亚麻酸对东海原甲藻的克生效应及细胞凋亡机制的研究

发布时间：2018-07-25 12:14

【摘要】：赤潮(Red tides)是一种海洋生态异常现象。近年来,随着我国工农业发展,水体富营养化加重,赤潮发生频率增高,尤其是有毒有害赤潮频发,对海洋养殖业、人类健康及海洋生态安全构成严重威胁。当前,赤潮的有效治理和防治已成为海洋环境领域的重要研究内容。赤潮的防治主要有物理法、化学法和生物法三种方法。其中,利用大型海藻分泌的克生物质抑制赤潮微藻的爆发独具优势,有着较强的可持续性和环境友好的特点,日益受到研究者的重视。本文采用实验生态学方法,以赤潮优势藻种东海原甲藻为研究对象,探究高克生活性物质亚麻酸胁迫下东海原甲藻的生理生化响应及细胞凋亡相关机制。本文的研究为防控以东海原甲藻为优势藻种的赤潮提供了理论基础。具体结果如下:1. 亚麻酸对东海原甲藻生长及生理生化的影响亚麻酸对东海原甲藻生长表现出显著的“低促高抑”现象。低浓度的亚麻酸对东海原甲藻的生长具有促进作用(负抑制),随着亚麻酸浓度升高,东海原甲藻生长量逐渐减小,亚麻酸对其生长表现为抑制作用。亚麻酸胁迫下,藻细胞可溶性蛋白含量表现出与生长量变化相对应的先上升后下降趋势;光合色素含量也出现先升后降的现象,影响了藻类的光合效率;硝酸还原酶活性出现“升—降”趋势。2.亚麻酸胁迫下东海原甲藻活性氧信号通路与钙离子信号通路的响应亚麻酸胁迫下,东海原甲藻细胞内抗氧化酶系统(CAT、SOD、GPx、GST、GR)与非酶系统(GSH/GSSG、As A/DHA)均呈现出“低促高抑”现象;细胞总活性氧(reactive oxygen species,ROS)水平升高,膜脂过氧化程度加剧,表明活性氧信号通路参与了亚麻酸对东海原甲藻的克生过程。亚麻酸胁迫下,东海原甲藻细胞内Ca~(2+)-ATPase活性出现先上升后下降趋势,表明钙离子信号通路在东海原甲藻对亚麻酸胁迫的响应中发挥了作用。亚麻酸胁迫下,外加钙效应剂Ca Cl_2增强了抗氧化酶GPx活性,而外加胞外钙螯合剂EGTA、质膜钙通道阻断剂La Cl_3、胞内钙螯合剂BAPTA,均显著抑制GPx活性。因此,亚麻酸胁迫下,钙离子参与了东海原甲藻的氧化应激反应,且胞质Ca~(2+)增加来自胞外Ca~(2+)内流和胞内钙库的离子释放。外加抗氧化剂NAC后,钙泵活性被显著抑制,推断东海原甲藻钙离子信号通路的激发有活性氧的参与。因此两条通路间存在交互关系。3.亚麻酸对东海原甲藻细胞凋亡机制的研究扫描电镜观察表明:亚麻酸处理后,藻细胞外形发生明显变化,出现破裂现象;细胞个体不分明,表面出现大量絮状物,此絮状物随着亚麻酸处理浓度升高而增加。透射电镜观察表明:亚麻酸处理组的东海原甲藻细胞胞质收缩,出现明显质壁分离现象,体积变小;细胞壁、细胞膜、液泡膜溶解断裂;叶绿体膜界限模糊,基粒片层结构松散甚至消失;多数细胞出现解体。利用带有绿色荧光的荧光探针FITC标记的Annexin V检测细胞凋亡时出现在细胞膜表面的磷酯酰丝氨酸,结果显示:亚麻酸处理东海原甲藻后,大部分细胞进入到凋亡晚期。亚麻酸胁迫下东海原甲藻线粒体膜电位降低,Cyt-C释放量增多,Bcl-2含量降低,Caspase-9,3活性升高等指标证实克生物质诱导细胞发生凋亡,且由线粒体途径介导。
[Abstract]:Red tides is a marine ecological anomaly. In recent years, with the development of industrial and agricultural development in China, the increase of eutrophication, the increasing frequency of red tide, especially the frequent toxic and harmful red tide, poses a serious threat to the marine aquaculture, human health and marine ecological safety. There are three main research contents in the field of environment. The prevention and control of red tide mainly include physical, chemical and biological methods. Among them, the use of large algae secreted by the gram biomass to inhibit the outbreak of red tide microalgae has a unique advantage, and has strong sustainability and environmental friendliness, and is increasingly being paid attention to by the researchers. This paper uses experimental ecology. In this study, the physiological and biochemical responses and apoptosis related mechanisms of proto alga in the East China Sea under linolenic acid stress were studied. This study provides a theoretical basis for the prevention and control of the red tide of the dominant alga in the East China Sea. The results are as follows: 1. linolenic acid is used in the original armour of the East China Sea The growth of algae and physiological and biochemical effects of linolenic acid showed a significant "low inhibition" phenomenon to the growth of prozina in the East China Sea. The low concentration of linolenic acid had a promoting effect on the growth of the East China Sea. With the increase of linolenic acid concentration, the growth of the primary alga in the East China Sea decreased and the linolenic acid inhibited its growth. Under the stress of hemp acid, the content of soluble protein in the algal cells showed a rising and descending trend relative to the change of growth. The