Isolation And Cloning,Expression Patterns And Regulation Mechanism Study Of Porcine Maternal Effect Gene PADI6

Maternal effect genes transcripts are abundantly transcribed during oogenesis and accumulate in the oocyte cytoplasm, and play an important role in the oogenesis, oocyte maturation and preimplantation embryo development. But after fertilization the maternal effect genes products are rapidly degraded along with the early embryo cleavage, and turn the embryo from maternal control to zygotic genome control. Most of the study on maternal effect genes has been done in the mouse and this type of model animals, but little is known about the maternal effect genes in pig and this kind of large animals, and the regulation mechanism of the unique expression pattern with maternal effect genes is also rarely known. Therefore, finding and identifying the maternal effect genes, understanding the genes function and exploring the mechanism of expression and regulation have great guiding significance to better know the oocyte growth and early embryo development, and have great reference value on improvement and development the high-end biotechnology, such as oocytes in vitro maturation, somatic cell clone and new genetically modified animals breeding. We aimed to investigate porcine PADI6 maternal effect gene, and carried out research on the expression pattern, gene function and regulatory mechanism of PADI6 gene. The results were as follows:1. The 2380 bp full-length cDNA of porcine PADI6 gene was isolated using common PCR and RACE in this study. The full-length cDNA contains 76 bp 5’UTR, 228 bp 3’UTR and 2076 bp open reading flame. The base G located at-76 bp upstream of the ATG was identified as the major transcriptional start site of porcine PADI6 gene by 5’RACE and RT-PCR.2. The expression patterns of the three porcine maternal effect genes PADI6, MATER and NPM2 in different tissues were analyzed and we found that all the three maternal effect genes were displaying a rather restricted tissue-specific expression pattern. They were mainly expressed in the ovary and accord with the expression feature of maternal effect gene. The expression patterns of the three maternal effect genes in the early embryos generated by parthenogenetic activation and in vitro fertilization were analyzed, and the results showed that the transcripts were abundantly accumulated in GV and MII oocytes, but rapidly degraded along with the cleavage and no transcripts could be detected in morula and blastocyst, and proved that all the three genes were regulated in a typical way of maternal effect genes. The expression pattern of PADI6 gene at different development stages of ovary was analyzed, and the result indicated that the 2 months ovary had the highest expression level of PADI6 gene. The expression level was rapidly decreased from 2 months to 4 months stage along with the development, and then reached a relatively stable level at 4 to 6 months sexual maturation stage.3. The overexpression experiment indicated that overexpression of PADI6 promoted the histone deacetylases HDAC1 and HDAC2 expression, and increased the DNA methylase DNMT2 expression. Moreover, overexpression of PADI6 could also increase the ratio of G1 phase cells and decrease the total number of S and G2 phase cells.4. The 1886bp(-1777/+109) 5’-flanking nucleotide sequence of porcine PADI6 was successfully isolated, and Via the fluorescence analysis of the constructed 5’ deletion vectors we identified the 153 bp core promoter located in the region from-85 bp to +68bp. Computational analysis of the core promoter region found that there were two Sp1 binding sites located at-56/-47 bp and-11/-4bp respectively, and the-56/-47 bp Sp1 binding site was highly conserved with the homologous sequence of mouse.5. Mutant fluorescent vectors of the two predicted Sp1 binding sites were constructed and the fluorescence activities were analyzed, we found only the-56/-47 bp Sp1 binding site could influence the promoter activity. The EMSA and ChIP experiments showed that Sp1 could not only bind to the-56/-47 Sp1 site in vitro, but also bind to it in vivo. The ChIP experiment also demonstrated the transcription factor Sp3 could bind to the-56/-47 Sp1 site in vivo.6. Overexpression of Sp1 or Sp3 were able to increase the PADI6 promoter activity and promote PADI6 gene expression. Accordingly, inhibition of Sp1 or Sp3 expression with specific siRNA could reduce the PADI6 promoter activity and suppresse the expression of PADI6. We inhibited Sp1 from binding to the GC box with Mithramycin A and found that both the promoter activity and the expression level of PADI6 were reduced with Mithramycin A treatment in a dose-dependent manner. The expression patterns of Sp1 and Sp3 at different development stages of ovary was analyzed, and the results demonstrated that the Sp1 and Sp3 shared very similar expression pattern with PADI6 gene in this period. Taken together, Sp1 and Sp3 transcription factors could cooperatively regulate the PADI6 gene expression by binding to the-56/-47 bp site.7. The pig and mouse 3’UTR pmirGLO fluorescent vectors were constructed respectively and co-transfected with mimics or inhibitor of the relevant miRNA, and identified that the activities of PADI6 3’UTR fluorescent vectors could be reduced by miR-320. The target sites of miR-320 in the PADI6 3’UTR region were confirmed by site mutation experiment of the predicted binding sites. The mimics and inhibitor of miR-320 were transfected into CHO cells, and found that miR-320 could suppresse the protein expression level of PADI6. Therefore we concluded that miR-320 could target the 3’UTR of PADI6 and inhibit the PADI6 expression.Through the above study, we had a deeper understanding about the molecular mechanisms of generation and degradation of maternal factors, and this would have great reference value for further exploration of the mechanisms of oocyte growth and early embryo development.