亞麻酸對東海原甲藻的克生效應(yīng)及細(xì)胞凋亡機(jī)制的研究
發(fā)布時間：2018-07-25 12:14
【摘要】：赤潮(Red tides)是一種海洋生態(tài)異常現(xiàn)象。近年來,隨著我國工農(nóng)業(yè)發(fā)展,水體富營養(yǎng)化加重,赤潮發(fā)生頻率增高,尤其是有毒有害赤潮頻發(fā),對海洋養(yǎng)殖業(yè)、人類健康及海洋生態(tài)安全構(gòu)成嚴(yán)重威脅。當(dāng)前,赤潮的有效治理和防治已成為海洋環(huán)境領(lǐng)域的重要研究內(nèi)容。赤潮的防治主要有物理法、化學(xué)法和生物法三種方法。其中,利用大型海藻分泌的克生物質(zhì)抑制赤潮微藻的爆發(fā)獨(dú)具優(yōu)勢,有著較強(qiáng)的可持續(xù)性和環(huán)境友好的特點(diǎn),日益受到研究者的重視。本文采用實(shí)驗(yàn)生態(tài)學(xué)方法,以赤潮優(yōu)勢藻種東海原甲藻為研究對象,探究高克生活性物質(zhì)亞麻酸脅迫下東海原甲藻的生理生化響應(yīng)及細(xì)胞凋亡相關(guān)機(jī)制。本文的研究為防控以東海原甲藻為優(yōu)勢藻種的赤潮提供了理論基礎(chǔ)。具體結(jié)果如下:1. 亞麻酸對東海原甲藻生長及生理生化的影響亞麻酸對東海原甲藻生長表現(xiàn)出顯著的“低促高抑”現(xiàn)象。低濃度的亞麻酸對東海原甲藻的生長具有促進(jìn)作用(負(fù)抑制),隨著亞麻酸濃度升高,東海原甲藻生長量逐漸減小,亞麻酸對其生長表現(xiàn)為抑制作用。亞麻酸脅迫下,藻細(xì)胞可溶性蛋白含量表現(xiàn)出與生長量變化相對應(yīng)的先上升后下降趨勢;光合色素含量也出現(xiàn)先升后降的現(xiàn)象,影響了藻類的光合效率;硝酸還原酶活性出現(xiàn)“升—降”趨勢。2.亞麻酸脅迫下東海原甲藻活性氧信號通路與鈣離子信號通路的響應(yīng)亞麻酸脅迫下,東海原甲藻細(xì)胞內(nèi)抗氧化酶系統(tǒng)(CAT、SOD、GPx、GST、GR)與非酶系統(tǒng)(GSH/GSSG、As A/DHA)均呈現(xiàn)出“低促高抑”現(xiàn)象;細(xì)胞總活性氧(reactive oxygen species,ROS)水平升高,膜脂過氧化程度加劇,表明活性氧信號通路參與了亞麻酸對東海原甲藻的克生過程。亞麻酸脅迫下,東海原甲藻細(xì)胞內(nèi)Ca~(2+)-ATPase活性出現(xiàn)先上升后下降趨勢,表明鈣離子信號通路在東海原甲藻對亞麻酸脅迫的響應(yīng)中發(fā)揮了作用。亞麻酸脅迫下,外加鈣效應(yīng)劑Ca Cl_2增強(qiáng)了抗氧化酶GPx活性,而外加胞外鈣螯合劑EGTA、質(zhì)膜鈣通道阻斷劑La Cl_3、胞內(nèi)鈣螯合劑BAPTA,均顯著抑制GPx活性。因此,亞麻酸脅迫下,鈣離子參與了東海原甲藻的氧化應(yīng)激反應(yīng),且胞質(zhì)Ca~(2+)增加來自胞外Ca~(2+)內(nèi)流和胞內(nèi)鈣庫的離子釋放。外加抗氧化劑NAC后,鈣泵活性被顯著抑制,推斷東海原甲藻鈣離子信號通路的激發(fā)有活性氧的參與。因此兩條通路間存在交互關(guān)系。3.亞麻酸對東海原甲藻細(xì)胞凋亡機(jī)制的研究掃描電鏡觀察表明:亞麻酸處理后,藻細(xì)胞外形發(fā)生明顯變化,出現(xiàn)破裂現(xiàn)象;細(xì)胞個體不分明,表面出現(xiàn)大量絮狀物,此絮狀物隨著亞麻酸處理濃度升高而增加。透射電鏡觀察表明:亞麻酸處理組的東海原甲藻細(xì)胞胞質(zhì)收縮,出現(xiàn)明顯質(zhì)壁分離現(xiàn)象,體積變小;細(xì)胞壁、細(xì)胞膜、液泡膜溶解斷裂;葉綠體膜界限模糊,基粒片層結(jié)構(gòu)松散甚至消失;多數(shù)細(xì)胞出現(xiàn)解體。利用帶有綠色熒光的熒光探針FITC標(biāo)記的Annexin V檢測細(xì)胞凋亡時出現(xiàn)在細(xì)胞膜表面的磷酯酰絲氨酸,結(jié)果顯示:亞麻酸處理東海原甲藻后,大部分細(xì)胞進(jìn)入到凋亡晚期。亞麻酸脅迫下東海原甲藻線粒體膜電位降低,Cyt-C釋放量增多,Bcl-2含量降低,Caspase-9,3活性升高等指標(biāo)證實(shí)克生物質(zhì)誘導(dǎo)細(xì)胞發(fā)生凋亡,且由線粒體途徑介導(dǎo)。
[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.
【學(xué)位授予單位】：曲阜師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X173
本文編號：2143790
[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.
【學(xué)位授予單位】：曲阜師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X173
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