photosynthetic pigment content also appeared to rise first and then descend, which affected the photosynthetic efficiency of algae; the activity of nitrate reductase was "rising and falling", and the active oxygen signal pathway of the.2. linolenic acid stress under the stress of linolenic acid under the stress of the East China Sea Under the response to linolenic acid stress, the intracellular antioxidant enzyme system (CAT, SOD, GPx, GST, GR) and the non enzyme system (GSH/GSSG, As A/DHA) in the cells of the alga of the East China Sea showed a "low growth inhibition" phenomenon, and the level of total active oxygen (reactive oxygen species, ROS) increased, and the degree of membrane lipid peroxidation increased, indicating the active oxygen signaling pathway. Under linolenic acid stress, the activity of Ca~ (2+) -ATPase in the cells of the East China Sea tozobacteria increased and then declined, indicating that the calcium ion signaling pathway played a role in the response to linolenic acid stress in the East China Sea. Under linolenic acid stress, the addition of calcium effect agent Ca Cl_2 enhanced the oxidation resistance. Enzyme GPx activity, external calcium chelating agent EGTA, plasma membrane calcium channel blocker La Cl_3 and intracellular calcium chelating agent BAPTA, all significantly inhibit GPx activity. Therefore, under linolenic acid stress, calcium ions participate in the oxidative stress reaction of the protozina of the East China Sea, and the cytoplasmic Ca~ (2+) increases from the extracellular Ca~ (2+) internal flow and intracellular calcium library ion release. Plus anti oxygen. After the chemical agent NAC, the activity of calcium pump was significantly inhibited. It was concluded that the activation of the calcium ion signaling pathway was involved in the activity of active oxygen. Therefore, there was a interaction relationship between the two pathways and the interaction of.3. linolenic acid on the mechanism of apoptosis in the cells of the East China Sea. Split phenomenon; a large number of flocs appeared on the surface of the cells. The floc increased with the increase in the concentration of linolenic acid. Transmission electron microscopy showed that the cytoplasm of the cells in the linolenic acid treatment group contracted with the cytoplasm of the cell wall, the cell wall, the cell membrane, the vacuolar membrane, and the boundary of the chloroplast membrane. The structure of the granal lamellae was loosely and even disappeared; most cells were disintegrated. The phosphonyl serine appeared on the surface of the cell membrane by Annexin V marked with a fluorescent probe FITC with green fluorescence. The results showed that after linolenic acid treatment of Dong Haiyuan, most cells entered the late stage of apoptosis. The mitochondrial membrane potential of the totalgam in the East China Sea decreased, the release of Cyt-C increased, the content of Bcl-2 decreased, and the higher activity of Caspase-9,3 confirmed that the cell apoptosis induced by KB was mediated by the mitochondrial pathway.
【学位授予单位】：曲阜师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X173

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