本文選題：mIRF-3 + 啟動子�。� 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：克隆小鼠干擾素調(diào)節(jié)因子3（murine Interferon regulatory factor3，mIRF-3）基因啟動子并尋找其核心功能區(qū)域，鑒定與mIRF-3基因啟動子結(jié)合的轉(zhuǎn)錄因子，為闡明其表達(dá)調(diào)控機(jī)制奠定基礎(chǔ)。 方法：以小鼠NIH3T3細(xì)胞DNA為模板，采用PCR方法獲取mIRF-3基因5’側(cè)翼序列，測序正確后插入pGL3-Basic載體。運(yùn)用雙熒光素酶報告基因檢測系統(tǒng)鑒定其啟動子活性。采用缺失分析法分別從mIRF-3基因5’側(cè)翼的5’端逐段缺失，克隆得到不同長度的截短片段，插入pGL3-Basic載體，使用雙熒光素酶檢測系統(tǒng)定位mIRF-3基因啟動子核心功能區(qū)域，鑒定其啟動子活性。利用點(diǎn)突變技術(shù)、染色質(zhì)免疫沉淀（ChIP）和RNA干擾等實(shí)驗，分析mIRF-3核心啟動子區(qū)域的轉(zhuǎn)錄因子結(jié)合位點(diǎn)。 結(jié)果：核酸序列測定結(jié)果顯示，克隆到的mIRF-3基因5’側(cè)翼序列與Genbank中記錄完全一致，熒光素酶活性分析結(jié)果表明其在NIH3T3細(xì)胞中具有較強(qiáng)的啟動子活性。結(jié)果表明，自5’端缺失的截短片段其活性與pGL3-Basic相比，表現(xiàn)為先升高后降低，-301～+1bp截短片段表現(xiàn)出接近最高的啟動子活性，而-196～+1bp截短片段的活性基本消失，提示mIRF-3的核心啟動子區(qū)域位于-301～-196bp之間。并且-301/-196區(qū)域間可能存在核心調(diào)控元件。生物信息學(xué)預(yù)測該區(qū)域包含了Sp1、IK1、E2F1、C-MYB、Egr2和YY1等結(jié)合位點(diǎn)。點(diǎn)突變結(jié)果表明Egr2和YY1具有維持mRF-3的基本轉(zhuǎn)錄活性的作用。RNA干擾實(shí)驗表明Egr2和YY1增強(qiáng)了mIRF-3的啟動子活性。染色質(zhì)免疫沉淀(chromatin immunoprecitation，ChIP)實(shí)驗結(jié)果表明YY1轉(zhuǎn)錄因子與mIRF-3的啟動子區(qū)有結(jié)合。 結(jié)論：mIRF-3基因的核心啟動子區(qū)域位于轉(zhuǎn)錄起始位點(diǎn)上游-301與-196bp之間，Egr2和YY1在核心啟動子調(diào)控方面具有關(guān)鍵的調(diào)控作用，，且二者起正調(diào)控作用。
[Abstract]:Aim: to clone the promoter of mouse interferon regulatory factor 3(murine Interferon regulatory factor3 mIRF-3 and to identify the transcription factors binding to the promoter of mIRF-3 gene, so as to lay a foundation for elucidating the mechanism of its expression and regulation. Methods: the 5 'flanking sequence of mIRF-3 gene was obtained by PCR using DNA of mouse NIH3T3 cells as template, and then inserted into pGL3-Basic vector after correct sequencing. The promoter activity was identified by double luciferase reporter gene detection system. Different length truncated fragments were cloned from 5'flanking end of mIRF-3 gene by deletion analysis and inserted into pGL3-Basic vector. The core functional region of promoter of mIRF-3 gene was located by double luciferase detection system. Its promoter activity was identified. Point mutation technique, chromatin immunoprecipitation and RNA interference were used to analyze transcription factor binding sites in the core promoter of mIRF-3. Results: the results of nucleic acid sequencing showed that the 5'flanking sequence of the cloned mIRF-3 gene was identical with that recorded in Genbank, and luciferase activity analysis showed that it had strong promoter activity in NIH3T3 cells. The results showed that the activity of the truncated fragment from 5 '-terminal deletion was higher than that of pGL3-Basic, and the activity of the truncated fragment from -301 to 1bp showed the highest promoter activity, while the activity of the truncated fragment of -196~ 1bp was almost disappeared. It suggested that the core promoter region of mIRF-3 was between -301 and 196 BP. And there may be core regulatory elements between-301 /-196 regions. Bioinformatics predicted that the region contained binding sites such as Sp1CIK1E2F1, C-MYBNEgr2 and YY1. The results of point mutation showed that Egr2 and YY1 could maintain the basic transcriptional activity of mRF-3. RNAi experiments showed that Egr2 and YY1 enhanced the promoter activity of mIRF-3. Chromatin immunoprecipitation assay revealed that YY1 transcription factors bind to the promoter region of mIRF-3. Conclusion the core promoter region of the 1: mIRF-3 gene is located between -301 and -196bp upstream of the transcription initiation site. Egr2 and YY1 play a key role in the regulation of the core promoter, and both play a positive role in the regulation of the core promoter.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R346

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