本文選題：河南華溪蟹　切入點(diǎn)：vasa基因　出處：《山西大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：vasa基因編碼一種ATP依賴的RNA解旋酶,屬于DEAD-box家族,最先在果蠅(Drosophila)中被發(fā)現(xiàn)。vasa基因在生殖細(xì)胞中特異表達(dá),在動(dòng)物生殖細(xì)胞發(fā)生和生殖調(diào)控中發(fā)揮著重要作用,被用作生殖細(xì)胞的分子標(biāo)記物,主要用于配子發(fā)生和原生殖細(xì)胞(primordial germ cells,PGCs)的起源、遷移和分化等方面的研究。本文通過(guò)RACE技術(shù)克隆得到了河南華溪蟹(Sinopotamon henanense)vasa基因cDNA序列,證實(shí)了vasa基因僅在生殖細(xì)胞中特異表達(dá);構(gòu)建了河南華溪蟹VASA蛋白原核表達(dá)載體Vexp-28a、Vexp-32a、Ve-28a及Ve-32a,并在大腸桿菌BL21(DE3)中進(jìn)行了原核表達(dá)。本文克隆得到的河南華溪蟹vasa基因cDNA序列長(zhǎng)2896bp,其中編碼區(qū)長(zhǎng)1329bp,編碼442個(gè)氨基酸的蛋白。Blast比對(duì)結(jié)果表明由該cDNA序列推測(cè)翻譯的蛋白氨基酸序列與魚(yú)類、兩棲類、鳥(niǎo)類、哺乳類以及一些無(wú)脊椎動(dòng)物的VASA蛋白氨基酸序列有較高的相似性;結(jié)構(gòu)域分析也表明該氨基酸序列具有DEAD-box家族共有的4個(gè)保守motif(motifⅠ、motifⅠa、motifⅠb、motifⅡ)、一個(gè)鋅指結(jié)構(gòu)和GG重復(fù)序列,還具有VASA亞家族基因?qū)Ｒ恍蕴卣鹘Y(jié)構(gòu),如Q-motif、RGG重復(fù)序列和甘氨酸富集區(qū),與DEAD-box家族蛋白具有的8個(gè)保守結(jié)構(gòu)域motifⅠ、motifⅠa、motifⅠb、motifⅡ、motifⅢ、motifⅣ、motifⅤ、motifⅥ以及兩個(gè)鋅指結(jié)構(gòu)、一個(gè)Q-motif,GG重復(fù)的結(jié)構(gòu)相比,本文獲得的蛋白氨基酸序列在motifⅡ之前(包括motifⅡ)的序列是正確的,但在motifⅡ之后的序列中,由于一些原因使蛋白翻譯提前終止,因此,我們所得到的Sh-vasa基因cDNA序列的3'端還待進(jìn)一步驗(yàn)證;多序列比對(duì)和系統(tǒng)進(jìn)化分析顯示該cDNA序列編碼的氨基酸序列與其它蝦蟹類的VASA蛋白進(jìn)化關(guān)系最近。以上結(jié)果均表明,本文所獲得的cDNA序列確定為河南華溪蟹vasa基因5'端及部分編碼區(qū)cDNA序列。通過(guò)半定量PCR(Sq-PCR)監(jiān)測(cè)河南華溪蟹vasa基因在不同組織中的表達(dá)情況,結(jié)果顯示,vasa基因在河南華溪蟹體內(nèi)僅在生殖細(xì)胞中特異表達(dá)。以得到的河南華溪蟹vasa基因cDNA序列為基礎(chǔ),設(shè)計(jì)兩對(duì)引物,通過(guò)PCR,在選定的vasa基因片段兩端加上EcoRⅠ和XhoⅠ酶切位點(diǎn),然后利用基因重組技術(shù)將獲得的vasa基因表達(dá)片段分別克隆至pET-28a、pET-32a原核表達(dá)載體,經(jīng)酶切鑒定和測(cè)序,成功構(gòu)建河南華溪蟹VASA蛋白原核表達(dá)載體Vexp-28a、Vexp-32a、Ve-28a及Ve-32a,并通過(guò)IPTG誘導(dǎo)進(jìn)行原核表達(dá),蛋白上清和包涵體沉淀分別經(jīng)過(guò)His·Tag親和層析柱分離、純化,通過(guò)SDS-PAGE分析以及Western blot鑒定,所得蛋白并非河南華溪蟹VASA融合蛋白,需在河南華溪蟹vasa基因cDNA序列內(nèi)重新選取表達(dá)片段,或重新選擇表達(dá)載體,改進(jìn)實(shí)驗(yàn)方案,再次實(shí)驗(yàn),以期獲得河南華溪蟹VASA融合蛋白。
[Abstract]:Vasa gene encodes a ATP dependent RNA helicase, belonging to the DEAD-box family. It was first found in Drosophila melanogaster that the RNA ASA gene is specifically expressed in germ cells and plays an important role in the genesis and reproductive regulation of animal germ cells. It is used as a molecular marker of germ cells, mainly used in the study of gametogenesis and the origin, migration and differentiation of primordial germ cells. The cDNA sequence of Sinopotamon henanense)vasa gene was cloned by RACE technique. It was confirmed that vasa gene was expressed only in germ cells. The prokaryotic expression vectors of Vexp-28a- Vexp-32aHe-28a and Ve-32a were constructed and expressed in E. coli BL21DE3. The cDNA sequence of vasa gene of Huaxi crab in Henan Province was 2896bp, in which the coding region was 1329bp, encoding 442 amino acids. The results of protein. Blast alignment showed that the translated amino acid sequence of protein and fish were deduced from the cDNA sequence. The amino acid sequences of VASA protein in amphibians, birds, mammals and some invertebrates are similar. Domain analysis also showed that the amino acid sequence had four conserved motif(motif 鈪，

本文編號(hào)：1581685