Tissue Expression Analysis Of Porcine Imprinted Gene MAGEL2and Cloning And Activity Analysis Of Its Promoter

Researches show that MAGEL2gene is a paternally expressed but maternally imprinted gene. It plays an important regulatory function in the hypothalamus. The abnormal gene expression leads to diminished capacity of taking care of children, biological rhythm regulation, and fertility in female rats, the male olfactory dysfunction in the sex, and increased suckling mortality. These results indicate MAGEL2gene has an important relationship with mammalian reproductive traits. In order to research the correlation of MAGEL2gene expression and maternal behavior in pigs, we chose Meishan and Duroc great maternal differences as the experimental animals, and analyzed tissue expression profile in the hypothalamus, placenta, etc. by quantitative real-time PCR. For further study MAGEL2molecular mechanism of gene expression, we had cloned MAGEL2gene promoter region for bioinformatic analysis, constructed the luciferase report gene expression vectors about the5’deletion promoter fragment, and analyzed promoter activity by transient transfection cell technology.This study achieved the following results:1. In order to investigate MAGEL2gene function in maternal behaviour, the expression level of MAGEL2in different species and newborn tissues were detected by real-time PCR. The results showed that MAGEL2gene expression was detected in all newborn tissues with the highest expression level in hypothalamus (P<0.01). The expression level in hypothalamus was significantly higher (P<0.01) in Meishan pigs than that in Duroc pigs. Although there was no significant difference during gestation period in Meishan placentas (P>0.05), MAGEL2was highly expressed in Meishan placentas than in Duroc placentas on days30of gesation (P<0.01), and undetected in shed placenta. The results indicated that MAGEL2gene might be participated in placental function and maternal behaviour in pigs. So it is probable a candidate gene in maternal behaviour.2. Through5’RACE technology, we got110bp5’UTR sequence of MAGEL2and identified the gene transcription start site. Comparing about2kb promoter sequence between Meishan and Duroc, results showed there were9bases variations including2bases missed in Meishan. The gene expression differences may be caused by these differences between different pig breeds. We did not find CpG island, but predicted2core promoter sequence, a total of18TATA box including7typical TATA box in1885bp promoter sequence,2GC box close to the transcription start site100bp range, and a lot of transcription factor binding site, for example Spl, GR, YY1, C/EBPβ and TFIID. It indicated that MAGEL2gene promoter could be a strong promoter, so we also studied promoter activity of MAGEL2.3. The5’deletion fragments of pig MAGEL2gene promoter were cloned and recombined into pGL3-Basic plasmids. The recombined vectors were pGL3-110, pGL3-261, pGL3-466and pGL3-1281. We tested their luciferase activity of5’ deletion series in HEK293cells. The results showed5’UTR of MAGEL2gene had no promoter activity, but the promoter sequence from0to-1171bp had strong promoter activity.