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题名: 微囊藻衰亡过程生理生化特征研究
作者: 李杰
答辩日期: 2008-06-15
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 微囊藻 ; 死亡 ; 生命力评价 ; MTT ; 浮力调控 ; 程序性死亡 ; Ca2+ ; Cu2+
其他题名: Studies on the Physiological and Biochemical Changes during Declining Process of Bloom-Forming Microcystis (Cyanobacteria)
摘要: 蓝藻水华因其对水环境水生态和人类健康的直接或潜在危害成为世界范围的热点问题。围绕蓝藻水华的研究,长期以来主要集中在水华的暴发过程和机理,很少关注蓝藻水华的衰亡过程及其机理。蓝藻水华有害有毒次生代谢产物引起的危害多发生在水华衰亡时期,因此,深入研究蓝藻水华的衰亡过程及其机理具有重要的理论和应用意义。基于以上认识,本论文对室内模拟胁迫和野外冬季微囊藻的衰退过程中微囊藻的生理生化特征进行了研究,探讨了不同条件下微囊藻的死亡特征,发展了微囊藻室内培养物和野外藻样的生命力评价方法,具体研究内容和研究结果如下: (1)设计了四组模拟胁迫条件,N胁迫、P胁迫、黑暗和4 ℃低温,比较研究了这四种模拟胁迫条件下Microcystis aeruginosa FACHB 905衰亡过程以及衰亡过程中生长生理与抗氧化酶系统的变化。结果表明,在黑暗和低温条件下,M.aeruginosa生长受到明显抑制,但黑暗条件下25天后M.aeruginosa衰亡,低温条件下仅需7天;与物理因子引起的胁迫相比,在营养胁迫条件下,M.aeruginosa在胁迫前期均表现出生长趋势。N饥饿条件下叶绿素a含量从开始胁迫后即下降,P饥饿20天后叶绿素a含量以0.1 mg.L-1.d-1急剧下降,在四种胁迫条件中下降速率最高,表明M.aeruginosa对P的长期耐受能力最差。Fv:Fm和ETRmax的下降发生在不同的生理阶段:在细胞转入胁迫条件后,ETRmax即开始下降,而Fv:Fm比值下降较晚。在低温胁迫条件下,Fv:Fm比值与ETRmax下降尤为明显。酯酶活性在低温胁迫条件下3天内即观察到显著明显,在N、P胁迫初期也观察到微量上升,仅在黑暗条件下逐步下降。这一结果表明,当外部环境对生长不利时,细胞可能增加特定酯酶活性改善其生存状况,只在特定情形如黑暗胁迫下,酯酶活性与细胞光合活性相关。M.aeruginosa在受到胁迫后,SOD和CAT活性都会上升,但两者上升过程表现方式不同,SOD在胁迫条件下平稳上升,而CAT在胁迫前期升幅较小,而在胁迫后期叶绿素含量下降阶段可观察到大幅度的上升,M.aeruginosa中的CAT可能为诱导酶。 (2)以M.aeruginosa FACHB 905为研究对象,比较了MTT法和FDA、Evan’s Blue染色法及自发荧光法在藻类生命力评价中的优缺点。对MTT法染色条件包括MTT浓度、反应时间、反应温度等进行了优化,对MTT法的灵敏度与可靠性进行了改进,包括:DMSO提取MTT还原产物甲臢时应在黑暗条件下进行以减少DMSO环境下甲臢的光降解, MTT法应通过阳性细胞比率反应样品生命力。MTT还原酶NADH氧还脱氢酶对胁迫条件的响应与酯酶类适,该酶在死亡微囊藻中的半衰期为3 h。MTT法可应用于蓝细菌、硅藻、红藻生命力评价。 (3)以滇池微囊藻为研究对象,比较水样中微囊藻漂浮细胞与沉降细胞差异,结果表明野外样品细胞的沉降与细胞生理状态有关,漂浮细胞具有更高的光合能力与MTT还原能力,漂浮细胞比沉降细胞健康。微囊藻的沉降比率与MTT法、Evan’s Blue法估测的生命力结果显著相关,可以将微囊藻的沉降比率应用于野外样品的生命力评价,特别是水体中不同形态的群体微囊藻生命力评价。 (4)滇池冬季微囊藻衰亡与沉降过程研究。滇池冬季以小群体微囊藻为主,群体直径比夏季群体直径小,但单个细胞直径比夏季单个细胞直径大。微囊藻水华衰退的平均气温为10.2 ℃,平均水温为14.3 ℃。微囊藻光合系统活性在冬季显著下降,沉降装置中收集到的微囊藻光合活性显著低于表层水样,装置底部样品MTT还原能力、Evan’s Blue阴性比率也显著低于表层水样。微囊藻的沉降是滇池微囊藻水华衰退的重要原因,沉降方式存在以藻细胞和以碎屑沉降两种方式;微囊藻至少存在两种死亡方式,一种死亡方式中细胞破裂而亡只剩下破裂后的堆积物,另一死亡方式中微囊藻细胞皱缩而亡,死亡过程中始终维持细胞形态;微囊藻的死亡过程在胶被内完成,水体里单细胞比例不足3%。 (5)研究了Ca2+ 作用下微囊藻的死亡方式。Ca2+引起微囊藻室内培养物M.aeruginosa FACHB 905死亡的最低作用浓度为0.1 mol.L-1,Ca2+作用下M.aeruginosa细胞膜通透性快速改变,先于光合系统、MTT还原能力的下降,但微囊藻毒素未见大量释放。Ca2+引起M.aeruginosa的死亡属程序性死亡,主要证据包括:细胞内含物的降解为有规律有步骤的降解,细胞彻底瓦解前只剩下类囊体搭建的细胞框架;细胞死亡过程中观测到DNA断裂与类Caspase-3活性表达;Ca2+引起的死亡可被4-16 μg.mL-1氯霉素抑制,低温光照条件也可以阻止Ca2+引起的死亡。但这种死亡方式与真核生物中经典的程序性死亡存在差异,Caspase-3抑制剂Z-VAD-FMK对M.aeruginosa细胞存在毒性,不仅不能抑制M.aeruginosa的死亡,反而加速细胞死亡进程。 (6)研究了0.25 mg.L-1 Cu2+作用下微囊藻的死亡方式。0.25 mg.L-1 Cu2+处理30 min后,转接到正常BG11培养基中已彻底丧失繁殖能力,但其生命特征包括光合作用、MTT还原能力等在8 h内仍有检测到,Evan’s Blue染色表明24 h仅20%的细胞丧失膜通透性,叶绿素48 h仅下降到58%,由于细胞壁不裂解,96 h细胞数仅稍有下降。微囊藻毒素在前4 h大量释放。0.25 mg.L-1 Cu2+作用下微囊藻仅少量细胞的死亡方式属程序性死亡,电镜观察表明细胞内含物的降解无选择性,氯霉素、低温黑暗等不能阻止Cu2+作用下微囊藻的死亡。0.25 mg.L-1 Cu2+下微囊藻的死亡为藻类生命力评价方法发展提供了一个理想的研究模型。
