草鱼两个GTPase基因的分子克隆及免疫功能分析

发布时间：2018-12-18 07:17

【摘要】：草鱼(Ctenopharyngodon idellus)作为我国最重要的养殖鱼类之一,能够提供巨大的经济价值以及营养价值。但是由于环境污染等一系列问题,草鱼容易被病原体感染,从而造成巨大的经济损失。所以从水产养殖的方面考虑,以选育良种的方式提高草鱼的抗病力是增加草鱼养殖产量的一项重要途径。而抗病良种选育的一项重要工作便是鉴定潜在具有免疫功能的相关基因。GTPase蛋白,小G蛋白在哺乳动物中被证明为一类能够参与细胞骨架调控、细胞吞噬作用以及免疫联级反应的蛋白。但是有关草鱼GTPase蛋白的鉴定以及功能分析的研究至今尚未出现。所以本文利用分子克隆的方法鉴定了草鱼中GTPase蛋白家族的两个重要成员,Rac1以及Cdc42的c DNA全长序列并对它们的免疫功能进行了探究。1.Rac1,在哺乳动物中具有关键性的免疫功能。本研究中,我们克隆了草鱼中存在的Rac1基因。草鱼Rac1基因c DNA全长为2023 bp,包含579 bp的开放阅读框序列,能够编码192个氨基酸并且包含一个保守的RHO结构域以及核定位信号。草鱼Rac1氨基酸序列与其他物种中的Rac1具有极高的相似度同时在系统发育树中与斑马鱼Rac1聚为一束。gc Rac1在草鱼的多个组织中广泛表达;同时能够于草鱼成体以及细胞中在嗜水气单胞菌感染时被激活。同时,gc Rac1的表达量也能够被其他细菌相关病原体激发。当gc Rac1过表达的时候,在细菌以及细菌相关PAMPs的刺激下,gc PAK1,gc IL-1β,gc TNF-α以及gc IFN的表达量都会不同程度的上升;而gc Rac1的敲降则会导致这些基因的表达量下降。gc Rac1的过表达能够抑制细菌入侵细胞的数量,同时gc Rac1的敲降促进了细菌的入侵。而且,gc Rac1能够激活NF-κB信号通路。2.作为一个在哺乳动物中与多种疾病发生相关的基因,Cdc42,首次在草鱼中被鉴定及功能性验证。我们得到的草鱼Cdc42 c DNA全长1955bp,包含能够编码191个氨基酸,长度为576 bp的开放阅读框序列。在其氨基酸序列中存在一个保守RHO结构域及核定位信号。与之相应,重组载体表达显示Ci Cdc42在细胞核区域表达量更高。Ci Cdc42组织表达谱结果显示其在草鱼的十个组织中广泛表达,同时在嗜水气单胞菌感染的情况下表达量上调。在草鱼细胞中,细菌以及细菌相关病原体均能刺激Ci Cdc42的表达量上升;而在这些病原体的感染下,Ci Cdc42的过表达基本能够刺激Ci IL-1β,Ci TNF-α以及Ci IFN的表达,但不会刺激Ci PAK1的表达;同时Ci Cdc42的RNA干扰会导致这三个基因的表达量下降。而且,Ci Cdc42的过表达减少了嗜水气单胞菌入侵细胞的数量,同时Ci Cdc42的敲降增加了细菌入侵细胞的效率。这些研究证明了Cdc42在草鱼抵抗病原体感染过程中的作用并且为草鱼先天免疫的研究提供了一个新的视角。
[Abstract]:As one of the most important cultured fish in China, grass carp (Ctenopharyngodon idellus) can provide great economic and nutritional value. However, due to environmental pollution and a series of problems, grass carp is liable to be infected by pathogens, resulting in huge economic losses. Therefore, from the point of view of aquaculture, it is an important way to increase the yield of grass carp to improve the disease resistance of grass carp by breeding improved species. And one of the important tasks in breeding disease-resistant varieties is to identify potentially immune-related genes. GTPase proteins, small G proteins, have been shown to be involved in cytoskeleton regulation in mammals. Phagocytosis and immunosynthesis of proteins. However, studies on the identification of GTPase protein and functional analysis of grass carp have not been reported. Therefore, two important members of GTPase protein family in grass carp, Rac1 and c DNA of Cdc42, were identified by molecular cloning and their immune function was studied. It has a key immune function in mammals. In this study, we cloned the Rac1 gene from grass carp. The full-length c DNA of grass carp Rac1 gene is 2023 bp, which contains an open reading frame sequence of 579 bp, encoding 192 amino acids and containing a conserved RHO domain and nuclear localization signal. The amino acid sequence of Rac1 of grass carp has high similarity with Rac1 of other species and Rac1 of zebrafish is clustered in phylogenetic tree. Gc Rac1 is widely expressed in many tissues of grass carp. It can also be activated by Aeromonas hydrophila infection in grass carp adults and cells. At the same time, the expression of gc Rac1 can also be stimulated by other bacteria-related pathogens. When gc Rac1 was overexpressed, the expression of gc PAK1,gc IL-1 尾, gc TNF- 伪 and gc IFN increased in varying degrees under the stimulation of bacteria and their related PAMPs. The overexpression of gc Rac1 can inhibit the number of bacterial invading cells, and the knock down of gc Rac1 can promote the invasion of bacteria. Moreover, gc Rac1 can activate the NF- 魏 B signaling pathway. 2. 2. As a gene associated with many diseases in mammals, Cdc42, was first identified and functionally verified in grass carp. The total length of grass carp Cdc42 c DNA is 195 BP, which contains an open reading frame sequence which encodes 191 amino acids and is 576 bp in length. There is a conserved RHO domain and nuclear localization signal in its amino acid sequence. Accordingly, the expression of recombinant vector showed that the expression of Ci Cdc42 in the nuclear region was higher. The results of Ci Cdc42 tissue expression profile showed that it was widely expressed in ten tissues of grass carp, and the expression level was up-regulated in the presence of Aeromonas hydrophila. In grass carp cells, bacteria and bacteria related pathogens can stimulate the expression of Ci Cdc42. The overexpression of Ci Cdc42 could stimulate the expression of Ci IL-1 尾, Ci TNF- 伪 and Ci IFN, but not the expression of Ci PAK1, and the RNA interference of Ci Cdc42 could decrease the expression of these three genes. Moreover, the overexpression of Ci Cdc42 reduced the number of invasive cells of Aeromonas hydrophila, and the knockout of Ci Cdc42 increased the efficiency of invading cells. These studies demonstrate the role of Cdc42 in the resistance of grass carp to pathogen infection and provide a new perspective for the study of innate immunity of grass carp.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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