used model organism with a clear genetic background and rapid reproduction.The optimal growth temperature of zebrafish is 28.5℃,and the temperature range of its natural survival is 16-40℃,so it is a good material for studying temperature adaptation.Our lab previously analyzed the dynamic changes of genomic DNA methylation in zebrafish embryonic fibroblast like cell line(ZF4)under low temperature,and found that the methylation level increased under short-term low temperature pressure(18℃,5d)and decreased under long-term low temperature pressure(18℃,30d).At the same time,we also found that the regions where the DNA methylation changed dramatically mainly focused on the promoter of gene which is associated with the cold adaption of fish,indicating that DNA methylation is of great importance during the cold adaption of fish.However,the upstream regulatory mechanism of DNA methylation and the impact under low temperature pressure remain unclear.This study detects the expression of dnmt1 of ZF4 cells under low temperature pressure by RT-q PCR.Then,we overexpress or knock down dnmt1 in ZF4 cells which are treated at low temperature and detect the expression of dnmt1,analyzing the regulation mechanism of dnmt1 gene of cold adaption in fish.In this study,ZF4 cells which is in good growth state was treated at 18℃ for 1,2,3,4 and 5 days,and the expression of dnmt1 in ZF4 cells under low temperature pressure was detected by RT-q PCR.The CDS region of zebrafish dnmt1 was cloned and the pc DNA3.1-FLAG-dnmt1 overexpression plasmid was constructed.pc DNA3.1-FLAGdnmt1 overexpression plasmid(FLAG-dnmt1)was transfected into ZF4 cells to overexpress dnmt1.p LKO.1-dnmt1-sh RNA(sh-dnmt1)knockdown plasmid and p LKO.1-negative control-sh RNA(sh-nc)were constructed and transfected into 293T/17 cells by co-transfection with lentiviral packaging plasmids p CMV-d R8.91 and p CMV-VSVG,respectively.The lentivirus of sh-dnmt1 was used to knock down dnmt1 in ZF4 cells.The dnmt1 overexpression cells and dnmt1 knockdown cells were cryogenically treated at 18℃ for 3 days.The genomic DNA methylation level was detected by ELISA,and trypan blue was used to detect the cell viability.Furthermore,RT-q PCR was used to detect the expressions of apoptosis related genes bax,bcl-2 and caspase-3.The results are as follows:1.Under low temperature pressure,the m RNA relative expression level of dnmt1 in zebrafish cells ZF4 increased;2.Under low temperature pressure,the genomic DNA methylation level of FLAG-dnmt1 overexpression group cells increased significantly than that of control cells while the cell viability of FLAG-dnmt1 overexpression group decreased significantly than that of control cells.The expressions of bax and caspase-3 of FLAG-dnmt1 overexpression group cells increased while the expression of bcl-2 was down-regulated in contrast to the control group cells;3.Under low temperature pressure,the level of the genomic DNA methylation of sh-dnmt1 knockdown group cells significantly decreased than that of sh-nc cells while the cell viability of sh-dnmt1 knockdown group increased significantly than that of sh-nc cells.The expressions of bax and caspase-3 of sh-dnmt1 knockdown group cells were down-regulated while the expression of bcl-2 of sh-dnmt1 knockdown group cells was up-regulated in contrast to the sh-nc group cells.In conclusion,this study suggested that zebrafish DNMT1 may regulate cell apoptosis by regulating DNA methylation level under low temperature pressure and participate in the biological process in response to low temperature.This study provides new clues for further research on the molecular mechanism of zebrafish cold adaptation.