铜锈微囊藻两种表型的生长生理特性及毒素组成比较分析

从滇池蓝藻水华中分离得到的铜锈微囊藻群体在实验室无机营养中解聚成单细胞,结果表明,群体微囊藻的生长速度明显低于单细胞微囊藻;前者具明显可见的胞外酸性多糖胶鞘,而单细胞则几乎没有;按常规方法分析比较两种细胞形态的毒性大小和毒素组成,发现群体微囊藻主要含有三种微囊藻毒素的异构体,而单细胞以MCLR为主;且单细胞微囊藻的毒性约为群体的10倍.二者的LDH和PGM同工酶酶谱也有差异.本研究为解释毒素的合成和调控机理提供了新的证据.       

滇池优势水华蓝藻微囊藻的环肽成分

从滇池优势水华蓝藻微囊藻的干藻粉中分离出一环肽化合物,经光谱数据分析鉴定为Anabeanopeptin F（1）,其结构为cyclo（Phe-MeAla-Hty-Ile—Lys）-NH—CO—NH—Arg。     

微囊藻毒素在滇池鱼体内的积累水平及分布特征

为了解富营养化水体中鱼体内微囊藻毒素(MC)的积累水平及其分布特征,2003年4月和9月份两次在滇池试验区采集了鲢、鳙和草鱼等鱼种,用ELISA方法对鱼体中肝、肾、空肠、胆、肌肉等不同组织中MC的含量进行了检测。结果表明,MC在所有样品中均能检测到,且主要分布在鱼体的肝肾脏和消化道等器官,而肌肉和非消化道器官中毒素含量相对较低。不同鱼种不同组织对MC的富集程度也明显不同,鲢鳙中肝脏和肾脏这两个主要的靶器官对MC的蓄积能力就远高于草鱼。同时,不同季节MC在鱼体内的积累水平也明显不同,4月份鱼样中MC的含量普遍低于9月份鱼样中MC的含量。最后按照WHO生活饮用水安全标准的建议进行推算,所有鱼肉中的MC均没有超过其推荐的人体每日可允许摄入量(≤0.04μg/kg人体重),初步推断鱼肉中MC暂时还未危及到人体健康,但仍具有潜在的风险性。     

微囊藻毒素对典型微生物生长及生理生化特性的影响

研究了不同浓度微囊藻毒素对典型微生物大肠杆菌和枯草芽孢杆菌生长及生理生化特性的影响。微囊藻毒素对大肠杆菌和枯草芽孢杆菌的生长和细胞活性具有一定的剂量效应,较高浓度微囊藻毒素对其生长和活性有短时间的抑制作用,随着处理时间的延长,细胞的生长和活性逐渐恢复。细胞内可溶性糖和可溶性蛋白的含量,处理组和对照组相比均有先上升后下降的趋势。结果表明,微囊藻毒素的处理对大肠杆菌和枯草芽孢杆菌具有一定的胁迫作用,细胞通过调节细胞内可溶性蛋白和可溶性糖的含量来抵抗外界胁迫,但随着处理时间的延长,细菌逐渐适应了这种胁迫,恢复正常的生长。     

微囊藻毒素对细长聚球藻生长及生理生化特性的影响

采用改良的微囊藻毒素提取、制备方法可获得一定纯度的MC。经HPLC分析 ,MC RR的含量在 95 %以上。用微囊藻毒素处理细长聚球藻 ,发现毒素能显著抑制聚球藻的生长 ,降低聚球藻的可溶性蛋白与可溶性、不可溶碳水化合物含量 ,改变PC/Chl比值 ,抑制光合系统PSⅡ活性 ,进而导致光合作用减弱 ,生化反应减慢 ,从而抑制该藻的细胞分裂 ,使生长受阻     

有机磷农药对滇池微囊藻生长和摄磷效应的影响

采集滇池水体作为铜绿微囊藻培养基,研究了两种有机磷农药(甲胺磷和辛硫磷)对微囊藻生长和摄磷效应的动力学规律。结果表明,在滇池水体中添加较低浓度的甲胺磷(0.8、1.6、3.2mg/L)和辛硫磷(0.02、0.06、0.1mg/L)均能不同程度地促进微囊藻的生长,且在HGZ培养基中抑制微囊藻生长的浓度在滇池水体中却能促进微囊藻的生长。微囊藻的生长取决于细胞内磷的浓度且对磷的吸收利用存在积累性,在微囊藻生长初期,摄取各形态磷的速率较快;随后微囊藻摄取各形态磷的速率较慢。总溶解磷(TSP)和溶解反应磷(SRP)是微囊藻优先摄取的磷形态,在生长过程中微囊藻利用了大量的溶解有机磷(DOP)作为磷源加速生长。这一特点对于微囊藻成为淡水湖泊富营养化发展过程中的一种重要优势种具有极为重要的作用。     

