【摘要】：花鱸(Lateolabrax maculatus)是隸屬于鱸形目(Perciformes)的肉食性的經(jīng)濟(jì)魚類,廣泛分布于我國近海水域,在廣鹽、廣溫的環(huán)境下具有生長(zhǎng)快和適應(yīng)力強(qiáng)等特點(diǎn)�；|海水養(yǎng)殖規(guī)模的增長(zhǎng),與之伴隨的是各類病毒性和細(xì)菌性疾病的不定期無規(guī)律爆發(fā),除了給養(yǎng)殖業(yè)造成經(jīng)濟(jì)和種質(zhì)的損失。而對(duì)花鱸免疫基因的研究可以幫助人們?cè)谏钊肓私怍~類免疫系統(tǒng)的同時(shí),更進(jìn)一步認(rèn)識(shí)到魚類當(dāng)中的免疫應(yīng)答機(jī)制。而通過結(jié)合部分特定序列,干擾素調(diào)節(jié)因子和來自干擾素基因或干擾素激活基因的轉(zhuǎn)錄調(diào)控區(qū)形成聚合物,誘導(dǎo)并調(diào)控干擾素及其信號(hào)通路中相關(guān)基因的表達(dá)以影響免疫應(yīng)答的作用。本文以花鱸為實(shí)驗(yàn)動(dòng)物,首先對(duì)花鱸脾臟組織結(jié)構(gòu)進(jìn)行HE染色,接著對(duì)花鱸脾臟轉(zhuǎn)錄組進(jìn)行生物信息學(xué)分析,從中篩選出與花鱸非特異性免疫相關(guān)的基因,并對(duì)篩選得到的部分免疫相關(guān)基因進(jìn)行表達(dá)和功能分析,主要包括如下三個(gè)方面的研究?jī)?nèi)容�；|脾臟石蠟切片HE染色結(jié)果顯示:被膜外包在花鱸脾臟的表面,脾臟組織結(jié)構(gòu)含有分界不清晰的紅髓和白髓,除了在脾臟組織間隙分布大量血管、淋巴細(xì)胞和血細(xì)胞外,花鱸脾臟的血管附近還有黑色素巨噬細(xì)胞中心。這些結(jié)果表明花鱸脾臟可能參與魚類的免疫和造血等方面的功能。對(duì)花鱸脾臟轉(zhuǎn)錄組的高通量測(cè)序,得到3.73GB的clean data,花鱸脾臟總unigenes為25380條,Q20為92.27%,Q30為87.66%,GC含量為52.60%。并分別對(duì)花鱸脾臟轉(zhuǎn)錄組進(jìn)行eggNOG Group分析、GO注釋和KEGG通路分析:eggNOG Group結(jié)果顯示功能主要注釋到Signal transduction mechanisms信號(hào)傳導(dǎo)機(jī)制(5317個(gè),21.15%),而注釋到轉(zhuǎn)錄Transcription、防御機(jī)制Defense mechanisms和RNA加工修飾(RNA processing and modification)的基因分別為2487、216和690個(gè);GO功能注釋到三大類,分別是分子功能(molecular function)、細(xì)胞位置(cellular component)、生物過程(biological process),以分子功能的注釋次數(shù)最高(48.11%,49629次),在注釋到‘生物過程’的當(dāng)中注釋次數(shù)最多的是biological_process,達(dá)到7234,占7.01%,而注釋到‘immune system process’次數(shù)有603次;KEGG代謝通路注釋結(jié)果表明,有17220條基因能在KEGG數(shù)據(jù)庫得到注釋,其中富集到Environmental Information Processing的Signal transduction通路上的基因數(shù)目最多,注釋到‘signal transduction’通路的共有2158個(gè)基因,占總Unigene的12.53%,注釋在‘immune system’通路下花鱸脾臟內(nèi)有15種免疫相關(guān)的信號(hào)通路,包括Toll樣受體信號(hào)通路等信號(hào)通路,在這當(dāng)中,Chemokine signaling pathway、Platelet activation和T cell receptor signaling pathway的表達(dá)量最高。目前在花鱸轉(zhuǎn)錄組測(cè)序數(shù)據(jù)當(dāng)中已經(jīng)發(fā)現(xiàn)的IRF家族包括IRF1-IRF9。本研究通過克隆首次得到花鱸干擾素調(diào)節(jié)因子1、2和3的序列,已獲得花鱸IRF1和IRF2的開放性閱讀框分別為899 bp和1007 bp,對(duì)應(yīng)編碼的氨基酸數(shù)分別是292個(gè)和328個(gè),將通過構(gòu)建的IRF1-pFLAG-CMV、IRF2-pFLAG-CMV和IRF3-pFLAG-CMV報(bào)告基因載體進(jìn)行表達(dá)分析。本實(shí)驗(yàn)克隆了得到花鱸IRF3的cDNA全長(zhǎng)1781 bp,根據(jù)1398 bp的開放閱讀框推測(cè)的編碼465個(gè)氨基酸蛋白。氨基酸比對(duì)結(jié)果顯示:花鱸IRF3和脊椎動(dòng)物IRF3同源蛋白質(zhì)在結(jié)構(gòu)上具有相似的保守性,如相同的DNA結(jié)合域、IRF關(guān)聯(lián)域、絲氨酸富集的C-末端結(jié)構(gòu)域和色氨酸殘基簇。系統(tǒng)進(jìn)化分析表明,花鱸IRF3隸屬于IRF3家族。通過qRT-PCR對(duì)比經(jīng)過poly I:C或PBS處理的花鱸各種組織中的SpIRF3 mRNA差異表達(dá),發(fā)現(xiàn)SpIRF3主要集中分布在腦、鰓、肝、脾和頭腎中表達(dá)。經(jīng)過poly I:C處理后,花鱸五個(gè)組織特別是頭腎和脾臟的IRF3轉(zhuǎn)錄表達(dá)成倍上調(diào)。FITC-anti-FLAG標(biāo)記的IRF3的熒光圖像的亞細(xì)胞定位分析表明,花鱸IRF3在未受到刺激時(shí)主要分布在細(xì)胞質(zhì)中,但是經(jīng)過Poly I:C處理后花鱸IRF3出現(xiàn)往細(xì)胞核中遷移聚集,Western blot實(shí)驗(yàn)結(jié)果也驗(yàn)證了以上結(jié)果。同時(shí)Western blot結(jié)果表明,SpIRF3在He La細(xì)胞中正常表達(dá)蛋白分子量的大小約51kDa。此外,IRF3作為調(diào)節(jié)因子能通過結(jié)合魚類IFN啟動(dòng)子、人IFN-β-啟動(dòng)子和ISRE順式元件啟動(dòng)子促使重組質(zhì)粒的熒光素酶報(bào)告基因的表達(dá)上調(diào)。
[Abstract]:Lateolabrax maculatus is a carnivorous economic fish belonging to the perch (Perciformes). It is widely distributed in the coastal waters of China. It has the characteristics of fast growth and adaptability under wide salt and wide temperature environment. The growth of sea bass aquaculture is accompanied by irregular and irregular viral and bacterial diseases. The outbreak, in addition to the loss of economic and Germplasm in the aquaculture industry, and the study of the immune genes of the bass can help people to understand the immune system of fish in depth, and further recognize the immune response mechanism in fish. By combining some specific sequences, interferon regulatory factors and interferon genes or interferon stimulated. The transcriptional regulation area of the living gene is formed to induce and regulate the expression of related genes in the interferon and its signaling pathway to affect the immune response. In this paper, bass was used as an experimental animal to dye the splenic structure of bass bass by HE staining, and then the bioinformatics analysis was carried out on the splenic transcriptional group of bass bass and the flowers were screened from the flowers. The non specific immune related genes of perch were expressed and functional analysis of the selected