猪MKRN3基因启动子区CpG岛在胚胎心脏组织甲基化模式分析

Methylation patterns of MKRN3 promoter CpG island in porcine embryo heart

以梅山猪-大白猪正反交为模型,以65日龄和100日龄的胚胎心脏组织为研究材料,对MKRN3基因启动子区CpG岛进行预测,根据预测的CpG岛设计引物,采用重亚硫酸盐测序法(BSP法),分析MKRN3基因启动子区CpG岛在胚胎心脏组织的甲基化程度。结果显示:对于65日龄胚胎,猪MKRN3基因启动子区CpG岛在大白×梅山和梅山×大白杂交后代的胚胎心脏组织中均表现高度甲基化(75.6%、76.4%);对于100日龄的胚胎,大白×梅山杂交后代的胚胎心脏组织表现高度甲基化(80%),而梅山×大白杂交后代的胚胎心脏组织呈现低甲基化(35.6%)。由此可见,猪MKRN3基因启动子区CpG岛的甲基化模式随着正反交、胚胎发育时期不同而呈现出一定变化,即在胚胎发育65日龄时,正反交子代均具有高度甲基化;而在胚胎发育至100日龄,正交子代呈现高度甲基化,反交子代呈现低甲基化,说明在胚胎发育晚期父本或母本等位基因可能会发生明显的去甲基化。

To identify the methylation pattern of the porcine MKRN3 promoter CpG island in the embryo heart,embryos of days 65 and 100 of gestation from the reciprocal crosses of Yorkshire-Meishan pig were collected.Firstly,CpG island in the promoter region of MKRN3 was predicted by utilizing bio-informatics method,and primers was designed based on this predicted CpG island.Bisulfite sequencing(BSP)was utilized to detect methylation level in the embryo heart from Yorkshire-Meishan reciprocal crosses.Results showed that,in the heart from embryo of days 65,the CpG island of porcine MKRN3 showed high level of methylation,with 75.6%from Yorkshire×Meishan and 76.4%from Meishan×Yorkshire.In the heart from embryo of 100 days,the CpG island of target gene showed high level of methylation(80%)from Yorkshire×Meishan,while low level of methylation(35.6%)from Meishan×Yorkshire crosses.Results above indicated that the methylation pattern of the porcine MKRN3 promoter CpG island would change according to the change of reciprocal crosses and developmental stages.For embryo of 65 days,reciprocal crosses of Yorkshire-Meishan represented high methylationlevel,but for embryos of 100 days,high methylation from Yorkshire×Meishan but low methylation from Meishan×Yorkshire were observed,indicating that de-methylation might take place in the paternal or maternal allele,during the late stage of embryonic development.