本文選題：長豐鯽 + 普通鯽�。� 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：長豐鯽(Chang Feng Carassius auratus gibelio)(品種登記號:GS-04-01-2015)肉質(zhì)嫩、生長快、遺傳穩(wěn)定,市場應用前景非常廣闊。但近年來,隨著我國水產(chǎn)養(yǎng)殖規(guī)模的日益擴大,鯽(Carassius auratus)大規(guī)模養(yǎng)殖過程中細菌性病害越來越嚴重。溶菌酶(Lysozyme,lys)具有廣譜的殺菌功能,在魚類非特異免疫中占有重要地位。本文以長豐鯽和普通鯽為研究對象,克隆長豐鯽c-lys和g-lys c DNA基因全長;檢測c-lys和g-lys基因在長豐鯽和普通鯽中的組織表達情況;兩種魚分別感染遲鈍愛德華氏菌、嗜水氣單胞菌和金黃色葡萄球菌后,分析兩種基因在長豐鯽和普通鯽肝臟、腎臟、脾臟和鰓中的表達變化特征,同時檢測四種組織的形態(tài)學變化,初步評價它們的免疫防御功能,為探討長豐鯽抗細菌性疾病能力提供一定的理論依據(jù)。本研究的實驗結(jié)果如下:1.長豐鯽c-lys和g-lys基因克隆長豐鯽c-lys全長698 bp,開放閱讀框(ORF)438 bp,編碼145個氨基酸。含有Glu 53和Asp 69兩個保守的氨基酸催化位點,八個保守的Cys,可形成四對二硫鍵。N端具有信號肽,是一種分泌蛋白;長豐鯽g-lys全長861 bp,開放閱讀框(ORF)558 bp,編碼185個氨基酸。含有Glu(第73位)、Pro(第86位)和Asp(第97位)三個保守的催化位點。存在一個Cys殘基,但不形成二硫鍵,不存在N端信號肽,是一種胞內(nèi)蛋白。氨基酸序列比對和進化樹分析結(jié)果表明:長豐鯽c-lys和g-lys基因在鯉形目魚類進化過程中均較為保守。2.三種細菌刺激后,長豐鯽和普通鯽溶菌酶基因組織表達變化c-lys和g-lys基因在長豐鯽和普通鯽所檢測組織中均有表達,且均在腎臟中表達量最高。感染細菌后,肝臟、腎臟、脾臟和鰓組織中,長豐鯽和普通鯽c-lys和g-lys基因表達量均呈現(xiàn)不同程度的上調(diào),但兩種基因在兩種魚同一組織中升幅、出現(xiàn)高峰的時間等均有差異。3.三種細菌感染后,長豐鯽和普通鯽各組織的病理變化長豐鯽和普通鯽發(fā)病后鱗片脫落,體表、肛門和鰭等部位伴有不同程度充血和出血現(xiàn)象,但兩種魚在體色和不同細菌感染剖檢后各組織顏色變化存在一定差異。三種細菌刺激后,長豐鯽和普通鯽出現(xiàn)癥狀相似:肝臟表現(xiàn)為肝細胞排列紊亂,分布不均,細胞核大小不一致,固縮,碎裂壞死,細胞腫脹,肝血竇和中央靜脈淤血,肝細胞滴狀玻璃樣變等癥狀;腎臟呈現(xiàn)出腎小球腫大,腎小管上皮細胞脫落,顆粒樣變,單核巨噬細胞增多,腎間質(zhì)炎性細胞浸潤等癥狀;脾臟組織紅髓白髓出現(xiàn)明顯分界,細胞核碎裂,固縮壞死,脾動脈周圍網(wǎng)狀細胞、淋巴細胞減少。鰓小片呼吸上皮細胞浮離,鰓絲變粗變短,血管擴張,鰓小片基部水腫,部分患病魚鰓小片組織細胞解體、脫落。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4;S943

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