immune related genes, including the following three aspects. The results of HE staining in paraffin splenic paraffin section showed that the membrane was coated on the surface of the spleens of the bass, and the splenic fabric contains the red and white pulp with unclear boundaries. In the splenic space, a large number of blood vessels, lymphocytes and blood cells, and the blood cells of the spleens of the bass have the center of the melanocytic macrophage. These results show that the spleens of the bass may be involved in the function of the immunization and hematopoiesis of the fish. The high throughput sequencing of the spleer transcriptional group of the bass bass, the 3.73GB clean data, the spleens of the bass spleens The total unigenes was 25380, the Q20 was 92.27%, the Q30 was 87.66%, the GC content was 52.60%., and the eggNOG Group analysis, GO annotation and KEGG pathway analysis were carried out respectively. The eggNOG Group results showed that the function was mainly annotated to the Signal signal transduction mechanism (5317, 21.15%), and the notes were transcribed to prevent it. The genes of Defense mechanisms and RNA processing modification (RNA processing and modification) were 2487216 and 690, respectively, and GO functions were annotated to three categories, respectively, molecular function (molecular function), cell position (cellular component), biological processes, with the highest number of annotations for molecular function (48.11%, 49629). The most annotated number of annotations to the 'biological process' was biological_process, 7234, 7.01%, and the number of' immune system process' 603 times; the KEGG metabolic pathway notes showed that 17220 genes could be annotated in the KEGG database, which was enriched in Environmental Information Processing. The number of genes on the Signal transduction pathway is the most, annotated to a total of 2158 genes in the 'signal transduction' pathway, accounting for 12.53% of the total Unigene. There are 15 immune related signaling pathways in the spleen of the bass under the 'immune system' pathway, including the Toll like receptor signaling pathway, in which Chemokine Si The expression of gnaling pathway, Platelet activation and T cell receptor signaling pathway is the highest. The IRF family, which has been found in the sequence data of the perch transcriptome, includes the sequence of 1,2 and 3 of the interferon regulatory factor of bass bass by cloning for the first time. The number of amino acids encoded by 899 BP and 1007 BP is 292 and 328 respectively. The expression analysis will be carried out through the constructed IRF1-pFLAG-CMV, IRF2-pFLAG-CMV and IRF3-pFLAG-CMV gene carrier. The experiment cloned the cDNA full length 1781 BP of the bass IRF3, and the 465 amino acid proteins deduced from the 1398 BP open reading frame. IRF3 and vertebrate IRF3 homologous proteins have similar conservatism in structure, such as the same DNA binding domain, the IRF associated domain, the serine enriched C- terminal domain and the tryptophan residue cluster. Phylogenetic analysis shows that the bass IRF3 is subordinate to the IRF3 family. The poly I:C or PBS through qRT-PCR is compared. The differential expression of SpIRF3 mRNA in various tissues of the treated perch found that SpIRF3 mainly concentrated in the brain, gills, liver, spleen and the head kidney. After poly I:C treatment, the subcellular localization analysis of the five tissues, especially the IRF3 of the head kidney and spleen, expressed the fluorescence image of the IRF3 that multiplied the.FITC-anti-FLAG marked IRF3. IRF3 was mainly distributed in the cytoplasm when unstimulated, but after Poly I:C treatment, the perch IRF3 migrated to the nucleus, and the results of Western blot test confirmed the above results. Meanwhile, the Western blot results showed that the normal expression of the egg white molecular weight of SpIRF3 in He La cells was about the size of the 51kDa.. The expression of the luciferase reporter gene of the recombinant plasmid is up-regulated by combining the IFN promoter with the fish, the human IFN- beta promoter and the ISRE cis element promoter.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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