| Min pig is a local pig breed formed under the cold conditions in Northeast China,and they have excellent cold resistance.It is of great significance to study the genetic mechanism of their cold resistance characteristics to promote the application of outstanding genetic resources of the pigs.Epigenetics are affected by environmental factors,which in turn alter the way genes are expressed.ST cells have important supportive functions for sperm development,and play an important role in cold resistance of Min pig and male passage.Therefore,exploring the molecular regulation mechanism of pig ST cells at low temperature is of reference value for studying the epigenetic mechanism of cold resistance in pig.In order to study the effect of low temperature on ST cells,this study took the pig ST cells cultured in vitro as a model.After culturing in low temperature(25℃)for different times(0h,24h,48h,72h),cell morphology was observed,apoptosis rate was detected,three genes related to apoptosis Caspase-3,C-myc,P53 and six temperature related miRNAs were detected.At the same time,two over-expressing ST cells lines of two important genes related to epigenetics,DNA methyltransferase 3A(DNMT3A)and DNA methyltransferase 3B(DNMT3B)were constructed.Scratch test was used to detect the migration ability of cells after transgenes were detected,and Real-time PCR method was used to detect downstream gene expression changes related to methylation,heat shock and apoptosis.The main research results obtained were as follows:(1)Apoptosis characteristics of ST cells appeared after 24 hours of low temperature treatment,and apoptosis characteristics were more obvious after 48 hours.Flow cytometry results also showed that with the extension of low temperature treatment time,the apoptosis rate increased significantly.(2)Real-time PCR showed that during low temperature treatment,the relative expression of Caspase-3 was significantly up-regulated(p<0.01),the relative expression of P53 was extremely down-regulated(p<0.01),and the relative expression of C-myc decreased significantly(p<0.01)and then increased significantly(p<0.05).(3)Real-time PCR was used to detect the changes in the relative expression of six miRNAs during low temperature treatment.The results showed that with the increase of treatment time,the relative expression levels of all detected miRNA increased significantly(p<0.05),among which miR-425-5p and miR-29b reached the highest point at 48h.(4)The complete coding region sequence of pig DNMT3A gene was successfully cloned,the size was 2070bp,and the eukaryotic expression vector of pcDNA3.1-DNMT3A was constructed.The complete coding region of pig DNMT3B gene was successfully cloned,with a size of 2382bp,and the eukaryotic expression vector of pcDNA3.1-DNMT3B was constructed.(5)Cells scratch test results showed that relative cells migration rate was inhibited in pig ST cells that over-expressed of DNMT3A gene compared with the control group,and the inhibition was the most significant at 24h(p<0.05).The results showed that relative cells migration rate was inhibited in pig ST cells that over-expressed of DNMT3B gene compared with the control group,and the inhibition was the most significant at 24、48h(p<0.05).(6)In the over-expressed DNMT3A and DNMT3B cells lines,the relative expression levels of EHMT2,HSP70,and HSPB1 were significantly reduced(p<0.05),and the relative expression levels of HDAC1,HMOX1,P53,and C-myc were significantly increased after overexpression of DNMT3A gene(p<0.05).The relative expression of DNMT3A,HSF1,P53,and C-myc increased significantly after overexpression of the DNMT3B gene(p<0.05). |
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