發(fā)布時間：2018-12-25 15:33

【摘要】：目的鑒定人不育-α-基序結(jié)構(gòu)域和組氨酸/天冬氨酸殘基雙聯(lián)體結(jié)構(gòu)域包涵蛋白1(SAMHD1)基因的核心啟動子區(qū)域,研究SAMHD1的轉(zhuǎn)錄調(diào)控機制。方法從Hep G2細胞中提取基因組DNA,PCR擴增SAMHD1基因翻譯起始位點上游937、667、553、399 bp片段。將擴增出的4個片段連入p MD19-T載體,雙酶切鑒定后將DNA條帶切膠回收,再連入p GL3-Basic載體中,雙酶切及DNA測序鑒定。將包含有4個片段的熒光素酶表達載體與pRL-TK共轉(zhuǎn)染Hep G2細胞,熒光素酶報告基因活性檢測并分析4個片段的啟動子活性。5'RACE鑒定SAMHD1在Hep G2細胞中的轉(zhuǎn)錄起始位點。結(jié)果電泳結(jié)果顯示已經(jīng)從Hep G細胞中提取出基因組DNA。PCR擴增后電泳結(jié)果顯示已經(jīng)擴增出937、667、553、399 bp片段。雙酶切后電泳結(jié)果顯示成功將擴增出的4個片段連入p MD19-T載體。雙酶切及DNA測序鑒定已成功構(gòu)建熒光素酶表達載體p GL3-937、p GL3-667、p GL3-553和p GL3-399。熒光素酶報告基因活性檢測顯示SAMHD1核心啟動子區(qū)域位于0~-399區(qū)域(ATG前一個堿基為-1)。5'RACE結(jié)果顯示SAMHD1在Hep G2細胞中轉(zhuǎn)錄起始位點位于-101。結(jié)論 SAMHD1的核心啟動子位于翻譯起始位點上游-101~-399區(qū)域,為深入研究肝細胞中SAMHD1轉(zhuǎn)錄調(diào)控機制奠定了基礎(chǔ)。
[Abstract]:Objective to identify the core promoter region of human infertility 偽 -motif domain and histidine / aspartic acid residue double conjoined domain (SAMHD1) gene, and to study the transcriptional regulation mechanism of SAMHD1. Methods Genomic DNA,PCR was extracted from Hep G2 cells to amplify the 937667553399 bp fragment upstream of the translation initiation site of SAMHD1 gene. The four amplified fragments were inserted into the p MD19-T vector. The DNA bands were identified by double enzyme digestion and then recovered into the p GL3-Basic vector. The results were confirmed by double enzyme digestion and DNA sequencing. The luciferase expression vector containing four fragments was cotransfected with pRL-TK into Hep G2 cells. The promoter activity of luciferase reporter gene was detected and analyzed. The transcription initiation site of SAMHD1 in Hep G2 cells was identified by 5'RACE. Results the genomic DNA.PCR was extracted from Hep G cells and then amplified. The results showed that 937667553399 bp fragment had been amplified. The results of double enzyme digestion electrophoresis showed that the four amplified fragments were successfully inserted into p MD19-T vector. Construction of luciferase expression vectors p GL3-937,p GL3-667,p GL3-553 and p GL3-399. by double enzyme digestion and DNA sequencing The detection of luciferase reporter gene activity showed that the core promoter of SAMHD1 was located in the 0- 399 region (the previous base of ATG was -1). The 5'RACE results showed that the transcription initiation site of SAMHD1 in Hep G2 cells was -101. Conclusion the core promoter of SAMHD1 is located in the region of -101 ~ 399 upstream of the translation initiation site, which lays a foundation for further study on the mechanism of transcriptional regulation of SAMHD1 in hepatocytes.
【作者單位】： 安徽醫(yī)科大學免疫學教研室;安徽醫(yī)科大學生物藥物研究所;
【基金】：國家自然科學基金(編號:81372576) 安徽省自然科學基金(編號:1508085MH158) 安徽省博士后研究人員科研活動經(jīng)費資助(編號:2015B066)
【分類號】：Q78

【相似文獻】

相關(guān)期刊論文 前9條

1 高繼平，酈永忠，，王宗陽，洪孟民;秈稻232蠟質(zhì)基因轉(zhuǎn)錄起始位點的鑒定[J];遺傳學報;1995年06期

2 杜耀華;王正志;倪青山;;基于滑動窗口的原核轉(zhuǎn)錄起始位點計算定位方法[J];生物物理學報;2006年05期

3 李志英;牟紅珍;高丁梅;丁國平;馬婷;王盛;;本氏煙Ⅰ型啟動子的克隆及其轉(zhuǎn)錄起始位點分析[J];中國生物工程雜志;2014年01期

4 王智;轉(zhuǎn)錄起始位點的測定[J];基礎(chǔ)醫(yī)學與臨床;1990年04期

5 李杰;張義全;高鶴;王麗;胡小許;周冬生;;利用優(yōu)化的5′-RACE實驗定位副溶血弧菌基因的轉(zhuǎn)錄起始位點[J];生物技術(shù)通訊;2014年03期

6 孫亮先;謝憲兵;桑慶亮;陳懷宇;陳朝陽;黃周英;;用5’LongSAGE標簽分析蜜蜂Yps基因轉(zhuǎn)錄起始位點的多樣性[J];泉州師范學院學報;2009年02期

7 莊海濱;朱景德;劉湘軍;;人類全基因組范圍的CpG島的預(yù)測與分析[J];生物物理學報;2006年05期

8 孫亮先;黃周英;鄭華軍;游燕琳;;西方蜜蜂表皮蛋白基因apd-like轉(zhuǎn)錄起始位點的定位及cDNA序列的分析[J];昆蟲學報;2011年02期

9 馮麗;任茂智;羅洪發(fā);何光華;;分離植物目的基因全長cDNA和啟動子的新方法——快速定位轉(zhuǎn)錄起始位點(RITIS)(英文)[J];分子植物育種;2006年01期

相關(guān)會議論文 前1條

1 孟繁疆;;從TSSP-TCM數(shù)據(jù)文件中提取轉(zhuǎn)錄起始位點[A];2007年中國農(nóng)業(yè)工程學會學術(shù)年會論文摘要集[C];2007年

相關(guān)碩士學位論文 前3條

1 王麗雪;TNFα影響有機陰離子轉(zhuǎn)運多肽1A2表達水平的分子機理研究[D];華南農(nóng)業(yè)大學;2016年

2 侯婧逸;裂殖酵母全基因組范圍內(nèi)轉(zhuǎn)錄起始位點研究[D];上海交通大學;2012年

3 周艾彬;BMPRⅡ基因轉(zhuǎn)錄起始位點的確定及其近側(cè)調(diào)控元件的鑒定[D];暨南大學;2007年

本文編號：2391305