花鲈IRF3基因表达与功能分析

发布时间：2018-07-27 11:04

【摘要】：花鲈(Lateolabrax maculatus)是隶属于鲈形目(Perciformes)的肉食性的经济鱼类,广泛分布于我国近海水域,在广盐、广温的环境下具有生长快和适应力强等特点。花鲈海水养殖规模的增长,与之伴随的是各类病毒性和细菌性疾病的不定期无规律爆发,除了给养殖业造成经济和种质的损失。而对花鲈免疫基因的研究可以帮助人们在深入了解鱼类免疫系统的同时,更进一步认识到鱼类当中的免疫应答机制。而通过结合部分特定序列,干扰素调节因子和来自干扰素基因或干扰素激活基因的转录调控区形成聚合物,诱导并调控干扰素及其信号通路中相关基因的表达以影响免疫应答的作用。本文以花鲈为实验动物,首先对花鲈脾脏组织结构进行HE染色,接着对花鲈脾脏转录组进行生物信息学分析,从中筛选出与花鲈非特异性免疫相关的基因,并对筛选得到的部分免疫相关基因进行表达和功能分析,主要包括如下三个方面的研究内容。花鲈脾脏石蜡切片HE染色结果显示:被膜外包在花鲈脾脏的表面,脾脏组织结构含有分界不清晰的红髓和白髓,除了在脾脏组织间隙分布大量血管、淋巴细胞和血细胞外,花鲈脾脏的血管附近还有黑色素巨噬细胞中心。这些结果表明花鲈脾脏可能参与鱼类的免疫和造血等方面的功能。对花鲈脾脏转录组的高通量测序,得到3.73GB的clean data,花鲈脾脏总unigenes为25380条,Q20为92.27%,Q30为87.66%,GC含量为52.60%。并分别对花鲈脾脏转录组进行eggNOG Group分析、GO注释和KEGG通路分析:eggNOG Group结果显示功能主要注释到Signal transduction mechanisms信号传导机制(5317个,21.15%),而注释到转录Transcription、防御机制Defense mechanisms和RNA加工修饰(RNA processing and modification)的基因分别为2487、216和690个;GO功能注释到三大类,分别是分子功能(molecular function)、细胞位置(cellular component)、生物过程(biological process),以分子功能的注释次数最高(48.11%,49629次),在注释到‘生物过程’的当中注释次数最多的是biological_process,达到7234,占7.01%,而注释到‘immune system process’次数有603次;KEGG代谢通路注释结果表明,有17220条基因能在KEGG数据库得到注释,其中富集到Environmental Information Processing的Signal transduction通路上的基因数目最多,注释到‘signal transduction’通路的共有2158个基因,占总Unigene的12.53%,注释在‘immune system’通路下花鲈脾脏内有15种免疫相关的信号通路,包括Toll样受体信号通路等信号通路,在这当中,Chemokine signaling pathway、Platelet activation和T cell receptor signaling pathway的表达量最高。目前在花鲈转录组测序数据当中已经发现的IRF家族包括IRF1-IRF9。本研究通过克隆首次得到花鲈干扰素调节因子1、2和3的序列,已获得花鲈IRF1和IRF2的开放性阅读框分别为899 bp和1007 bp,对应编码的氨基酸数分别是292个和328个,将通过构建的IRF1-pFLAG-CMV、IRF2-pFLAG-CMV和IRF3-pFLAG-CMV报告基因载体进行表达分析。本实验克隆了得到花鲈IRF3的cDNA全长1781 bp,根据1398 bp的开放阅读框推测的编码465个氨基酸蛋白。氨基酸比对结果显示:花鲈IRF3和脊椎动物IRF3同源蛋白质在结构上具有相似的保守性,如相同的DNA结合域、IRF关联域、丝氨酸富集的C-末端结构域和色氨酸残基簇。系统进化分析表明,花鲈IRF3隶属于IRF3家族。通过qRT-PCR对比经过poly I:C或PBS处理的花鲈各种组织中的SpIRF3 mRNA差异表达,发现SpIRF3主要集中分布在脑、鳃、肝、脾和头肾中表达。经过poly I:C处理后,花鲈五个组织特别是头肾和脾脏的IRF3转录表达成倍上调。FITC-anti-FLAG标记的IRF3的荧光图像的亚细胞定位分析表明,花鲈IRF3在未受到刺激时主要分布在细胞质中,但是经过Poly I:C处理后花鲈IRF3出现往细胞核中迁移聚集,Western blot实验结果也验证了以上结果。同时Western blot结果表明,SpIRF3在He La细胞中正常表达蛋白分子量的大小约51kDa。此外,IRF3作为调节因子能通过结合鱼类IFN启动子、人IFN-β-启动子和ISRE顺式元件启动子促使重组质粒的荧光素酶报告基因的表达上调。
[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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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