微囊藻毒素对油菜幼苗生长及抗氧化酶活性的影响

以'秦优9号'油菜为材料,研究不同浓度藻毒素(MC)对油菜种子萌发和幼苗生长、叶绿素含量和抗氧化酶活性的影响,以及并对MC-LR和-RR在幼苗体内的积累进行分析.结果表明:仅5 000 ng/mL MC处理的油菜种子发芽率和发芽势显著低于对照和其余浓度处理,而其他处理及对照间无显著差异;在高浓度(1 000～5 000 ng/mL)MC处理下,幼苗出现植株矮化、叶片呈黄褐色、叶面积减小,以及主根变短、变粗且根系呈现黄褐色等毒害症状;叶绿素含量随着藻毒素浓度的递增表现为先上升后下降趋势;MC对SOD活性具有抑制作用,但对POD的活性具有促进作用,CAT活性随MC浓度的增加表现为先上升后下降的趋势;单位鲜重幼苗体内MC-LR和-RR含量随MC处理浓度的升高而增加,但MC-LR和-RR的积累率随MC浓度的增加而减小.研究发现,高浓度微囊藻毒素能显著抑制油菜幼苗生长,同时显著改变其体内保护酶活性,且MC可被油菜植株吸收并在其体内积累.     

一种快速提取分析微囊藻毒素的方法

以五种群体和单细胞微囊藻及自然微囊藻水华为材料,在沸水浴中经不同时间处理,过滤后直接进行HPLC—UV检测,发现12min的沸水浴处理就足以达到抽提目的。研究中发现,去离子水比蒸馏水是更有效的抽提溶剂。传统的甲醇抽提结果与沸水浴处理的相对误差主要在0．2％—16．59％之间。结果还显示,群体微囊藻需要比单细胞微囊藻抽提更长时间。本研究提供了一种经过改进的高效、廉价和快速的微囊藻毒素抽提分析方法。     

滇池微囊藻病毒溶藻的研究

微囊藻在养殖中有时会发生藻体溶解现象 ,其特征为藻体在生长旺盛期时如有病毒污染会迅速将藻体彻底溶解 ,由绿色变为清澈的淡黄色或无色透明状。此种病状尚无文献报道。因此 ,对此病状进行了分离及电镜观察和核酸分析。分离的噬藻体有 2种形态 ,一种呈蝌蚪型 ,具有多面体的头部和长而不收缩的长尾。头部 6 0 .9nm× 6 1.4nm ,尾部 2 88nm× 10nm。另一种头部呈多面体 ,尾部明显短 ,有 2个刺突状结构 ,头部5 6nm× 5 5nm ,尾部 15 6nm× 12nm。噬藻体核酸经酶解证明为DNA ,分子量均在 2 9.8× 10 6u。     

微囊藻毒素转运酶MlrD结构及生物学特性分析

微囊藻毒素(Microcystins,MCs)是一类由有毒蓝藻产生的具有肝毒性的环状七肽,生物降解能有效且持续的去除MCs,其中Mlr 降解途径在 MCs 的生物降解过程中发挥了重要作用。转运酶MlrD被认为负责MCs及其降解产物的转运,但对于MlrD功能及其结构的研究依然缺乏。该文分析了MlrD的保守性并构建了其三级结构。结果表明,MlrD在MCs降解菌之间高度保守, MlrD二级结构由α-螺旋和无规则卷曲组成。进一步分析其活性位点为位于第2和3跨膜螺旋连接处的Gly69、Ala73、Asp74、Gly78、Arg79、Ala82、Ile83以及位于第5跨膜螺旋上的Phe142、Tyr143、Ala146、Val147、Gly150,可以通过突变或敲除活性位点研究其功能。该文为进一步研究MlrD的结构功能提供了理论基础,有助于深入了解MCs的转运及代谢机制。     

二种淡水微囊藻rDNA16S-235基因间隔区的序列测定与分析

本研究采用PCR及序列测定的方法,对我国淡水铜绿微囊藻有毒株(M8641)和另一低毒的种类惠氏微囊藻(M574)rDNA16S-23S基因间隔区进行了序列的测定和分析,结果表明:rDNA16S-23S基因间隔区可以作为一个精细且稳定的指标,用于微囊藻的分类和鉴定。并从分子水平提出了铜绿微囊藻与惠氏微囊藻在种系发生上有较近缘的关系。本文首次对微囊藻属Microcystis rDNA基因间隔区全序列作了报道,为微囊藻属的鉴定及系统学研究提供了分子基础。       

