蒙古羊多胸椎性状与Hoxc8基因DNA甲基化的相关性研究
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摘要
蒙古羊的多脊椎性状具有巨大的应用价值,但是,对蒙古羊一直没有开展系统深入的遗传研究。本研究从表观遗传学角度探索蒙古羊多胸椎变异的分子发育遗传学机制。利用PCR扩增并分析多脊椎蒙古羊与正常蒙古羊的Hoxc8外显子序列,并对其核苷酸和氨基酸进行了生物信息学分析,包括序列同源性比较,以及该区域编码的氨基酸;将实时定量PCR技术与甲基化DNA免疫共沉淀(MeDIP)技术结合,对Hoxc8 exon-1中的甲基化水平进行定性检测;利用Bisulfite Sequencing PCR技术对Hoxc8 exon-1中的甲基化水平进行定位分析,对其中的CpG岛进行精确制图,确定其甲基化发生的位置。结果如下:
(1)测序得到正常和多脊椎蒙古羊Hoxc8的exon-1(432bp)和exon-2(273bp),并已提交Genebank(登录号EU817489和FJ905472)。经序列比对二者的DNA序列除两侧个别碱基有差异,中间序列完全一致。蒙古羊Hoxc8的exon-1和exon-2序列分别与其他物种进行同源性比对,结果表明蒙古羊Hoxc8 exon-1与人、小鼠、大鼠、犬的同源性达到96%以上,与斑马鱼的同源性75.8%;exon-2与大猩猩、犬、人、小鼠和大鼠的同源性达到91%以上,与斑马鱼的同源性74%。
(2)设计两对引物,扩增多脊椎蒙古羊Hoxc8 exon-1中的两段序列,进行甲基化DNA免疫共沉淀-实时定量PCR实验,结果表明:用引物P2扩增,6只多脊椎蒙古羊和4只正常的蒙古羊样本校正后的甲基化DNA的浓度分别为11.50%;11.00%;5.28%;4.49%;2.89%;2.41%;1.20%;0.60%;0.35%;0.03%,两实验组差异显著(P=0.017<0.05)。而用引物P1扩增,两实验组甲基化DNA浓度差异不显著(P=0.953>0.05)。
(3)用Bisulfite Seqnencing PCR方法对Hoxc8 exon-1内的CpG岛中的CpG位点的甲基化模式进行精确制图,在引物扩增的293bp区域中,包含27个CpG二核苷酸,经重亚硫酸盐转化后,正常蒙古羊30号、28号和27号样本的CpG岛中CpG二核苷酸中的“C”分别有6个、3个、3个没有被转化成“T”,即这些“C”发生了甲基化;而多脊椎蒙古羊12号、13号和14号样本的CpG岛中CpG二核苷酸中的“C”分别有23个、20个、21个发生了甲基化。正常与多脊椎蒙古羊的甲基化的CpG二核苷酸的比例分别为22.2%±0.179,11.1%±0.103,22.2%±0.179和92%±0.077,74.1%±0.199,77.8%±0.179,正常蒙古羊实验组甲基化的CpG二核苷酸比例的均值为18.50±0.064%,多脊椎蒙古羊实验组为79.03±0.056%,经t检验,两实验组差异极显著(P=0.002<0.01)。这一区域内多脊椎蒙古羊的甲基化水平远远高于正常蒙古羊。并且每个CpG位点间的距离非常近,CpG二核苷酸非常密集。
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.
(1)测序得到正常和多脊椎蒙古羊Hoxc8的exon-1(432bp)和exon-2(273bp),并已提交Genebank(登录号EU817489和FJ905472)。经序列比对二者的DNA序列除两侧个别碱基有差异,中间序列完全一致。蒙古羊Hoxc8的exon-1和exon-2序列分别与其他物种进行同源性比对,结果表明蒙古羊Hoxc8 exon-1与人、小鼠、大鼠、犬的同源性达到96%以上,与斑马鱼的同源性75.8%;exon-2与大猩猩、犬、人、小鼠和大鼠的同源性达到91%以上,与斑马鱼的同源性74%。
(2)设计两对引物,扩增多脊椎蒙古羊Hoxc8 exon-1中的两段序列,进行甲基化DNA免疫共沉淀-实时定量PCR实验,结果表明:用引物P2扩增,6只多脊椎蒙古羊和4只正常的蒙古羊样本校正后的甲基化DNA的浓度分别为11.50%;11.00%;5.28%;4.49%;2.89%;2.41%;1.20%;0.60%;0.35%;0.03%,两实验组差异显著(P=0.017<0.05)。而用引物P1扩增,两实验组甲基化DNA浓度差异不显著(P=0.953>0.05)。
(3)用Bisulfite Seqnencing PCR方法对Hoxc8 exon-1内的CpG岛中的CpG位点的甲基化模式进行精确制图,在引物扩增的293bp区域中,包含27个CpG二核苷酸,经重亚硫酸盐转化后,正常蒙古羊30号、28号和27号样本的CpG岛中CpG二核苷酸中的“C”分别有6个、3个、3个没有被转化成“T”,即这些“C”发生了甲基化;而多脊椎蒙古羊12号、13号和14号样本的CpG岛中CpG二核苷酸中的“C”分别有23个、20个、21个发生了甲基化。正常与多脊椎蒙古羊的甲基化的CpG二核苷酸的比例分别为22.2%±0.179,11.1%±0.103,22.2%±0.179和92%±0.077,74.1%±0.199,77.8%±0.179,正常蒙古羊实验组甲基化的CpG二核苷酸比例的均值为18.50±0.064%,多脊椎蒙古羊实验组为79.03±0.056%,经t检验,两实验组差异极显著(P=0.002<0.01)。这一区域内多脊椎蒙古羊的甲基化水平远远高于正常蒙古羊。并且每个CpG位点间的距离非常近,CpG二核苷酸非常密集。
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.
引文
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