湖羊BMP7啟動(dòng)子區(qū)轉(zhuǎn)錄調(diào)控的研究
發(fā)布時(shí)間：2018-08-17 08:44
【摘要】：前期在湖羊羔皮毛囊中利用基因芯片技術(shù)篩選出不同花紋間差異表達(dá)基因BMP7,結(jié)合湖羊羔皮毛囊組織學(xué)特性關(guān)聯(lián)分析,確定BMP7作為候選基因,BMP7蛋白是TGF-β超家族中最大的分泌型信號(hào)傳導(dǎo)分子之一,BMP7除了與骨形成發(fā)生有關(guān),還被發(fā)現(xiàn)與羽毛胚芽的大小和空間分布有關(guān),在毛囊的發(fā)育與毛發(fā)生的生長(zhǎng)中起著重要作用,但本身的調(diào)控機(jī)制并不清楚。為此,本研究對(duì)BMP7的轉(zhuǎn)錄調(diào)控機(jī)制進(jìn)行初步研究,首先對(duì)湖羊不同組織進(jìn)行BMP7表達(dá)水平分析,其次克隆BMP7近端啟動(dòng)子進(jìn)行生物信息學(xué)分析,接著根據(jù)生物信息學(xué)分析結(jié)果與近端啟動(dòng)子序列構(gòu)建系列缺失載體雙熒光素活性檢測(cè),最后對(duì)轉(zhuǎn)錄因子結(jié)合位點(diǎn)定點(diǎn)突變和轉(zhuǎn)錄因子過(guò)表達(dá)驗(yàn)證,從而揭開(kāi)BMP7啟動(dòng)子區(qū)的轉(zhuǎn)錄調(diào)控機(jī)制,為后續(xù)的研究提供依據(jù)。本研究主要通過(guò)以下幾個(gè)方面研究BMP7的轉(zhuǎn)錄調(diào)控機(jī)制:(1)利用RT-qPCR對(duì)BMP7在湖羊的各個(gè)組織表達(dá)情況進(jìn)行分析,發(fā)現(xiàn)在羔皮毛囊組織中高表達(dá)。(2)通過(guò)NCBI數(shù)據(jù)庫(kù)得到BMP7轉(zhuǎn)錄起始點(diǎn)上游大約2000bp下游500bp的調(diào)控序列,以2500bp的序列為基礎(chǔ)進(jìn)行近端啟動(dòng)子克隆,獲得了 1757bp的序列。對(duì)BMP7近端啟動(dòng)子上游轉(zhuǎn)錄調(diào)控區(qū)域進(jìn)行了生物信息學(xué)預(yù)測(cè)分析,發(fā)現(xiàn)在BMP7近端啟動(dòng)子上游調(diào)控區(qū)存在-1216bp—-1166bp與-632bp—-582bp兩個(gè)轉(zhuǎn)錄起始位點(diǎn);對(duì)CpG島分布情況分析,發(fā)現(xiàn)-549bp—+295bp處富含CpG島,進(jìn)行轉(zhuǎn)錄因子結(jié)合位點(diǎn)預(yù)測(cè),發(fā)現(xiàn)有SP1、EGR1、NRF1、TFAP2B等轉(zhuǎn)錄因子結(jié)合位點(diǎn)。(3)根據(jù)BMP7近端啟動(dòng)子序列設(shè)計(jì)啟動(dòng)子系列缺失片段,將各個(gè)缺失片段構(gòu)建到雙熒光素酶報(bào)告基因載體上,轉(zhuǎn)染293T細(xì)胞進(jìn)行雙熒光酶檢測(cè)不同缺失片段活性并進(jìn)行顯著性比較,尋找雙熒光素酶活性最高的序列片段,發(fā)現(xiàn)核心區(qū)域-758bp—-545bp之間活性較高,對(duì)其進(jìn)行生物信息學(xué)預(yù)測(cè),發(fā)現(xiàn)存在轉(zhuǎn)錄因子SP1、EGR1可能結(jié)合位點(diǎn),對(duì)BMP7啟動(dòng)子轉(zhuǎn)錄調(diào)控起著一定作用。(4)為了進(jìn)一步鑒定BMP7近端啟動(dòng)子上游轉(zhuǎn)錄調(diào)控區(qū)域的轉(zhuǎn)錄因子結(jié)合位點(diǎn),根據(jù)生物信息學(xué)結(jié)果篩選出EGR1和SP1,對(duì)其轉(zhuǎn)錄因子結(jié)合位點(diǎn)進(jìn)行定點(diǎn)突變,同時(shí)構(gòu)建BMP7的轉(zhuǎn)錄因子EGR1進(jìn)行過(guò)表達(dá)載體進(jìn)一步驗(yàn)證。結(jié)果顯示,突變后的BMP7近端啟動(dòng)子上游調(diào)控區(qū)的活性呈顯著性降低;過(guò)表達(dá)EGR1轉(zhuǎn)錄因子,BMP7上游調(diào)控區(qū)活性呈顯著性上升,初步推斷EGR1與SP1對(duì)BMP7轉(zhuǎn)錄調(diào)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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S826
本文編號(hào)：2187084
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S826
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