Study On Correlation Of DNA Methylation In Hoxc8 And Multi-Thoracic Vertebrae Characters Of Mongolia Sheep

The trait of Multi-Vertebrae of Mongolia sheep was worth to study because of it's excellent meat performance. However, there was not system genetic study on Mongolia sheep. In this study, the development genetics mechanism of Multi-Thoracic Vertebrae variant of Mongolia sheep was explored from epigenetics. The sequences of Hoxc8 exon-1 and exon-2 were obtained by PCR and bioinformatics analysis on nucleotides and amino acids were performed, included sequence homology and composition of amino acids and so on; methylation level of Hoxc8 exon-1 was detected by RT-PCR combined Methylation DNA Immuno-Precipitation; CpG islands were mapped by Bisulfite Sequencing PCR to determin the numbers and status of CpG sites. The results were as follows:(1) The sequences of Hoxc8 exon-1 (432bp) and exon-2 (273bp) of normal and Multi-Thoracic Vertebrae Mongolia sheep were obtained by PCR and the Genebank accession number EU817489 and FJ905472 were achieved. Alignment results of them were that the sequences were conformity except a little difference in two side of sequences. Hoxc8 exon-1and exon-2 were aligned with other species and the results were that higher homology were above 96%( exon-1) and 91%( exon-2) with other mammals(including human,dog,mouse,rat and chimpanzee ) and lower homology with zebrafish were 75.8% and 74%.(2) After immuno-precipitation, the real-time quantitative PCR was performed using two pairs of primers designed according Hoxc8 exon-1 of the Mongolia sheep.The results were the corrected methylated DNA concentrations of the samples in 6 Multi-Thoracic Vertebrae and 4 normal Mongolia sheep using P2 were 11.50%, 11.00%, 5.28%, 4.49%, 2.89%, 2.41%, 1.20%, 0.60%, 0.35% and 0.03% respectively. P = 0.017 of two experimental group by t-test. However, P = 0.953 by amplification using P1. It indicated that the methylated DNA level of the normal Mongolia sheep was lower than that of the Multi-Thoracic Vertebrae Mongolia sheep amplificated by P2.(3) Mapped the methylation status and numbers of CpG site in Hoxc8 exon-1 of normal and Multi-Thoracic Vertebrae Mongolia sheep by Bisulfite Sequencing PCR (BSP) technology The result indicated that CpG site in Hoxc8 exon-1 of normal and Multi-Thoracic Vertebrae Mongolia sheep had different methylation patterns and the methylated DNA level of the normal Mongolia sheep was much lower than that of the Multi-Thoracic Vertebrae Mongolia sheep. In CpG islands of 293bp region, there were respectively 6,3,6"C'in CpG islands methylated in normal Mongolia sheep and 23,20,21 in Multi-Thoracic Vertebrae Mongolia sheep respectively. The ratio of methylated CpG dinucleotide of normal and Multi-Thoracic Vertebrae Mongolia sheep was 22.2%±0.179, 11.1%±0.103, 22. %±0.179, 92%±0.077,74.1%±0.199, 77.8%±0.179 respectively and the mean of two experiment group were 18.50±0.064% and 79.03±0.056%(P = 0.002<0.01).And also, the distance of CpG dinucleotide was very closed.