Analysis Of The Methylation Status Of Porcine UCP3 Gene Promoter And Its MRNA Expression Regulation

To explore the influence of promoter methylation status of uncoupling protein 3 in PuTian Black Pig among different growth and development stages and different tissues on gene expression,Bisulfite Genomic Sequencing PCR has been used in our study to detect the methylation status of UCP3 promoter in heart,pectoralis,leg muscle,fat,longissimus dorsi and ear tissues of PuTian Black Pig among 1,90 and 210 days and in 90 days leg muscle of Duroc and DuPu Pig.Meanwhile fluorescence quantitive real-time PCR has been used to detect their mRNA expression.and in order to know about the relationship between DNA methylation and heterosis,BSP method has been used to detect the methylation status of UCP3 promoter in leg muscle from 90 days PuTian Black Pig,Duroc and their hybrid DuPu Pig.And fluorescence quantitive real-time PCR has been used to detect their mRNA expression,which can help us get insight into the regulation mechanism of UCP3 gene expression by DNA methylation and how it works on the formation of hybrid vigor.The main research process and result showed as follows:(1)Refers to the original sequence of UCP3 gene promoter,use biology software BIQ-Analyzer to analyze the UCP3 gene promoter sequence among different growth and development stages and different tissues modified by bisulfite.The results show that,there existed 9 CpG loci in the target fragment from sequencing,respectively located in 173bp,186bp,201bp,208bp,227bp,234bp,241bp,247bp and 250bp in the origin sequence.Through analyzed and compared by DNAssist software,we found that,compared to GenBank sequence,all unmethylated cytimidine in the sequence modified by bisulfite were translated into thymine,and "A?T"and "G?A" base mutation were respectively detect in 32bp and 62bp,in addition,the rest base remained unchanged.(2)Using BSP to detect the methylation status of CpG loci in UCP3 gene promoter,the result shows that,the methylation level of CpG loci 1 is 100%,while CpG loci 5 and 6 are unmethylated,and the rest CpG loci shows low methylation level and dynamic change.(3)Using BSP to detect the methylation status of UCP3 gene promoter in different tissues of PuTian Black Pig,The result shows that,the methylation levels of UCP3 gene promoter in leg muscle are all lowest,in heart follows and in ear tissue are highest,while there exist no significant difference between different tissues(P>0.05).(4)Using BSP to detect the methylation status of UCP3 gene promoter in different days of PuTian Black Pig,The result shows that,the methylation level of UCP3 gene promoter in 90 days are all lowest,and in 210 days are highest,while there exist no significant difference between different days(P>0.05).(5)Using BSP to detect the methylation status of UCP3 gene promoter in leg muscle of 90 days PuTian Black Pig,Duroc and DuPu Pig,The result shows that,the methylation level of UCP3 gene promoter in leg muscle of 90 days PuTian Black Pig,Duroc and DuPu Pig respectively is 14.81%,11.11%and 18.52%.The methylation level of UCP3 gene promoter in DuPu Pig is lowest,in PuTian Black Pig follows and in Duroc is highest.(6)The methylation level of UCP3 gene promoter in leg muscle of 90 days DuPu Pig is close to PuTian Black Pig,there is no significant difference between DuPu Pig and its parent(P>0.05),but demethylation was occured to its CpG loci 8 and 9 except locil,which led to the significantly increase of UCP3 gene expression(P<0.05).It is observed that the variation of methylation level in the specific loci has remarkable effect on heterosis.(7)Using fluorescence quantitive real-time PCR to detect the expression of mRNA of UCP3 gene promoter of PuTian Black Pig among different tissues and different days.The result shows that,the expression of mRNA of UCP3 gene promoter of heart in 90 days is significantly higher than in 210 days(P<0.05),the expression of UCP3 gene promoter of leg muscle,longissimus dorsi and ear tissue in 90 days is significantly higher than in 1 days and in 210 days(P<0.05),the expression of UCP3 gene promoter of pectoralis and fat tissue shows no significantly difference between different days(P>0.05).For the PuTian Black Pig in 1 days and 210 days,the expression of UCP3 gene promoter of leg muscle and heart is significantly higher than longissimus dorsi,pectoralis and fat tissue(P<0.05),while the expression of UCP3 gene promoter of longissimus dorsi,pectoralis and fat tissue is significantly higher than ear tissue(P<0.05).For the PuTian Black Pig in 90 days,the expression of UCP3 gene promoter of leg muscle is significantly higher than heart(P<0.05),while the expression of UCP3 gene promoter of heart is significantly higher than longissimus dorsi,pectoralis and fat tissue(P<0.05),and the expression level of UCP3 gene in longissimus dorsi,pectoralis and fat tissue is significantly higher than ear tissue(P<0.05).(8)Using fluorescence quantitive real-time PCR to detect the expression level of mRNA of UCP3 gene in leg muscle of 90 days PuTian Black Pig,Duroc and DuPu Pig.The result shows that,the expression level of UCP3 gene in leg muscle of 90 days PuTian Black Pig,Duroc and DuPu Pig shows as 1.16±0.03?1.3±0.03 and 1.16±0.04 respectively.The expression of UCP3 gene promoter in leg muscle of 90 days is significantly higher than PuTian Black Pig and Duroc(P<0.05).