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通过体细胞核移植技术获得克隆哺乳动物的种类在不断增加,但成功率仍然较低,常表现为围产期的高死亡率.其中,肺发育异常是最常见的发育缺陷之一.哺乳动物肺器官的发生受一系列蛋白因子的调控,其中隶属于同源结构域转录因子家族的TTF-1参与肺的发生和出生后功能的维持,是一种重要的转录因子,其表达水平的异常很可能与肺的发育缺陷有关.SCNT胚胎异常的甲基化可能影响发育关键基因的表达.本研究克隆了牛TTF-1基因,包括启动子区(921bp)、外显子1(373bp)、内含子1(932bp)和外显子2的一部分(273bp)在内约2.5kb的核苷酸序列,发现3头肺发育有缺陷的SCNT牛TTF-1mRNA的表达量均显著低于肺发育正常的SCNT牛及对照牛(P<0.05).对表达量最低的个体和对照牛肺组织TTF-1基因启动子区63个CpG位点的甲基化状态进行了分析,发现在大多数位点上,二者均处于去甲基化状态,甲基化百分比差异不显著(P>0.05),说明肺发育缺陷的体细胞核移植牛TTF-1mRNA的低表达与启动子区CpG的甲基化无关.
The number of cloned mammals acquired by somatic cell nuclear transfer technology is increasing, but the success rate is still low, often manifested as high perinatal mortality, of which lung dysplasia is one of the most common developmental defects. The development of organs is regulated by a series of protein factors. TTF-1, which belongs to the homology domain transcription factor family, is involved in the maintenance of lung function and postnatal function. It is an important transcription factor and its expression level is very abnormal May be related to the developmental defects of the lung.The abnormal methylation of SCNT embryos may affect the expression of developmental key genes.This study cloned the bovine TTF-1 gene, including the promoter region (921bp), exon 1 (373bp) A nucleotide sequence of about 2.5 kb containing a portion of 1 (932 bp) and a portion of exon 2 (273 bp) showed that expression levels of 3 TTF-deficient SCNT bovine TTF-1 mRNAs were significantly lower than those of normal lung development (P <0.05) .The methylation status of 63 CpG sites in the TTF-1 promoter region of the lowest expressing and control bovine lung tissues was analyzed and found that at most sites Both are in a demethylated state, methylated No significant difference compared (P> 0.05), irrespective of lung developmental defects described SCNT bovine TTF-1mRNA low expression promoter region CpG methylation.