英文摘要: Phytoplankton evolved in the Archaean oceans more than 2.8 billion years ago and are of crucial importance in regulating aquatic food webs, biogeochemical cycles and the Earth’s climate. For a better understanding of phytoplankton dynamics, it is important to know the processes that affect both the increase and decline of a population. In addition, release of intracellular content, such as Microcystin, odours compound, during the cell death, contaminated the drinking water, posing a threat to human and livestock health. However, loss via cell death has been poorly understood. This paper focused on the cell death process of harmful bloom algae Microcystis (Cyanobacteria), characterized the physiological change when Microcystis suffered from different artificial or natural stresses to death, and developed viability assessment methods for Microcystis pure culture and filed colonies. The main results are as followings: 1. The decaying process of M.aeruginosa FACHB 905 under nitrogen starvation, phosphorus starvation, dark or low temperature (10 ℃) was investigated. The decrease of biomass and the concomitant change of photosynthetic activity, esterase activity and antioxidant system, including superoxide dismutase (SOD) and catalase (CAT) activities were studied. The results indicated that the growth was significantly inhibited under dark and low temperature. Compared with the physical stress, nutrition stress such as nitrogen and phosphorus starvation did not limit the growth effectively in the early stage. After being kept in the phosphorus free medium for 20 days, Chlorophyll a reached peak, about the twice of the primary concentration, and then decreased at a rate of 0.1 mg.L-1.d-1, the highest decaying rate among the four stress factors, which indicated its significance of available phosphorus for Microcystis long survival. Esterase activity increased significantly under low temperature, also increased slightly under nitrogen and phosphorus stresses. Only under dark stress, the decrease of esterase activity correlated with the decrease of photosystem activity. The increase of SOD and CAT activity presented the similar behaviour as the decrease of Fv:Fm and ETRmax. SOD activity increased in the early stress stage, while CAT activity did not increase significantly until the content of Chlorophyll a decreased. 2. Several assay methods were screened for viability assessment in cyanobacteria using M.aeruginosa FACHB 905. Compared with FDA, Evan’s Blue and autofluorescence, the MTT assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. The MTT assay was also found to be applicable to other Cyanobacteria and Diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. 