【摘要】：草魚(Ctenopharyngodon idellus)作為我國最重要的養(yǎng)殖魚類之一,能夠提供巨大的經(jīng)濟(jì)價(jià)值以及營養(yǎng)價(jià)值。但是由于環(huán)境污染等一系列問題,草魚容易被病原體感染,從而造成巨大的經(jīng)濟(jì)損失。所以從水產(chǎn)養(yǎng)殖的方面考慮,以選育良種的方式提高草魚的抗病力是增加草魚養(yǎng)殖產(chǎn)量的一項(xiàng)重要途徑。而抗病良種選育的一項(xiàng)重要工作便是鑒定潛在具有免疫功能的相關(guān)基因。GTPase蛋白,小G蛋白在哺乳動物中被證明為一類能夠參與細(xì)胞骨架調(diào)控、細(xì)胞吞噬作用以及免疫聯(lián)級反應(yīng)的蛋白。但是有關(guān)草魚GTPase蛋白的鑒定以及功能分析的研究至今尚未出現(xiàn)。所以本文利用分子克隆的方法鑒定了草魚中GTPase蛋白家族的兩個(gè)重要成員,Rac1以及Cdc42的c DNA全長序列并對它們的免疫功能進(jìn)行了探究。1.Rac1,在哺乳動物中具有關(guān)鍵性的免疫功能。本研究中,我們克隆了草魚中存在的Rac1基因。草魚Rac1基因c DNA全長為2023 bp,包含579 bp的開放閱讀框序列,能夠編碼192個(gè)氨基酸并且包含一個(gè)保守的RHO結(jié)構(gòu)域以及核定位信號。草魚Rac1氨基酸序列與其他物種中的Rac1具有極高的相似度同時(shí)在系統(tǒng)發(fā)育樹中與斑馬魚Rac1聚為一束。gc Rac1在草魚的多個(gè)組織中廣泛表達(dá);同時(shí)能夠于草魚成體以及細(xì)胞中在嗜水氣單胞菌感染時(shí)被激活。同時(shí),gc Rac1的表達(dá)量也能夠被其他細(xì)菌相關(guān)病原體激發(fā)。當(dāng)gc Rac1過表達(dá)的時(shí)候,在細(xì)菌以及細(xì)菌相關(guān)PAMPs的刺激下,gc PAK1,gc IL-1β,gc TNF-α以及gc IFN的表達(dá)量都會不同程度的上升;而gc Rac1的敲降則會導(dǎo)致這些基因的表達(dá)量下降。gc Rac1的過表達(dá)能夠抑制細(xì)菌入侵細(xì)胞的數(shù)量,同時(shí)gc Rac1的敲降促進(jìn)了細(xì)菌的入侵。而且,gc Rac1能夠激活NF-κB信號通路。2.作為一個(gè)在哺乳動物中與多種疾病發(fā)生相關(guān)的基因,Cdc42,首次在草魚中被鑒定及功能性驗(yàn)證。我們得到的草魚Cdc42 c DNA全長1955bp,包含能夠編碼191個(gè)氨基酸,長度為576 bp的開放閱讀框序列。在其氨基酸序列中存在一個(gè)保守RHO結(jié)構(gòu)域及核定位信號。與之相應(yīng),重組載體表達(dá)顯示Ci Cdc42在細(xì)胞核區(qū)域表達(dá)量更高。Ci Cdc42組織表達(dá)譜結(jié)果顯示其在草魚的十個(gè)組織中廣泛表達(dá),同時(shí)在嗜水氣單胞菌感染的情況下表達(dá)量上調(diào)。在草魚細(xì)胞中,細(xì)菌以及細(xì)菌相關(guān)病原體均能刺激Ci Cdc42的表達(dá)量上升;而在這些病原體的感染下,Ci Cdc42的過表達(dá)基本能夠刺激Ci IL-1β,Ci TNF-α以及Ci IFN的表達(dá),但不會刺激Ci PAK1的表達(dá);同時(shí)Ci Cdc42的RNA干擾會導(dǎo)致這三個(gè)基因的表達(dá)量下降。而且,Ci Cdc42的過表達(dá)減少了嗜水氣單胞菌入侵細(xì)胞的數(shù)量,同時(shí)Ci Cdc42的敲降增加了細(xì)菌入侵細(xì)胞的效率。這些研究證明了Cdc42在草魚抵抗病原體感染過程中的作用并且為草魚先天免疫的研究提供了一個(gè)新的視角。
[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- 偽 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- 偽 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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S917.4

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