本文關(guān)鍵詞：SLC22A3基因負(fù)性調(diào)控元件的定位　出處：《重慶醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 冠心病 SLC22A3基因 負(fù)性調(diào)控元件 熒光素酶活性

【摘要】：目的擬尋找人第6號染色體q26-q27區(qū)域的SLC22A3基因intron7序列中具有重要負(fù)性調(diào)控功能的核心調(diào)控元件的具體序列位置。方法用基因重組的方法,分別構(gòu)建intron7截短型野生重組質(zhì)粒,分段查找該負(fù)性調(diào)控元件的具體序列位置。以包含人SLC22A3基因全長序列的細(xì)菌人工染色體為模板,PCR擴(kuò)增所需截短型片段,分別插入熒光素酶報告基因載體pGL3-Promoter,將重組質(zhì)粒與內(nèi)參質(zhì)粒pRL-SV40以50:1的比例共轉(zhuǎn)染HEK293T細(xì)胞,24h后檢測熒光素酶活性(Luciferase activity),通過對比重組質(zhì)粒和pGL3-Promoter熒光素酶活性,判斷插入片段是否具有調(diào)控作用,隨后通過實時定量PCR對Luciferase基因mRNA水平進(jìn)行檢測,并且驗證其調(diào)控元件作用與其位置的相關(guān)性,最后對發(fā)現(xiàn)元件進(jìn)行轉(zhuǎn)錄因子結(jié)合位點預(yù)測。結(jié)果截短型野生重組質(zhì)粒pGL3-pro-SLCi7-NO6,pGL3-pro-SLCi7-NO10,pGL3-pro-SLCi7-NO6.2,p GL3-pro-SLCi7-NO6.3,p-GL3-pro-SLCi7-NO10.2,pGL3-pro-SLCi7-NO6.21,p GL3-pro-SLCi7-NO10.22各組熒光素酶活性較pGL3-Promoter組降低(P0.05);而截短型野生重組質(zhì)粒pGL3-pro-SLCi7-NO6.21-300 bp(P0.05)和p GL3-pro-SLCi7-NO10.22-300 bp(P0.05)組熒光素酶活性較pGL3-Promoter組無統(tǒng)計學(xué)差異,實時定量PCR驗證mRNA水平與蛋白表達(dá)水平相一致,且調(diào)控元件發(fā)揮作用與其所處位置無相關(guān)。同時,通過生物軟件預(yù)測發(fā)現(xiàn)與負(fù)性調(diào)控元件結(jié)合的四個轉(zhuǎn)錄因子。結(jié)論人類第6號染色體q26-q27區(qū)域的SLC22A3基因intron7序列中存在2個負(fù)性調(diào)控元件,為今后進(jìn)一步對SLC22A3基因表達(dá)調(diào)控方面的研究提供了新的理論依據(jù)。
[Abstract]:Objective to search for the specific position of the core regulatory elements in the intron7 sequence of the SLC22A3 gene in the q26-q27 region of human chromosome 6, which have important negative regulatory functions. The method of restructuring. Intron7 truncated wild recombinant plasmids were constructed, and the specific sequence location of the negative regulatory element was found. The artificial chromosome of bacteria containing the full-length sequence of human SLC22A3 gene was used as template. The truncated PCR fragments were inserted into the luciferase reporter gene vector pGL3-Promoter respectively. The recombinant plasmid pRL-SV40 was co-transfected into HEK293T cells at the ratio of 50: 1. Luciferase activity and luciferase activity of luciferase activity were detected 24 hours later. The recombinant plasmid and pGL3-Promoter luciferase activity were compared. Then the mRNA level of Luciferase gene was detected by real-time quantitative PCR, and the correlation between the role of its regulatory element and its position was verified. Finally, transcription factor binding sites were predicted. Results truncated wild recombinant plasmid pGL3-pro-SLCi7-NO6 + pGL3-pro-SLCi7-NO10. PGL3-pro-SLCi7-NO6.2pGL3-pro-SLCi7-NO6.3pGL3-pro-SLCi7-NO10.2. PGL3-pro-SLCi7-NO6.21. The luciferase activity of P GL3-pro-SLCi7-NO10.22 group was lower than that of pGL3-Promoter group. The truncated wild recombinant plasmid pGL3-pro-SLCi7-NO6.21-300 bpn. P0. 05). There was no significant difference in luciferase activity between the two groups compared with the pGL3-Promoter group. Real-time quantitative PCR was used to verify that the level of mRNA was consistent with the level of protein expression, and the role of regulatory elements was not related to their position. Four transcription factors combined with negative regulatory elements were found by biosoftware prediction. Conclusion there are two SLC22A3 gene intron7 sequences in the q26-q27 region of human chromosome 6. Negative regulatory elements. It provides a new theoretical basis for further research on the regulation of SLC22A3 gene expression.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R541.4

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1 黃博;SLC22A3基因負(fù)性調(diào)控元件的定位[D];重慶醫(yī)科大學(xué);2016年

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