TOXIN EXTRACTION FROM THE BLUE-GREEN ALGA MICROCYSTIS AERUGINOSA BY DIFFERENT EXTRACTION MEDIA

SUMMARY

The extractability of toxin from Microcystis aeruginosa isolate UV-006 by different extraction media such as O, 1N HC1, 2N CH₃COOH, Triton X-100 and water at different pH levels was investigated. The best recovery of toxin was achieved with water at a pH of 10 while the recoveries with the more acidic media tended to be lower.     

FATE OF THE TOXIC CYCLIC HEPTAPEPTIDES,THE MICROCYSTINS,FROM BLOOMS OF MICROCYSTIS (CYANOBACTERIA) IN A HYPERTROPHIC LAKE1

The in situ fate of the toxic cyclic heptapeptides, the microcystins, produced by blooms of Microcystis was examined at two stations in a hypertrophic Japanese lake. Microcystins were detected in all samples of Microcystis with quantities varying seasonally and spatially (230–950 μg · g dry wt^?1 at St. 1 and 160–746 μg · g dry wt^?1 at St. 2) and composed of microcystin-LR, -RR, and-YR. Microcystin-RR was the dominant toxin in most samples. A large amount of microcystin (1.1 μg · L^?1) was detected in only one sample of filtered lake water. Accumulation of microcystin in zooplankton was indirectly estimated from a newly developed equation model. Large amounts of microcystin (75–1387 μg · g dry wt^?1) were accumulated in the zooplankton community, which consisted of two cladocerans, Bosmina fatalis Burckhardt and Diaphanosoma brachyurum Lieve, and a copepod, Cyclops vicinus Uljanin, that co-occurred with the toxic Microcystis blooms. The maximum percent of microcystin content in zooplankton to that in Microcystis was 202%. Among the three species of zooplankton, only B. fatalis seemed to be responsible for accumulation of the microcystins because C. vicinus appeared to avoid contact with Microcystis cells and D. brachyurum did not consume colonies of Microcystis. Microcystins may be transferred to higher trophic levels through B. fatalis.     

CHANGES IN THE MORPHOLOGY AND POLYSACCHARIDE CONTENT OF MICROCYSTIS AERUGINOSA (CYANOBACTERIA) DURING FLAGELLATE GRAZING1

To investigate the changes in the morphology and polysaccharide content of Microcystis aeruginosa (Kütz.) Kütz. during flagellate grazing, cultures of M. aeruginosa were exposed to grazing Ochromonas sp. for a period of 9 d under controlled laboratory conditions. M. aeruginosa responded actively to flagellate grazing and formed colonies, most of which were made up of several or dozens of cells, suggesting that flagellate grazing may be one of the biotic factors responsible for colony formation in M. aeruginosa. When colonies were formed, the cell surface ultrastructure changed, and the polysaccharide layer on the surface of the cell wall became thicker. This change indicated that synthesis and secretion of extracellular polysaccharide (EPS) of M. aeruginosa cells increased under flagellate grazing pressure. The contents of soluble extracellular polysaccharide (sEPS), bound extracellular polysaccharide (bEPS), and total polysaccharide (TPS) in colonial cells of M. aeruginosa increased significantly compared with those in single cells. This finding suggested that the increased amount of EPS on the cell surface may play a role in keeping M. aeruginosa cells together to form colonies.     

