【摘要】：草魚(Ctenopharyngodon idellus)是我國重要的淡水經(jīng)濟(jì)魚類,在我國多個(gè)地區(qū)大規(guī)模養(yǎng)殖。但是隨著草魚養(yǎng)殖業(yè)的大力發(fā)展,草魚的各類疾病頻繁爆發(fā),目前尚無健康有效的治療方法,這成為草魚養(yǎng)殖難以突破的瓶頸,嚴(yán)重阻礙了草魚養(yǎng)殖業(yè)的可持續(xù)發(fā)展。在抵御各種病原體的入侵時(shí),天然免疫系統(tǒng)發(fā)揮著至關(guān)重要的作用。TRIM(tripartite-motif protein)家族是機(jī)體先天性免疫的重要成員,在細(xì)胞的增殖、分化、腫瘤的抑制、病毒的抑制等方面發(fā)揮著重要的調(diào)控作用。TRIM家族蛋白,也稱為RBCC家族蛋白,從N端到C端依次是RING結(jié)構(gòu)域、B-box結(jié)構(gòu)域、Coiled-coil結(jié)構(gòu)域。在哺乳動(dòng)物中TRIM32的RING結(jié)構(gòu)域具有E3泛素連接酶的作用,可以調(diào)節(jié)信號(hào)通路的傳導(dǎo),參與機(jī)體的天然免疫反應(yīng)。本論文以草魚TRIM32為主要研究對(duì)象,旨在為草魚疾病的防御與治療方面提供進(jìn)一步的理論依據(jù)。主要研究內(nèi)容包括以下幾個(gè)方面:1.利用PCR技術(shù)從草魚的肝臟組織中克隆草魚TRIM32基因的編碼區(qū)全長,并翻譯成對(duì)應(yīng)的氨基酸序列。結(jié)果顯示,草魚TRIM32基因共1980 bp,編碼660個(gè)氨基酸。2.對(duì)草魚TRIM32進(jìn)行結(jié)構(gòu)域分析、核苷酸序列比對(duì)以及遺傳進(jìn)化樹分析。結(jié)果顯示,草魚TRIM32具有TRIM家族3個(gè)典型的結(jié)構(gòu)域,與斑馬魚TRIM32的核苷酸序列同源性較高,且遺傳進(jìn)化樹分析也與斑馬魚TRIM32聚為一支。3.對(duì)草魚TRIM32進(jìn)行真核表達(dá)載體的構(gòu)建,并通過間接免疫熒光和Western blotting分析草魚TRIM32的表達(dá)與亞定位情況。結(jié)果顯示草魚TRIM32可以在EPC細(xì)胞中成功表達(dá),主要以點(diǎn)狀分布在細(xì)胞質(zhì)中,而細(xì)胞核中的表達(dá)量較少。4.采用q PT-PCR檢測(cè)草魚TRIM32的組織分布,分析結(jié)果顯示草魚TRIM32在被檢測(cè)的11個(gè)組織中均有表達(dá),其中在頭腎組織中的表達(dá)量明顯高于其他組織。5.采用q PT-PCR檢測(cè)草魚TRIM32在不同胚胎發(fā)育時(shí)期的表達(dá)豐度,分析結(jié)果顯示,草魚TRIM32在受精卵時(shí)期、卵裂期和囊胚期的表達(dá)量顯著高于其他時(shí)期(p0.05),隨后表達(dá)量下降,直到出膜后表達(dá)量再次顯著性升高。6.利用雙熒光素酶報(bào)告系統(tǒng)檢測(cè)草魚TRIM32對(duì)NF-κB信號(hào)通路的影響。檢測(cè)結(jié)果顯示,實(shí)驗(yàn)組是對(duì)照組的1.66倍,說明草魚TRIM32能夠激活NF-κB信號(hào)通路,誘導(dǎo)下游的炎癥反應(yīng)。7.構(gòu)建了草魚TRIM32原核表達(dá)載體,并制備了高效價(jià)的抗草魚TRIM32的多克隆抗體。
[Abstract]:Grass carp (Ctenopharyngodon idellus) is an important freshwater economic fish in China. However, with the vigorous development of grass carp culture, various diseases of grass carp frequently erupt, and there is no healthy and effective treatment, which has become the bottleneck of grass carp breeding, which seriously hinders the sustainable development of grass carp breeding. The innate immune system plays an important role in resisting the invasion of various pathogens. TRIM (tripartite-motif protein) family is an important member of innate immunity in the cell proliferation, differentiation, tumor suppression. TRIM family proteins, also known as RBCC family proteins, are RING domain, B-box domain and Coiled-coil domain from N-terminal to C-terminal. In mammals, the RING domain of TRIM32 has the function of E3 ubiquitin ligase, which can regulate the signaling pathway and participate in the innate immune response of the body. The purpose of this thesis is to provide further theoretical basis for the prevention and treatment of grass carp disease. The main research contents include the following aspects: 1. The coding region of TRIM32 gene of grass carp was cloned from liver tissue of grass carp by PCR technique and translated into corresponding amino acid sequence. The results showed that the TRIM32 gene of grass carp encodes 660amino acids with a total of 1980 bp,. Domain analysis, nucleotide alignment and genetic phylogenetic tree analysis of grass carp TRIM32 were carried out. The results showed that grass carp TRIM32 had three typical domains of TRIM family, and had high homology with zebrafish TRIM32, and the genetic evolutionary tree analysis was also associated with zebrafish TRIM32. The eukaryotic expression vector of grass carp TRIM32 was constructed, and the expression and sublocalization of TRIM32 in grass carp were analyzed by indirect immunofluorescence and Western blotting. The results showed that grass carp TRIM32 could be successfully expressed in EPC cells, mainly in the cytoplasm, but only in the nucleus. Q PT-PCR was used to detect the tissue distribution of grass carp TRIM32. The results showed that the expression of grass carp TRIM32 was detected in 11 tissues, and the expression level in head and kidney tissues was significantly higher than that in other tissues. Q PT-PCR was used to detect the abundance of TRIM32 in grass carp at different embryonic stages. The results showed that the expression of TRIM32 in fertilized egg, cleavage and blastocyst was significantly higher than that in other stages (p0.05), and then decreased. The expression level was significantly increased again until after membrane exudation. 6. 6%. The effects of grass carp TRIM32 on NF- 魏 B signaling pathway were detected by double luciferase reporting system. The results showed that the experimental group was 1. 66 times higher than the control group, indicating that grass carp TRIM32 could activate the NF- 魏 B signaling pathway and induce the downstream inflammatory reaction. The prokaryotic expression vector of grass carp TRIM32 was constructed and the polyclonal antibody against grass carp TRIM32 was prepared.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;S917.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張青海;;草魚高產(chǎn)養(yǎng)殖技術(shù)及病害防治技術(shù)[J];農(nóng)業(yè)與技術(shù);2016年22期