3. The differences of floating and sediment Microcystis colonies from water surface were compared. The floating colony presented healthier color green, owned higher photosynthetic activity and MTT reducing ability. The ratio of the sediment in the mixed sample correlated significantly with the viability tested by the MTT, Evan’s Blue staining. The results suggested that the sediment of Microcystis should be related to the physiological status. The ratio of the sediment could be employed to estimate the viability of field Microcystis without disaggregating the colony. 4. Decline and sedimentation of Microcystis in Dianchi Lake was investigated in the winter of 2007. Photosynthetic activity decreased significantly in winter. Microcystis sank in two types, as structured or blank colony. Based on the observation, Microcystis died at least in two ways: cell broke down and left intracellular content or shrunk and died. The death process was completed in the capsule, which was speculated from the fact that the capsule remains kept the colony shape and the percentage of the single cell in the water body was less than 3%. 5. Cell death of M.aeruginosa FACHB 905 upon Ca2+ exposure was studied. Upon 0.1 mol.L-1 Ca2+ exposure, cell membrane permeability changed quickly before the decrease of photosynthetic activity and MTT reducing ability, however, intracellular microcystin release was not significant. The death of M.aeruginosa FACHB 905 should belong to programmed cell death since the markers of programmed cell death-DNA fragmentation and the Caspase-3 like activity were detected. In addition, the death could be blocked by the protein synthesis inhibitor chloromycin, also inhibited under low temperature (4 ℃) condition with light. The specific Caspase-3 inhibitor Z-VAD-FMK did not block the cell death, on the contrary, it accelerated the cell death even without excess Ca2+ exposure. 6. Cell death of M.aeruginosa FACHB 905 upon 0.25 mg.L-1 Cu2+ exposure was studied. After 30 min in BG11 medium containing 0.25 mg.L-1 Cu2+, all of the cells were unable to reproduce. The indicator of viable cell including photosynthetic activity, MTT reducing ability lost till 8 h later. The total content of Chlorophyll a decreased to 42% in 48 h, but the cell number did not changed significantly since the remains kept the cell shape. Microcystin was released dramatically in the early 4 hours, but the change of membrane permeability tested by the Evan’s Blue staining was not as significant as expected, and only 20% of the cells presented ruptured membrane. The results suggested that the death of M.aeruginosa upon 0.25 mg.L-1 Cu2+ exposure would be a perfect model to study the essence of prokaryotic life and to develop more accurate viability assessment methods.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12306
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
微囊藻衰亡过程生理生化特征研究.李杰[d].中国科学院水生生物研究所,2008.20-25
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