THE ACCLIMATIVE CHANGES IN PHOTOCHEMISTRY AFTER COLONY FORMATION OF THE CYANOBACTERIA MICROCYSTIS AERUGINOSA1

Microcystis aeruginosa (Kütz.) Kütz. commonly occurs as single cells at early recruitment but forms large colonies in summer. Colony formation will induce many acclimative changes. In this study, we demonstrated the photochemical changes before and after colony formation. In the laboratory, light curves showed that colonies were more responsive to high light than single cells. The values of the maximal slope of electron transport rate (ETR)—light curve (α), relative maximal electron transport rate (rETR_max), and onset of light saturation (I_k) of colonies were significantly higher than those of single cells (P < 0.05), indicating that colonies have higher photosynthetic capability than single cells, especially in high light, where values of rETR_max and I_k of colonies were 2.32 and 2.41 times those of single cells. Moreover, the dark‐light experiments showed that colonial cells can more effectively resist darkness damage. In addition, pigments of colonial cells were higher than those of single cells (P < 0.05). The higher pigment contents probably contribute to higher photosynthetic capability. In the field, the inhibition rate of F_v/F_m in single cells increased significantly faster than that of colonies as light increased (P < 0.05), but nonphotochemical quenching (NPQ) value of colonies was higher (32.4%) than that of single cells at noon, which indicated colonial cells can more effectively resist high‐light inhibition than single cells (P < 0.05). Polysaccharides of colonies were significantly higher compared to those in unicellular cells (P < 0.05) based on their contents and ultrastructural characteristics. This finding implies that colonies could not effectively decrease photoinhibition by negative buoyancy regulation. In fact, NPQ may be an important mechanism for avoiding photodamage. All of these phenomena can help explain the ecological success of colonial M. aeruginosa in eutrophic water.     

滇池水体理化环境状况时空分布格局研究

在滇池全湖设置了 4 0个采样点 ,从 2 0 0 2年 9月至 2 0 0 3年 8月 ,按每月一次的频度对滇池水体的pH、溶解氧、透明度、总磷、溶解性总磷、溶解性磷、总氮、氨氮、Chla进行了为期一年的监测。结果表明 ,滇池外海水质已属V类及超V类水质 ,重富营养程度 ,只能满足工业和农业供水水质要求。滇池外海水质存在区域性强的特点 ,其污染程度与该地区的工农业分布格局相关 ,西北部水质污染情况最为严重 ,北部次之 ,南部最轻 ;在不同月份水质存在明显差异 ,在汛期 (5— 9月份 )水质污染最为严重 ,冬春季节则较轻。本研究初步揭示了滇池污染的现状及时空分布规律 ,为分区域治理滇池水污染提供了科学依据     

COMPARISON OF RESISTANCE TO LIGHT STRESS IN TOXIC AND NON‐TOXIC STRAINS OF MICROCYSTIS AERUGINOSA (CYANOPHYTA)1

Blooms of Microcystis aeruginosa (Kützing) Kützing occur frequently in many freshwater ecosystems around the world, but the role of environmental factors in promoting the growth and determining the proportion of toxic and non‐toxic strains still requires more investigation. In this study, four strains (toxic CPCC299 & FACHB905 and non‐toxic CPCC632 & FACHB315) were exposed to high light (HL) condition, similar to light intensity found at the surface of a bloom, to evaluate their sensitivity to photoinhibition. We also estimated their capacity to recover from this HL stress. For all strains, our results showed an increased inhibition of the photosynthetic activity with HL treatment time. When comparing the extent of photoinhibition between strains, both toxic strains were more resistant to the treatment and recovered completely their photosynthetic capacity after 3 h, while non‐toxic strains needed more time to recover. For toxic strains, the rETR under HL was higher compared to the rETR under low light (LL) control condition despite 50% photoinhibition. This suggests that the detrimental effect of high light (HL; up to 2 h) is outweighed by their higher photosynthetic potential. This conclusion did not stand for non‐toxic strains, and indicates their preference for LL environment. We also demonstrated that a LL/HL cycle induced a 259% increase in cell yield for a toxic strain and a decrease by 22% for a non‐toxic strain. This also indicates that toxic strains have higher tolerance to HL in a fluctuating light environment. Our data demonstrated that difference of sensitivity to HL between strains can modify the competitive outcome between toxic and non‐toxic strains and may affect bloom toxicity.     

大漂提取物抑制铜绿微囊藻的试验研究

为了确定高效脱氮除磷的水生漂浮植物大漂的主要化感活性物质, 本文通过乙醇超声波提取获得大漂的化感粗提物, 利用溶剂萃取法从粗提物中分离得到4种能抑制铜绿微囊藻(FACHB-911)生长的化感萃取物,当其浓度均为0.1 g/L时共培养5d后抑藻率分别为40%、69%、40%、35%。将抑藻率最高的化感萃取物通过酸性氧化铝柱层析法进一步分离纯化, 分离得到4组抑藻作用较弱的(5d后抑藻率分别为12%、11%、33%、41%)和1组抑藻作用较强的化感层析流分(5d后抑藻率高达80%)。将抑藻作用最强的化感层析流分通过气相色谱-质谱联用(GC-MS)方法进行成分检测, 发现大漂提取物中含有对甲苯磺酸甲酯、硬脂酸酰胺两种具有化感潜力的化合物。