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

【摘要】：【目的】分析山羊PRNP基因啟動(dòng)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(hào):EU870890)為模板,設(shè)計(jì)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(yáng)性的克隆進(jìn)行測(cè)序;利用生物信息學(xué)方法和在線工具進(jìn)行啟動(dòng)子區(qū)域和轉(zhuǎn)錄因子結(jié)合位點(diǎn)的預(yù)測(cè);利用缺失突變技術(shù)擴(kuò)增啟動(dòng)子區(qū)不同長(zhǎng)度的片段11個(gè),并克隆至p EASY-T3載體后,鑒定為陽(yáng)性的質(zhì)粒和pGL3-Basic載體分別用限制性?xún)?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(yáng)性的熒光素酶報(bào)告基因重組質(zhì)粒進(jìn)行測(cè)序,并提取無(wú)內(nèi)毒素質(zhì)粒,用脂質(zhì)體轉(zhuǎn)染法瞬時(shí)轉(zhuǎn)染至SH-SY5Y細(xì)胞,轉(zhuǎn)染48h后,利用雙熒光素酶檢測(cè)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(dòng)活性檢測(cè);【結(jié)果】成功克隆了山羊PRNP基因5′側(cè)翼區(qū)片段,長(zhǎng)度為2 332 bp,且該片段含有預(yù)測(cè)的啟動(dòng)子活性區(qū)域、保守的motifs和多個(gè)轉(zhuǎn)錄因子的結(jié)合位點(diǎn);成功克隆了11個(gè)含有不同長(zhǎng)度啟動(dòng)子的片段,并與熒光素酶報(bào)告基因連接,并構(gòu)建了目的片段與熒光素酶報(bào)告基因的重組質(zhì)粒;轉(zhuǎn)染時(shí)脂質(zhì)體與DNA的比例為1㑳0.5,螢火蟲(chóng)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(dòng)子,啟動(dòng)子活性最強(qiáng)的區(qū)域?yàn)?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(duì)啟動(dòng)子活性中起重要的調(diào)控作用;4個(gè)motifs可能為正調(diào)控元件結(jié)合位點(diǎn);在強(qiáng)啟動(dòng)子活性區(qū)存在10個(gè)Sp1結(jié)合位點(diǎn),2個(gè)AP-2 alpha結(jié)合位點(diǎn)和1個(gè)AP-1結(jié)合位點(diǎn);山羊PRNP基因motif 3和motif 4分別預(yù)測(cè)為轉(zhuǎn)錄因子Foxp3和COE1的結(jié)合位點(diǎn)�！窘Y(jié)論】確定了山羊PRNP基因啟動(dòng)子的核心區(qū)域(-519—+82bp),外顯子1對(duì)啟動(dòng)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(dòng)物科技學(xué)院;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(31201775) 河北省首批青年拔尖人才支持計(jì)劃 河北農(nóng)業(yè)大學(xué)中青年骨干教師境外研修項(xiàng)目
【分類(lèi)號(hào)】：S827

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王洪梅;張利博;侯明海;王長(zhǎng)法;王玲玲;孫濤;何洪彬;仲躋峰;;牛Nramp1基因啟動(dòng)子的克隆及其活性分析[J];中國(guó)農(nóng)業(yè)科學(xué);2011年05期

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 周榮艷;魏彥輝;錫建中;李蘭會(huì);陳輝;高立杰;張振紅;;山羊PRNP基因啟動(dòng)子轉(zhuǎn)錄活性研究[J];中國(guó)農(nóng)業(yè)科學(xué);2016年10期

2 張俊珍;劉_g;姬凱元;楊?yuàn)檴?胡帥鵬;劉學(xué)賢;范瑞文;;小鼠角蛋白10(K10)基因啟動(dòng)子的克隆及活性分析[J];中國(guó)農(nóng)業(yè)科學(xué);2016年09期

3 馬曉菁;易新萍;谷文喜;葉鋒;鐘旗;;新疆3個(gè)不同品種牛SLC11A1基因啟動(dòng)子克隆及序列分析[J];新疆農(nóng)業(yè)科學(xué);2015年11期

4 胡倩;曹允考;佟慧麗;張偉偉;趙丹丹;王彬;高朋;嚴(yán)云勤;;牛骨骼肌α-actin基因5′上游調(diào)控元件及內(nèi)含子功能的初步分析[J];中國(guó)獸醫(yī)學(xué)報(bào);2015年07期

5 韋光輝;李東;左其生;張亞妮;朱睿;張蕾;劉志永;邱峰龍;李碧春;;徐淮山羊Oct4啟動(dòng)子功能的初步分析[J];遺傳;2014年08期

6 韋光輝;左其生;李東;張亞妮;劉志永;朱睿;邱峰龍;李碧春;;徐淮山羊c-Myc基因啟動(dòng)子的克隆及其功能的初步分析[J];畜牧獸醫(yī)學(xué)報(bào);2014年04期

7 張遠(yuǎn);楊旬旬;吳風(fēng)瑞;劉勇;丁彪;王彩紅;黃繼昌;李文雍;;小鼠Dnmt1啟動(dòng)子熒光素酶報(bào)告基因的構(gòu)建及活性分析[J];生物技術(shù)通報(bào);2013年10期

8 顧克翠;王歡歡;謝周瑞;徐銀學(xué);陳杰;;豬Nramp1基因啟動(dòng)子克隆及其組織表達(dá)活性分析[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2013年02期

9 趙曼;陳寧博;杜芳;馬云;;家畜基因啟動(dòng)子調(diào)控功能的研究進(jìn)展[J];家畜生態(tài)學(xué)報(bào);2012年06期

10 徐琪;陳陽(yáng);黃正洋;張揚(yáng);陳昌義;趙榮雪;李秀;段修軍;陳國(guó)宏;;鴨CD8α基因啟動(dòng)子的分析及其轉(zhuǎn)錄活性[J];中國(guó)農(nóng)業(yè)科學(xué);2012年12期

【二級(jí)參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 張利博;王洪梅;王長(zhǎng)法;李秋玲;黃金明;仲躋峰;劉松財(cái);;牛Nramp1基因5′調(diào)控區(qū)序列的克隆及序列分析[J];中國(guó)獸醫(yī)學(xué)報(bào);2009年09期

2 胡海川;王洪梅;李建斌;王長(zhǎng)法;賴(lài)松家;李秋玲;仲躋峰;;荷斯坦牛Nramp1基因遺傳多態(tài)性及其與乳房炎相關(guān)性的研究[J];遺傳;2009年01期

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