本文關(guān)鍵詞： PMEL基因 啟動(dòng)子 瞬時(shí)表達(dá) 點(diǎn)突變 轉(zhuǎn)錄調(diào)控元件　出處：《畜牧獸醫(yī)學(xué)報(bào)》2017年05期 　論文類型：期刊論文

【摘要】：旨在篩選調(diào)控山羊毛色基因PMEL的啟動(dòng)子活性區(qū)域及轉(zhuǎn)錄因子,為探究該基因的表達(dá)調(diào)控機(jī)制提供理論依據(jù),并為彩色山羊的育種和改良提供思路。以山羊基因組DNA為模板,PCR擴(kuò)增PMEL基因不同長(zhǎng)度的啟動(dòng)子缺失片段,定向克隆至pGL3-basic載體,將重組質(zhì)粒轉(zhuǎn)染到293T和A375細(xì)胞,通過(guò)雙熒光素酶檢測(cè)系統(tǒng)測(cè)定相對(duì)熒光素酶活性值;利用生物信息學(xué)方法對(duì)PMEL基因核心啟動(dòng)子區(qū)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)進(jìn)行預(yù)測(cè),隨后利用重疊延伸PCR分別對(duì)pGL3-327質(zhì)粒上預(yù)測(cè)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)進(jìn)行點(diǎn)突變并構(gòu)建突變載體,利用雙熒光素酶檢測(cè)系統(tǒng)進(jìn)行活性驗(yàn)證。結(jié)果顯示,本研究成功構(gòu)建了7個(gè)不同長(zhǎng)度的啟動(dòng)子片段,其中6個(gè)片段具有明顯的啟動(dòng)子活性。經(jīng)過(guò)雙熒光素酶活性檢測(cè)發(fā)現(xiàn)山羊PMEL基因-251/+76區(qū)域?yàn)楹诵膯?dòng)子區(qū)域。通過(guò)不同長(zhǎng)度的啟動(dòng)子片段的活性比較發(fā)現(xiàn),-251/-62區(qū)域的缺失造成啟動(dòng)子活性從最高到消失,表明該區(qū)域?qū)ι窖騊MEL基因轉(zhuǎn)錄調(diào)控有重要影響,生物信息學(xué)分析發(fā)現(xiàn)該區(qū)域存在5個(gè)轉(zhuǎn)錄因子結(jié)合位點(diǎn),利用點(diǎn)突變構(gòu)建了5個(gè)突變載體,經(jīng)過(guò)雙熒光素酶檢測(cè)發(fā)現(xiàn)5個(gè)突變載體的活性均顯著下降。提示這5個(gè)轉(zhuǎn)錄因子是山羊PMEL基因轉(zhuǎn)錄的正調(diào)控元件。本研究確定了山羊PMEL基因啟動(dòng)子核心區(qū)域?yàn)?251/+76,NF-1(-206/-197)、Sp1(-186/-174)、Sp1(-151/-139)、CREB(-91/-82)和Sp1(-82/-71)結(jié)合位點(diǎn)為山羊PMEL基因轉(zhuǎn)錄的正調(diào)控元件。
[Abstract]:The aim of this study was to screen the promoter active regions and transcription factors of goat coat color gene PMEL, and to provide theoretical basis for exploring the mechanism of expression and regulation of goat coat color gene. The genomic DNA of goat was used as template to amplify the promoter deletion fragments of different length of PMEL gene, and then cloned into pGL3-basic vector. The recombinant plasmid was transfected into 293T and A375 cells, and the recombinant plasmid was transfected into 293T and A375 cells. The relative luciferase activity was measured by double luciferase detection system, and the transcription factor binding sites in the core promoter of PMEL gene were predicted by bioinformatics. Then the point mutation of the predicted transcription factor binding site on the pGL3-327 plasmid was carried out by overlapping extended PCR and the mutant vector was constructed. The activity was verified by double luciferase detection system. In this study, seven promoter fragments of different lengths were successfully constructed. Six of the fragments showed significant promoter activity. The goat PMEL gene -251 / 76 region was found to be the core promoter region by double luciferase activity test, and the activity of different length promoter fragments was found to be -251 / -62 region. The loss of promoter activity caused the promoter activity to peak to disappear, The results indicated that this region had an important effect on the transcriptional regulation of goat PMEL gene. Bioinformatics analysis showed that there were five transcription factor binding sites in the region and five mutants were constructed by point mutation. After double luciferase detection, it was found that the activity of the five mutant vectors was significantly decreased, which suggested that the five transcription factors were positive regulatory elements of goat PMEL gene transcription. The core region of goat PMEL gene promoter was -251 / 2. The binding sites of 76-NF-1T -206 / -197 / Sp1C1-186-174C / Sp1-151- 151- 139C ~ (-82) and Sp1+ -82 / -71) are positive regulatory elements for the transcription of goat PMEL gene.
【作者單位】： 河北科技師范學(xué)院;河北農(nóng)業(yè)大學(xué);
【基金】：河北省高校創(chuàng)新團(tuán)隊(duì)領(lǐng)軍人才培育計(jì)劃項(xiàng)目(LJRC004) 河北省應(yīng)用基礎(chǔ)研究計(jì)劃重點(diǎn)基礎(chǔ)研究項(xiàng)目(15962901D) 河北省高等學(xué)校青年拔尖人才計(jì)劃項(xiàng)目(BJ2016026) 秦皇島市科技支撐計(jì)劃項(xiàng)目(201502A058)
【分類號(hào)】：Q78;S827

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