miR-127和miR-136在转基因克隆绵羊胎盘中的表达分析及其靶基因的鉴定

克隆动物胎盘发育缺陷是造成动物克隆效率低下的一个重要原因。目前认为克隆胎盘发育异常通常是由于一些基因表达的异常所致,与表观遗传修饰有关。microRNA是一种重要的表观遗传修饰方式,对动物胚胎发育和胎盘的形成有着重要的调控作用。为研究miR-127和miR-136在克隆动物胎盘中的表达情况及其与克隆动物发育缺陷的关系,本实验运用荧光定量PCR分析了死亡克隆绵羊胎盘和同期普通绵羊胎盘组织中miR-127和miR-136的相对表达量,并鉴定了miR-127和miR-136的靶基因及靶基因在胎盘中的表达情况。结果显示,miR-127和miR-136在克隆绵羊胎盘中的表达量分别增加了3.1和2.8倍。EGFP荧光敲除实验证实,胎盘发育相关基因Rtl1是miR-127和miR-136的靶基因,同时定量PCR分析发现Rtl1基因在克隆绵羊胎盘中的表达量降低了3/5。结果说明,miR-127和miR-136在克隆胎盘中的异常表达可导致Rtl1基因的低表达,这很可能是导致克隆动物死亡的一个重要原因。

Expression and target analysis of miR-127 and miR-136 in placentas of cloned transgenic sheep

Abnormal development of cloned animal placenta is one of the main causes of the low efficiency of somatic cell nuclear transfer（SCNT）,which was caused by abnormal gene expession and involved in epigenetic modification of the genome.microRNA is a major epigenetic modification of the genome and plays a crucial role in animal embryo and placenta development.However,the expression and function of microRNA in cloned placenta has not been reported.In order to study the correlation between expression of miR-127 and miR-136 in cloned placenta development and the development abnormality of cloned sheep,expression of miR-127 and miR-136 in placenta of the dead cloned sheep fetus and the age-matched normal sheep fetus（control）were detected by using real time PCR,and targets of miR-127 and miR-136 were tested by using EGFP fluorescence reporting system.Results indicated that expression levels of miR-127 and miR-136 in cloned sheep placenta were 3.1 and 2.8 times more than that of normal sheep placenta.EGFP fluorescence knockdown assay confirmed that Rtl1 was the target of miR-127 and miR-136.Moreover,expression levels of Rtl1 in cloned sheep placenta were 2.5 time less than that of normal sheep placenta.These finding suggested that abnormal expression of miR-127 and miR-136 resulted in down-regulation of Rtl1 expression,which may be one of the main causes of death of the transgenic cloned animal.