湖羊BMP7启动子区转录调控的研究

发布时间：2018-08-17 08:44

【摘要】：前期在湖羊羔皮毛囊中利用基因芯片技术筛选出不同花纹间差异表达基因BMP7,结合湖羊羔皮毛囊组织学特性关联分析,确定BMP7作为候选基因,BMP7蛋白是TGF-β超家族中最大的分泌型信号传导分子之一,BMP7除了与骨形成发生有关,还被发现与羽毛胚芽的大小和空间分布有关,在毛囊的发育与毛发生的生长中起着重要作用,但本身的调控机制并不清楚。为此,本研究对BMP7的转录调控机制进行初步研究,首先对湖羊不同组织进行BMP7表达水平分析,其次克隆BMP7近端启动子进行生物信息学分析,接着根据生物信息学分析结果与近端启动子序列构建系列缺失载体双荧光素活性检测,最后对转录因子结合位点定点突变和转录因子过表达验证,从而揭开BMP7启动子区的转录调控机制,为后续的研究提供依据。本研究主要通过以下几个方面研究BMP7的转录调控机制:(1)利用RT-qPCR对BMP7在湖羊的各个组织表达情况进行分析,发现在羔皮毛囊组织中高表达。(2)通过NCBI数据库得到BMP7转录起始点上游大约2000bp下游500bp的调控序列,以2500bp的序列为基础进行近端启动子克隆,获得了 1757bp的序列。对BMP7近端启动子上游转录调控区域进行了生物信息学预测分析,发现在BMP7近端启动子上游调控区存在-1216bp—-1166bp与-632bp—-582bp两个转录起始位点;对CpG岛分布情况分析,发现-549bp—+295bp处富含CpG岛,进行转录因子结合位点预测,发现有SP1、EGR1、NRF1、TFAP2B等转录因子结合位点。(3)根据BMP7近端启动子序列设计启动子系列缺失片段,将各个缺失片段构建到双荧光素酶报告基因载体上,转染293T细胞进行双荧光酶检测不同缺失片段活性并进行显著性比较,寻找双荧光素酶活性最高的序列片段,发现核心区域-758bp—-545bp之间活性较高,对其进行生物信息学预测,发现存在转录因子SP1、EGR1可能结合位点,对BMP7启动子转录调控起着一定作用。(4)为了进一步鉴定BMP7近端启动子上游转录调控区域的转录因子结合位点,根据生物信息学结果筛选出EGR1和SP1,对其转录因子结合位点进行定点突变,同时构建BMP7的转录因子EGR1进行过表达载体进一步验证。结果显示,突变后的BMP7近端启动子上游调控区的活性呈显著性降低;过表达EGR1转录因子,BMP7上游调控区活性呈显著性上升,初步推断EGR1与SP1对BMP7转录调控具有重要的作用。
[Abstract]:In the early stage, the differentially expressed gene BMP7 was screened by gene chip technique in the fur sacs of lake lamb, and combined with the correlation analysis of histopathological characteristics of wool follicles of sheep lambskin. It is confirmed that BMP7 is one of the largest secretory signal transduction molecules in the TGF- 尾 superfamily, and it is also found to be related to the size and spatial distribution of plumage germ, in addition to its involvement in bone formation. It plays an important role in the development and growth of hair follicles, but its regulatory mechanism is not clear. Therefore, the transcriptional regulation mechanism of BMP7 was studied in this study. Firstly, the expression level of BMP7 was analyzed in different tissues of Hu sheep, and then the proximal promoter of BMP7 was cloned for bioinformatics analysis. Based on the results of bioinformatics analysis and proximal promoter sequence, a series of deletion vectors were constructed to detect the double fluorescein activity. Finally, the site-directed mutation of transcription factor binding site and overexpression of transcription factor were verified. Thus, the transcriptional regulation mechanism of BMP7 promoter region was revealed, which provided the basis for further research. In this study, the transcriptional regulation mechanism of BMP7 was studied in the following aspects: (1) RT-qPCR was used to analyze the expression of BMP7 in various tissues of Hu sheep. High expression was found in lambskin hair follicles. (2) the regulatory sequence of 500bp in the upstream of BMP7 transcription initiation site was obtained from NCBI database, and the 500bp sequence was cloned from the proximal promoter of 2500bp. The 1757bp sequence was obtained. The upstream transcriptional regulation region of BMP7 proximal promoter was predicted by bioinformatics. It was found that there were two transcriptional initiation sites -1216bp-1166bp and -632bp-582bp in the upstream regulatory region of BMP7 proximal promoter, and the distribution of CpG island was analyzed. It was found that the site of -549bp- 295bp was rich in CpG islands, and the transcription factor binding sites were predicted, and there were transcription factor binding sites such as SP1, EGR1, NRF1, TFAP2B, etc. (3) Promoter series deletion fragments were designed according to the proximal promoter sequence of BMP7. Each deletion fragment was constructed into a double luciferase reporter gene vector and transfected into 293T cells to detect the activity of different deletion fragments and to find the sequence fragment with the highest double luciferase activity. It was found that the core region -758bp 545bp was highly active. Bioinformatics prediction showed that the transcription factor SP1, EGR1, might bind to it. (4) in order to further identify the transcription factor binding sites in the upstream transcriptional regulatory region of BMP7 proximal promoter, According to the bioinformatics results, EGR1 and SP1 were screened out, and their transcription factor binding sites were mutated at the same time, the transcription factor EGR1 of BMP7 was constructed to further verify the expression vector. The results showed that the activity of the upstream regulatory region of the proximal promoter of BMP7 was significantly decreased after mutation, and the activity of the upstream regulatory region of overexpression of EGR1 transcription factor was significantly increased. It was preliminarily concluded that EGR1 and SP1 play an important role in the regulation of BMP7 transcription.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S826

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