Isolation, Imprinting Identification And Promoter Analysis Of Mest Gene In Different Development Stage Of The Porcine Embryo

Epigenetics is one part of genetics, which can be defined as heritable changes in development and cell differentiation process that are not caused by the changes in DNA sequence. Epigenetics mainly includes DNA methylation, histone acetylation and non-coding RNA. Genomic imprinting as a result of DNA methylation has been a hot spot in development biology. Genomic imprinting is the differential expression of alleles from both patents, only the paternal (or maternal) allele is expressed and the other parent’s allele is not expressed or rarely expressed. In mammals, genomic imprinting with a specific parental characteristic in fetal growth, placental function and behacior after birth plays an important regulatory role. In addition, studies have shown that imprinted genes often associated with a number of congenital diseases, genetic diseasas, some cancers and tumors. Now, most studies on genomic imprinting have focused on human and mouse, livestock especially pigs in the study is relatively small. Therefore, our research includes three main aspects:first, in different fetal development stage of pigs, we detected the imprinted status of MEST gene with RT-PCR-RFLP. Second, so as to analyze the feasibility of imprinted gene as molecular mark in breeding, we analyzed the association of MEST gene and the important economic traits with PCR-RFLP. Third, we cloned the promoter of MEST gene in the pig, and detected the activity. The main results are as follows:1. Under the imprinted status and physiolyogical function of human and mouse, we selected MEST gene as candidate imprinted gene. According to Genbank accession number EF619473, we obtained cDNA sequence 1248bp. We found a SNP in 133bp through sequence alignment, which is G/A transition mutation.2. Using bioinformatics, we assigned the location of MEST gene in pigs, analyzed the protein character, structure and function.3. We analyzed the imprinted status of MEST gene in different development stage of pigs with Large White and Meishan reciprocal crosses model and RT-PCR-RFLP. (1) On days 30 of gestation, MEST gene is paternally expressed. (2) On days 65 of gestation, MEST gene is biallelically expressed in heart, liver, spleen, lung and small intestine, but paternally expressed in kidney, stomach, skeletal muscle, brain and placenta. (3) On days 100 of gestation, MEST gene is biallelically expressed in spleen and small intestine, but paternally expressed in heart, liver, lung, kidney, stomach, skeletal muscle, brain and placenta. 4. In seven pigs breed, we conducted a genotype frequency and gene frequency statistics; Association analysis was performed in relatived groups. The result showed that for G133A-RsaⅠ-RFLP polymorphism of MEST gene, highly significant effects were observed on Loin Eye Width, and significant effects were observed on Loin Eye Height, Drip Loss Rate, Uterus Weight, Total number of piglet born.5. In order to study potential functions of MEST gene, temporal and spatial expression profiles were analyzed using semi-quantitative RT-PCR. The results of RT-PCR displayed that MEST gene is expressed in all detected tissues. On days 65 of gestation of Meishan boar×Large White sow, there is relatively higher expression in skeletal muscle, kidney, placenta and spleen, until to the day 100, there is relatively higher expression in liver, kidney and heart, lower expression in placenta and lung. On days 65 of gestation of Large White boar×Meishan sow, there is relatively higher expression in placenta and kidney, lower expression in liver, brain and spleen, until to the day 100, there is relatively higher expression in skeletal muscle, then is the heart and placenta. Detected MEST gene expression pattern in adult Meishan pig found that in the tissue of uterus has relatively higher expression, next is the lung, the expression level in the other tissues is similar.6. Using e!Ensembl predict MEST whole genomic sequence, according the sequence, we designed primers to clone, recycle and sequencing. The NNPP, Meth Primer, TFSEARCH WERE used to analyze the proximal promoter region, transcriptional start site, CpG island and predict the transcription factors.7. Dual luciferase reporter gene system detected recombinants activity. The result showed that except construct 419 and 934, all the recombinants of MEST were significant difference with pGL3-Basic. Construct 1134 contains highest activity, which conform to the prediction of website. From the 1448 to 1262,1134 to 934,587 to 419, the promoter activity was reduced which indicate that this region may contain positive regulatory sites. From the 1601 to 1448,1262 to 1134,934 to 587,419 to 238, the promoter activity was enhaced which indicate that this region may contain negative regulatory sites.