本文選題：山羊 + PRNP基因　； 參考：《中國農(nóng)業(yè)科學(xué)》2016年10期

【摘要】：【目的】分析山羊PRNP基因啟動子活性區(qū)域,旨在篩選調(diào)節(jié)朊蛋白表達(dá)水平的關(guān)鍵區(qū)域或轉(zhuǎn)錄因子,為闡明山羊PRNP基因的表達(dá)調(diào)控提供理論依據(jù),并為從遺傳學(xué)角度降低朊蛋白病的發(fā)生提供思路;【方法】以山羊PRNP基因序列(Gen Bank登錄號:EU870890)為模板,設(shè)計特異性引物,擴(kuò)增山羊PRNP基因5′側(cè)翼區(qū)片段,并將擴(kuò)增片段克隆至p EASY-T3載體,鑒定為陽性的克隆進(jìn)行測序;利用生物信息學(xué)方法和在線工具進(jìn)行啟動子區(qū)域和轉(zhuǎn)錄因子結(jié)合位點的預(yù)測;利用缺失突變技術(shù)擴(kuò)增啟動子區(qū)不同長度的片段11個,并克隆至p EASY-T3載體后,鑒定為陽性的質(zhì)粒和pGL3-Basic載體分別用限制性內(nèi)切酶Mlu I和Bgl II進(jìn)行酶切,并回收酶切產(chǎn)物;利用T4連接酶進(jìn)行目的片段與pGL3-Basic連接,鑒定為陽性的熒光素酶報告基因重組質(zhì)粒進(jìn)行測序,并提取無內(nèi)毒素質(zhì)粒,用脂質(zhì)體轉(zhuǎn)染法瞬時轉(zhuǎn)染至SH-SY5Y細(xì)胞,轉(zhuǎn)染48h后,利用雙熒光素酶檢測試劑盒進(jìn)行各缺失突變重組質(zhì)粒在細(xì)胞內(nèi)的啟動活性檢測;【結(jié)果】成功克隆了山羊PRNP基因5′側(cè)翼區(qū)片段,長度為2 332 bp,且該片段含有預(yù)測的啟動子活性區(qū)域、保守的motifs和多個轉(zhuǎn)錄因子的結(jié)合位點;成功克隆了11個含有不同長度啟動子的片段,并與熒光素酶報告基因連接,并構(gòu)建了目的片段與熒光素酶報告基因的重組質(zhì)粒;轉(zhuǎn)染時脂質(zhì)體與DNA的比例為1㑳0.5,螢火蟲熒光素酶載體與海腎熒光素酶比例為50㑳1;山羊PRNP基因5′側(cè)翼區(qū)存在著核心啟動子,啟動子活性最強(qiáng)的區(qū)域為-519—+82 bp,且在-220—+59 bp這一區(qū)域存在著正調(diào)控元件,外顯子1對啟動子活性中起重要的調(diào)控作用;4個motifs可能為正調(diào)控元件結(jié)合位點;在強(qiáng)啟動子活性區(qū)存在10個Sp1結(jié)合位點,2個AP-2 alpha結(jié)合位點和1個AP-1結(jié)合位點;山羊PRNP基因motif 3和motif 4分別預(yù)測為轉(zhuǎn)錄因子Foxp3和COE1的結(jié)合位點。【結(jié)論】確定了山羊PRNP基因啟動子的核心區(qū)域(-519—+82bp),外顯子1對啟動子活性起重要的調(diào)控作用。
[Abstract]:[objective] to analyze the active region of goat PRNP gene promoter in order to screen the key regions or transcription factors regulating the expression of PRNP gene, and to provide theoretical basis for elucidating the regulation of goat PRNP gene expression. [methods] Goat PRNP gene 5'flanking region was amplified using goat PRNP gene sequence Gen Bank accession number: EU870890 as template, and specific primers were designed to amplify the 5'flanking region of goat PRNP gene. The amplified fragments were cloned into p EASY-T3 vector and identified as positive clones for sequencing. The promoter regions and transcription factor binding sites were predicted by bioinformatics and online tools. Eleven fragments of different length of promoter region were amplified by deletion mutation technique and cloned into p EASY-T3 vector. The positive plasmid and pGL3-Basic vector were digested with restriction endonuclease Mlu I and Bgl II, respectively, and the products were recovered. The target fragment was ligated with pGL3-Basic by T4 ligase. The recombinant plasmid of luciferase reporter gene identified as positive was sequenced, and the non-endotoxin plasmid was extracted. The recombinant plasmid was transiently transfected into SH-SY5Y cells by liposome transfection and transfected into SH-SY5Y cells for 48 h. Double luciferase assay kit was used to detect the priming activity of the deletion mutant recombinant plasmid. [results] the 5'flanking region of goat PRNP gene was cloned successfully. The length of the fragment was 2 332 BP, and the fragment contained predicted promoter active region, conserved motifs and binding sites of multiple transcription factors. 11 fragments with different length promoters were cloned and linked to luciferase reporter gene. The recombinant plasmid of the target fragment and luciferase reporter gene was constructed, the ratio of liposome to DNA was 1: 0. 5, the ratio of luciferase vector to luciferase was 50? 1, and there was a core promoter in 5 'flanking region of goat PRNP gene. The most active region of promoter is -519-82 BP, and there are positive regulatory elements in the region of -220-59 BP, exon 1 plays an important role in the activity of promoter, and four motifs may be positive regulatory element binding sites. There were 10 Sp1 binding sites, 2 AP-2 alpha binding sites and 1 AP-1 binding site in the active region of strong promoter. Goat PRNP gene motif 3 and motif 4 were predicted to be binding sites of transcription factor Foxp3 and COE1 respectively. [conclusion] the core region of goat PRNP gene promoter is identified, and exon 1 plays an important role in the regulation of promoter activity.
【作者單位】： 河北農(nóng)業(yè)大學(xué)動物科技學(xué)院;
【基金】：國家自然科學(xué)基金項目(31201775) 河北省首批青年拔尖人才支持計劃 河北農(nóng)業(yè)大學(xué)中青年骨干教師境外研修項目
【分類號】：S827

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