猪DLK1基因序列特征、印记状况及组织表达分析

Characterization,Imprinting Status and Tissue Distribution of Porcine DLK1 Gene

提取了2月龄大白猪、梅山猪及其正反交F1代个体的DNA或RNA样品,采用RT-PCR、SNP和相对定量分析的方法研究了猪DLK1基因的不同剪接体,印记状况和组织特异性表达谱.结果表明:猪DLK1氨基酸具有糖基化位点,表皮生长因子重复结构特征和delta/notch/serrate信号分子家族高度相关的二级结构,并且与其他哺乳动物的氨基酸序列具有高度同源性;在2月龄猪的心脏、肝脏、脾脏、肺、肾、胃、舌头、小肠、骨骼肌和脂肪组织中除了表达DLK1-B和DLK1-C2外,还发现了新的剪接体DLK1-A,并且3种剪接体在所检测的组织中均只表达父源的等位基因;DLK1基因总的表达量在各组织中存在显著性差异(p<0.01),其中骨骼肌和肺表达量最高,其次是舌、脾和脂肪,心脏和肝脏最低(p<0.05).因此,DLK1基因在哺乳动物间高度保守;猪在生长发育的各个阶段主要表达DLK1-C2剪接体,在某阶段也表达DLK1-A或DLK1-B;DLK1基因在猪生长发育的不同阶段和不同组织中只表达父源等位基因,并且对猪各个组织生长发育起着必要的作用,但总的表达量在不同生长发育时期组织中存在差异.

In this study, we isolated DNA or RNA samples from parents and F1 animals generated with reciprocal crosses between Large White and Meishan breeds to investigate the alternative splicing isoforms, imprinting status and tissue-specific expression of porcine DLK1 gene with RT-PCR, SNP and relative quantitative PCR. The sequence analysis showed that porcine DLK1 amino acid residues had N-linked glycosylation sites, high similarity to other mammalian species homology and contained epidermal growth factor （EGF） repeat motifs, and secondary structure features closely related to those present in the delta/ notch/serrate family of signaling molecules. The new alternative isform, DLK1-A, as well as DLK1-B and DLK1-C2 was found expressed in the liver, heart, tongue, muscle, lung, spleen, fat, small intestine, stomach, and kidney of two-mouth-old pigs. Imprinting analysis showed that the three alternative splicing isoforms were all paternally expressed in the above tissues. The expression levels of DLK1 across tissues were significantly different （p〈0.01）. The lung and skeletal muscle had the highest DLK1 transcript abundance, followed by the tongue, spleen, and fat, which had significantly greater transcript abundance than the heart and liver did （p〈0.05）. These results indicate that the DLK1 gene is conserved in mammalians. While DLK1-A or DLK1-B is expressd at some point in development, DLK1-C2 is dominantly ex- pressed throughout the life cycle in the pig. The porcine DLK1 gene is paternally expressed in all tissues examined during development. In addition, the gene plays a necessary role in growth and development of various bodily tissues and has tissue-specific expressions.