2 徐先棟;付輝云;饒毅;盛銀平;王海華;傅義龍;李彩剛;周永燦;;草魚暴發(fā)性出血病病原分離、毒力基因檢測(cè)與藥敏分析[J];上海海洋大學(xué)學(xué)報(bào);2016年03期

3 姜文志;;草魚池塘養(yǎng)殖的要點(diǎn)[J];現(xiàn)代畜牧科技;2016年01期

4 方珍珍;蘇文;陶秉春;;感染小瓜蟲草魚幼魚組織病理學(xué)變化[J];水產(chǎn)科學(xué);2015年07期

5 董鋒;廖蘭杰;朱作言;汪亞平;;草魚免疫相關(guān)基因Prkrip1的克隆和表達(dá)分析[J];水生生物學(xué)報(bào);2013年04期

6 楊淞;吳淑勤;李寧求;石存斌;鄧國成;林文輝;林強(qiáng);;草魚出血病發(fā)生風(fēng)險(xiǎn)半定量評(píng)估模型的構(gòu)建[J];中國水產(chǎn)科學(xué);2012年03期

7 劉永奎;王慶;曾偉偉;石存斌;張超;陳道印;吳淑勤;;草魚呼腸孤病毒JX-0902株的分離和鑒定[J];中國水產(chǎn)科學(xué);2011年05期

8 王海華;馬保新;黃江峰;盛銀平;;草魚免疫防疫技術(shù)[J];科學(xué)養(yǎng)魚;2009年12期

9 楊先樂;曹海鵬;錢云云;;噬菌蛭弧菌——水產(chǎn)動(dòng)物病害生物防治的新工具[J];淡水漁業(yè);2006年02期

10 姚榮華;;草魚腸炎病的防治[J];中國水產(chǎn);1981年03期

相關(guān)會(huì)議論文 前1條

1 孫琪;胡鯤;楊先樂;;殼聚糖對(duì)草魚人工感染水霉的影響[A];中國水產(chǎn)學(xué)會(huì)魚病專業(yè)委員會(huì)2013年學(xué)術(shù)研討會(huì)論文摘要匯編[C];2013年

，

本文編號(hào)：2385360