长丰鲫c型和g型溶菌酶基因的克隆及其在细菌感染条件下的表达分析

发布时间：2018-05-23 14:28

本文选题：长丰鲫 + 普通鲫　；参考：《华中农业大学》2017年硕士论文

【摘要】：长丰鲫(Chang Feng Carassius auratus gibelio)(品种登记号:GS-04-01-2015)肉质嫩、生长快、遗传稳定,市场应用前景非常广阔。但近年来,随着我国水产养殖规模的日益扩大,鲫(Carassius auratus)大规模养殖过程中细菌性病害越来越严重。溶菌酶(Lysozyme,lys)具有广谱的杀菌功能,在鱼类非特异免疫中占有重要地位。本文以长丰鲫和普通鲫为研究对象,克隆长丰鲫c-lys和g-lys c DNA基因全长;检测c-lys和g-lys基因在长丰鲫和普通鲫中的组织表达情况;两种鱼分别感染迟钝爱德华氏菌、嗜水气单胞菌和金黄色葡萄球菌后,分析两种基因在长丰鲫和普通鲫肝脏、肾脏、脾脏和鳃中的表达变化特征,同时检测四种组织的形态学变化,初步评价它们的免疫防御功能,为探讨长丰鲫抗细菌性疾病能力提供一定的理论依据。本研究的实验结果如下:1.长丰鲫c-lys和g-lys基因克隆长丰鲫c-lys全长698 bp,开放阅读框(ORF)438 bp,编码145个氨基酸。含有Glu 53和Asp 69两个保守的氨基酸催化位点,八个保守的Cys,可形成四对二硫键。N端具有信号肽,是一种分泌蛋白;长丰鲫g-lys全长861 bp,开放阅读框(ORF)558 bp,编码185个氨基酸。含有Glu(第73位)、Pro(第86位)和Asp(第97位)三个保守的催化位点。存在一个Cys残基,但不形成二硫键,不存在N端信号肽,是一种胞内蛋白。氨基酸序列比对和进化树分析结果表明:长丰鲫c-lys和g-lys基因在鲤形目鱼类进化过程中均较为保守。2.三种细菌刺激后,长丰鲫和普通鲫溶菌酶基因组织表达变化c-lys和g-lys基因在长丰鲫和普通鲫所检测组织中均有表达,且均在肾脏中表达量最高。感染细菌后,肝脏、肾脏、脾脏和鳃组织中,长丰鲫和普通鲫c-lys和g-lys基因表达量均呈现不同程度的上调,但两种基因在两种鱼同一组织中升幅、出现高峰的时间等均有差异。3.三种细菌感染后,长丰鲫和普通鲫各组织的病理变化长丰鲫和普通鲫发病后鳞片脱落,体表、肛门和鳍等部位伴有不同程度充血和出血现象,但两种鱼在体色和不同细菌感染剖检后各组织颜色变化存在一定差异。三种细菌刺激后,长丰鲫和普通鲫出现症状相似:肝脏表现为肝细胞排列紊乱,分布不均,细胞核大小不一致,固缩,碎裂坏死,细胞肿胀,肝血窦和中央静脉淤血,肝细胞滴状玻璃样变等症状;肾脏呈现出肾小球肿大,肾小管上皮细胞脱落,颗粒样变,单核巨噬细胞增多,肾间质炎性细胞浸润等症状;脾脏组织红髓白髓出现明显分界,细胞核碎裂,固缩坏死,脾动脉周围网状细胞、淋巴细胞减少。鳃小片呼吸上皮细胞浮离,鳃丝变粗变短,血管扩张,鳃小片基部水肿,部分患病鱼鳃小片组织细胞解体、脱落。
[Abstract]:Chang Feng Carassius auratus gibelioc (variety registration no.: GS-04-01-2015) has the advantages of tender meat, fast growth, stable heredity and broad market application prospect. But in recent years, with the increasing scale of aquaculture in China, the bacterial diseases of Carassius auratus were more and more serious. Lysozymelysase has broad spectrum bactericidal function and plays an important role in fish nonspecific immunity. In this paper, the full-length c-lys and g-lys c DNA genes of Changfeng crucian carp and common crucian carp were cloned, and the tissue expression of c-lys and g-lys genes in Changfeng crucian carp and common crucian carp were detected. After Aeromonas hydrophila and Staphylococcus aureus, the expression of the two genes in liver, kidney, spleen and Gill of crucian carp were analyzed, and the morphological changes of four tissues were detected. The preliminary evaluation of their immune defense function provides a theoretical basis for the study of the ability of resistance to bacterial diseases in Changfeng crucian carp. The experimental results of this study are as follows: 1. The c-lys and g-lys genes of c-lys and g-lys in Changfeng crucian carp were cloned into 698 BP, ORF 438bp, encoding 145 amino acids. It contains two conserved amino acid catalytic sites, Glu 53 and Asp 69, and eight conserved cysts, which can form four pairs of disulfide bonds. The N-terminal signal peptide is a secretory protein, and the g-lys of Changfeng crucian carp is 861 BP in length and ORF 558 BP in open reading frame, encoding 185 amino acids. There are three conserved catalytic sites: Glu73, Prop (86) and Asp97 (97). There is a Cys residue, but no disulfide bond, no N-terminal signal peptide, is a intracellular protein. The results of amino acid sequence alignment and phylogenetic tree analysis showed that c-lys and g-lys genes were conserved in the evolution of Cyprinidae. Changes of lysozyme gene expression in Changfeng crucian carp and common crucian carp after three kinds of bacteria stimulation c-lys and g-lys genes were expressed in the tissues detected by Changfeng crucian carp and common crucian carp and the highest expression was found in kidney. After infection, the expression of c-lys and g-lys genes in liver, kidney, spleen and Gill were all up-regulated in Changfeng crucian carp and common crucian carp, but the two genes increased in the same tissue and appeared peak time. Pathological changes of tissues in Changfeng crucian carp and common crucian carp after infection of three kinds of bacteria, the scales of Changfeng crucian carp and common crucian carp fell off, and the body surface, anus and fins were accompanied with congestion and bleeding in varying degrees. However, there were some differences in color changes between the two species of fish after body color and bacterial infection. After three kinds of bacteria stimulation, the symptoms of Changfeng crucian carp and common crucian carp were similar: liver showed hepatocyte arrangement disorder, uneven distribution, inconsistent nuclear size, pyknosis, fragmentation and necrosis, cell swelling, hepatic sinusoidal and central vein congestion. The kidney showed glomerulomegaly, tubular epithelial cells shedding, granulocyte, mononuclear macrophage increasing, renal interstitial inflammatory cell infiltration and so on. Nuclear fragmentation, pyknotic necrosis, reticular cells around splenic artery, and lymphocytosis. The branchial filaments became thicker and shorter, vasodilation, edema at the base of the branchial segments, and the disassembly and shedding of some diseased Gill tissue cells.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4;S943

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