本文選題：熱敏感　切入點(diǎn)：生精作用　出處：《大連醫(yī)科大學(xué)》2016年碩士論文

【摘要】：Esx1(Extra-embryonic tissue-spermatogenesis-homeobox gene 1)系一X性聯(lián)同源箱(X-linked homeobox)蛋白基因,具有三個(gè)mRNA的異構(gòu)型,分別是a型、b型及x型。序列比對發(fā)現(xiàn),彼此差異主要在5'端的非轉(zhuǎn)譯區(qū)(5'-untranslated regions)與部分下游序列。這些不同異構(gòu)型的表現(xiàn)明顯地是受到不同的啟動(dòng)子區(qū)域所調(diào)節(jié),而此調(diào)節(jié)又高度地對熱敏感。為了了解Esx1的組織特異性表現(xiàn),分別抽取小鼠wj丸及胎盤的總RNA進(jìn)行定量反轉(zhuǎn)錄聚合酶連鎖反應(yīng)(quantitative RT-PCR analyses)。結(jié)果顯示在wj丸中主要表現(xiàn)a型及x型mRNA,而胎盤中主要表現(xiàn)x型。為了解存在于啟動(dòng)子區(qū)域(promoter region)內(nèi)的近端作用因子(cis-acting elements),首先透過短暫性轉(zhuǎn)染(transient transfection)分析小鼠Esx1基因的整個(gè)5'端鄰近區(qū)域(5'-flanking sequences)(核苷酸序列從-1493到+464),其后再利用泳動(dòng)遲滯試驗(yàn)(electrophoretic mobility shift assay,EMSA)及DNase I足跡試驗(yàn)(DNase I footprinting analysis,DFA)作佐證。報(bào)導(dǎo)基因分析結(jié)果顯示核苷酸序列-1015到-459可在分離的初級精母細(xì)胞(primary spermatocytes)及圓形精細(xì)胞(round spermatids)內(nèi)驅(qū)動(dòng)下游的熒光酵素基因大量表現(xiàn);核苷酸序列從+14到+189可在分離的精原細(xì)胞(spermatogonia)內(nèi)啟動(dòng)報(bào)導(dǎo)基因的基礎(chǔ)表現(xiàn)。DFA及EMSA則進(jìn)一步地證實(shí)序列-901到-882能夠?qū)Ｒ坏嘏cwj丸及圓形精細(xì)胞總核蛋白萃取液結(jié)合,唯與胎盤、腦及肝臟總核蛋白質(zhì)萃取液則否。利用TESS(transcription element search system)數(shù)據(jù)庫比對此20個(gè)鹼基發(fā)現(xiàn),CACCC結(jié)合蛋白可能為調(diào)控wj丸中Esx1基因選擇性轉(zhuǎn)錄的重要轉(zhuǎn)錄因子之一。刪除此20 bp序列可使啟動(dòng)子活性降低23%。在未來,則可利用RNA干擾技術(shù)(RNA interference)進(jìn)一步地分析CACCC結(jié)合蛋白在Esx1表現(xiàn)時(shí)的角色。
[Abstract]:Esx1(Extra-embryonic tissue-spermatogenesis-homeobox gene 1 is a X-linked homeobox) protein gene with three mRNA isomorphisms, type a and type x, respectively.Sequence alignment showed that the differences were mainly in the 5'-terminal non-translated regions (5'-untranslated regions) and some downstream sequences.The performance of these different isomerism is obviously regulated by different promoter regions and this regulation is highly sensitive to heat.In order to understand the tissue specificity of Esx1, the total RNA of mouse WJ pill and placenta were extracted for quantitative reverse transcription polymerase chain reaction (QRT) and quantitative RT-PCR analysis.The results showed that type a and type x mRNAs were mainly present in WJ pills, while those in placenta were mainly of type x.In order to understand the proximal action factor cis-acting elements in promoter region, the whole 5'terminal adjacent region of mouse Esx1 gene was analyzed by transient transfection.Then the nucleotide sequence ranged from -1493 to 464N.Electrophoretic mobility shift assayma (EMSA) and DNase I footprint test (DNase I footprinting analysis) were used as evidence.The results of gene analysis showed that nucleotide sequences -1015 to -459 could drive a large number of fluorescein genes downstream in isolated primary spermatocytes (primary spermatocytes) and round spermatocytes.Nucleotide sequences ranging from 14 to 189 could initiate the basic expression of the reporter gene in isolated spermatogonia. DFA and EMSA further confirmed that sequences -901 to -882 could specifically bind to WJ pill and circular spermatocyte total nuclear protein extract, but only to placenta.Brain and liver total nuclear protein extracts were not.Using the TESS(transcription element search system database, it was found that the CACCC-binding protein might be one of the important transcription factors regulating the selective transcription of Esx1 gene in WJ pills.Deletion of the 20 BP sequence reduced the promoter activity by 23%.In the future, RNA interference technique can be used to further analyze the role of CACCC binding proteins in the expression of Esx1.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R339.21

【相似文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王文彬;;絕育手術(shù)[J];科學(xué)大眾;1958年02期

2 ;[J];;年期

相關(guān)會(huì)議論文 前1條

1 _5珉;付秋林;，

本文編號：1720100