本文關(guān)鍵詞： 大鱗副泥鰍 尾鰭再生 Gig2基因 Frr2基因 原位雜交　出處：《河南師范大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：發(fā)育成熟的個(gè)體在受損后可以在原有組織基礎(chǔ)上重建已丟失的組織,這種現(xiàn)象稱為再生。所有生物體對(duì)組織損傷會(huì)做出相應(yīng)的生理反應(yīng),同時(shí)與疾病的發(fā)生又密切相關(guān),因此研究再生的過(guò)程和機(jī)制具有重要的意義。哺乳類動(dòng)物再生能力非常有限,而以蠑螈為代表的有尾兩棲類和以斑馬魚為代表的硬骨魚能夠在附肢及魚鰭截?cái)嗪笸ㄟ^(guò)割處再生完成結(jié)構(gòu)和功能的重建。但兩棲類繁殖速度慢,實(shí)驗(yàn)室不易飼養(yǎng),且缺乏相應(yīng)的遺傳操作技術(shù),不能成為理想的研究動(dòng)物。魚鰭在切斷后卻能夠完整的恢復(fù)原有結(jié)構(gòu),并且相對(duì)簡(jiǎn)單,易獲得,損傷后不危害機(jī)體生命,更具意義的是硬骨魚魚鰭與人類在內(nèi)的四足動(dòng)物肢體早期發(fā)育極其相似,但二者再生能力卻大不相同,因此魚鰭可以作為解析分子再生機(jī)制的良好體系。本研究先是從已經(jīng)建立的抑制性消減雜交文庫(kù)中篩選得到兩個(gè)魚鰭再生差異表達(dá)明顯很大的ESTs,在這兩個(gè)ESTs的基礎(chǔ)上,采用RACE的方法進(jìn)行擴(kuò)增,得到兩個(gè)cDNA全長(zhǎng)序列。通過(guò)Blast比對(duì)后,其中一個(gè)基因認(rèn)為是Gig2基因,另一個(gè)Blast結(jié)果并未發(fā)現(xiàn)有與高度同源的,認(rèn)為是一個(gè)新基因,將其命名為Frr2基因。隨后利用DNAMAN,MEGA6.0,sigmaplot等軟件對(duì)這兩個(gè)基因進(jìn)行生物學(xué)分析,還測(cè)定了這兩個(gè)基因在不同組織中的表達(dá)。Gig2基因是草魚出血性病毒誘導(dǎo)的基因2,它首先是從紫外失活的GCHV處理的鯽魚囊胚細(xì)胞中鑒定獲得的一個(gè)新基因。它在魚類干擾素抗病毒反應(yīng)中起著重要作用,而在再生中能夠調(diào)節(jié)再生則可能是一個(gè)新發(fā)現(xiàn)。它c(diǎn)DNA全長(zhǎng)是712bp,共編碼156個(gè)氨基酸,相對(duì)分子質(zhì)量為17.324KDa,等電點(diǎn)(pI)8.69。46個(gè)極性氨基酸,71個(gè)非極性疏水氨基酸,18個(gè)酸性氨基酸,21個(gè)堿性氨基酸。蛋白質(zhì)二級(jí)結(jié)構(gòu)發(fā)現(xiàn)α-螺旋占23.72%,延伸鏈占27.56%,β-轉(zhuǎn)角占16.03%,無(wú)規(guī)卷曲占32.69%。通過(guò)半定量以及熒光定量檢測(cè)發(fā)現(xiàn):在再生后4d表達(dá)達(dá)到最高水平,在胚胎發(fā)育時(shí)期的出膜期表達(dá)量最高,還發(fā)現(xiàn)在精巢組織表達(dá)也是最大的。根據(jù)Gig2的表達(dá)模式我們首次發(fā)現(xiàn)了該基因參與到了魚鰭再生的過(guò)程,它可能在魚鰭結(jié)構(gòu)形成過(guò)程中起著調(diào)控作用。同源度結(jié)果分析發(fā)現(xiàn)Gig2基因與其他物種的同源度相對(duì)并不是很大,這也使得研究其進(jìn)化關(guān)系引起很大興趣。Frr2(fin regeneration related gene2)基因是一個(gè)未知基因,它是一個(gè)與魚鰭再生相關(guān)的一個(gè)基因。cDNA全長(zhǎng)為1036bp,編碼了有88個(gè)氨基酸,二級(jí)結(jié)構(gòu)發(fā)現(xiàn)其中α螺旋占46.59%,β-轉(zhuǎn)角占9.09%,無(wú)規(guī)卷曲占32.95%,延伸鏈占11.36%。半定量以及熒光定量檢測(cè)發(fā)現(xiàn)在再生不同時(shí)期(0dpa,2dpa,3dpa,4dpa,5dpa,6dpa)的第四天表達(dá)最高,在不同組織(肝、心、腦、精巢、腎、卵巢)中心臟表達(dá)水平較高,胚胎不同發(fā)育時(shí)期(囊胚期,原腸胚,神經(jīng)胚,尾芽期,出膜期)原腸胚期的基因表達(dá)水平最高。冰凍切片把處于再生不同時(shí)期的尾鰭進(jìn)行原位雜交。原位雜交結(jié)果顯示:發(fā)現(xiàn)在魚鰭內(nèi)部成骨細(xì)胞中有表達(dá),在4dpa表達(dá)信號(hào)最強(qiáng)。我們推測(cè)Frr2基因可能參與到了成骨細(xì)胞的增殖和分化過(guò)程中,與鰭條的形成有關(guān)。這兩個(gè)基因在尾鰭再生過(guò)程中表達(dá)量發(fā)生變化并在再生后期表達(dá)量最高,預(yù)測(cè)在大鱗副泥鰍尾鰭再生過(guò)程中起著一定作用,它們可能調(diào)控再生過(guò)程中魚鰭結(jié)構(gòu)的形成,而對(duì)于他們更多的功能研究仍需進(jìn)一步的深入研究。
[Abstract]:Mature individuals in the damaged lost tissue can be reconstructed based on the original organization, this phenomenon is called regeneration. All organisms make corresponding physiological response to tissue injury, and disease are closely related, so the research of the regeneration process and mechanism is of great significance. The regeneration ability of mammalian animal is very limited, and as the representative of the salamander amphibians and zebrafish as the representative of the teleost fish in appendages and fins cut by rebuilding the epimorphosis complete structure and function. But the amphibian breeding is slow, laboratories are not easy to raise, and the lack of genetic manipulation of related techniques, can not become a research animal ideal. But after cutting the fins can restore the original structure intact, and relatively simple, easy to obtain, after the injury does not harm the body of life, but more significant is the teleost fin and human The quadruped animal limb early development is very similar, but the two regeneration ability is different, therefore can be used as a good system of fin regeneration mechanism. The analysis of molecular research is established from suppression subtractive hybridization library screened two differentially expressed very obvious fin regeneration of ESTs, based on the two ESTs, by the way of RACE were amplified from two cDNA sequences by Blast. After the comparison, one of the genes that Gig2 gene, another Blast results have not been found and highly homologous, that is a new gene named Frr2 gene. Then the use of DNAMAN. MEGA6.0, SigmaPlot and other biological software analysis of these two genes, also measured the expression of.Gig2 gene of the two genes in different tissues is grass carp hemorrhagic virus induced gene 2, it is first of all from the UV inactivation A new gene was identified GCHV live carp blastula cells in processing. It plays an important role in interferon antiviral response, and in regeneration can regulate regeneration may be a new discovery. Its full length cDNA is 712bp, encoding 156 amino acids, molecular weight was 17.324KDa, isoelectric point (pI) 8.69.46 polar amino acids, 71 non-polar hydrophobic amino acids, 18 amino acids, 21 basic amino acids. Protein two alpha helix structure discovery extension chain accounted for 23.72%, accounting for 27.56%, p-turns accounted for 16.03%, accounting for 32.69%. random coil by semi quantitative and quantitative testing found: the expression of 4D reached the highest level after regeneration, in the period of embryo development hatching the highest expression, also found expression in the testis is also the largest. According to the expression pattern of Gig2 for the first time we found the genes involved in the regeneration of the fin The process, it may play a role in the formation of the fin structure. Gig2 gene was found with other species identity is not large relative analysis of identity results, it also makes research on the evolutionary relationship of.Frr2 (fin regeneration caused great interest in related Gene2) gene is an unknown gene, it is a fin regeneration a related.CDNA gene was 1036bp, encoding 88 amino acids, two level structure found among the alpha helix beta turn accounted for 46.59%, accounting for 9.09%, accounting for 32.95% of random coil, extension chain accounted for semi quantitative 11.36%. and fluorescence quantitative detection in regeneration in different periods (0dpa, 2dpa, 3dpa, 4dpa. 5dpa, 6dpa) on the fourth day of the highest expression in different tissues (liver, heart, brain, kidney, testis, ovary) cardiac expression levels were higher in embryos at different developmental stages (blastula, gastrula, neurula, tail bud stage, hatching) gastrulation base Because the highest expression level. Frozen sections put in different periods of regeneration of the caudal fin in situ hybridization. In situ hybridization results showed that found in osteoblasts with internal fins expression, expression of the strongest signal in 4dpa. We speculated that Frr2 gene may be involved in osteoblast proliferation and differentiation, and fin two. The gene expression changes in regeneration late the highest expression in the caudal fin regeneration process, prediction plays a role in the process of fin regeneration of loach, they may form the regeneration process in the regulation of fin structure, and for their more functional studies still need further research.

【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S917.4

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