Proteomics of a toxic dinoflagellate Alexandrium catenella DH01: Detection and identification of cell surface proteins using fluorescent labeling

Alexandrium catenella DH01 is a toxic dinoflagellate species that is able to not only produce paralytic shellfish toxins,but also cause harmful algal blooms along the coast of China.In this study,we presented a new protocol for specific labeling and detection of the cell surface proteins(CSPs) of A.catenella DH01 cells using CyDye difference gel electrophoresis(DIGE) fluor minimal dyes.CSPs were identified using two-dimensional gel electrophoresis(2-DE) and MALDI TOF-TOF mass spectrometry(MS).The results showed that the fluorescent cyanine dye Cy3 could specifically label the CSPs of A.catenella DH01,with minimal labeling of intracellular proteins.Among three protein extraction methods evaluated,the Trizol method was the most efficient to extract CSPs with respect to protein spot number and resolution.Forty-one CSPs were separated and identified from A.catenella DH01 by 2-DE,in which 14 were identified in the protein database using MALDI TOF-TOF MS analysis.This work represents the first attempt to investigate the CSPs of A.catenella using the CyDye DIGE fluor dyeing method that provides a potentially important tool for future comprehensive characterization of CSPs and elucidation of the physiological functions of CSPs in dinoflagellates.     

BP神经网络水华预测模型的敏感性分析

敏感性分析能够定量地评价模型输入变量的变化对输出结果产生的影响,是揭示模型蕴含规律的有效途径.本文将敏感分析方法应用于BP神经网络巢湖水华预测模型中,分析结果表明巢湖水华形成受诸多环境因子共同影响,水温、溶解氧、浊度、气温、光照强度等环境因子变化与藻类质量浓度变化相关,其中气温是最大影响因素,相对贡献率达到17.01%;气压的上升则不利用于藻类质量浓度的增加;pH值的上升对藻类质量浓度的影响有正有负.     

Using quantile regression to analyze the stressor–response relationships between nutrient levels and algal biomass in three shallow lakes of the Lake Taihu Basin,China

Understanding the stressor–response relationship between nutrient levels and algal biomass is a prerequisite for the management of eutrophication in lakes. In this study, a quantile regression(QR) approach was used to interpret the stressor–response relationships between nutrient(e.g., phosphorus, nitrogen) concentrations and algal biomass as measured by chlorophyll-a(Chl-a) levels. QR results indicated that Lake Dianshan and Lake Changdang,which are both heavily eutrophicated, were P-limited only.In contrast, Lake Kuilei, which has significantly lower nutrient levels and algal biomass than the other two lakes,was P- and N-limited. Moreover, in Lake Kuilei, N and P levels had significant interaction effects on the algal biomass at the upper quantiles(s [ 0.68). The degree to which the lakes were P-limited increased with rises in the mean total P concentration in the lakes. QR has many advantages over ordinary least squares regression for discriminating limiting factors and, in particular, allows us to estimate changes near the upper extremes of distributions associated with limiting factors. QR is adapted to more specialized risk management problems, such as early warnings of the risk of algal blooms.The probable value-at-risk of harmful algal blooms for Lake Kuilei, Lake Dianshan and Lake Changdang is s = 0.76(Chl-a = 9 mg/m3), s = 0.87(Chl-a = 24 mg/m3) and s = 0.72(Chl-a = 35 mg/m3), respectively. Given the results of the stressor–response relationship analysis, we conclude that reductions in P input represent the most feasible and least costly approach for managing eutrophication in the shallow lakes of the Lake Taihu Basin. To control the magnitude and duration of algal blooms in shallow lakes,reductions in P and N inputs are required. The 95 % CI bounds of slopes indicated that the interactions of nonnutrient factors with nutrients had strong impacts on the algal biomass in lightly eutrophicated Lake Kuilei. It is suggested that, in addition to reductions in nutrient loads,several ecological measures, such as an increase in the biomass of submerged macrophytes and the reduction of hydraulic retention time by flushing, could represent important components of an integrated approach to eutrophication management in the Lake Taihu Basin.     

基于氮磷比的赤潮灾害危险度评估方法研究

 为了定量表示氮磷比对赤潮灾害危险度的影响,在赤潮藻类最大比生长速率的氮磷比耐受性模型和赤潮暴发基准细胞密度的基础上,建立了氮磷比与赤潮暴发时间的关系模型,并据此划分危险度等级且归一化得到危险度值,并以中肋骨条藻为例进行了应用分析。结果表明基于氮磷比的赤潮暴发时间模型受初始密度、氮磷比的影响,且随初始密度、氮磷比的变化呈倒高斯形态变化。依据赤潮藻类在不同氮磷比下的耐受特性,赤潮灾害危险度可划分为高危险度、中危险度、低危险度三个等级。在高危险度区,最大比生长速率大,赤潮暴发时间最短,赤潮发生的可能性最高;在中危险度区,最大比生长速率小,赤潮暴发时间长,赤潮发生可能性低;在低危险度区,最大比生长速率趋于初始比生长速率,赤潮暴发时间最长,赤潮发生可能性最低。     

BP神经网络在水华短期预测中的应用

为解决影响因素多、作用关系复杂的水华预测问题,将BP神经网与水体环境因子的高频实测数据相结合,构建了巢湖水华的短期动态预测模型,该模型准确地预测了每次水华发生的时间,预测值与实际观测值相关系数可达0.608 4;在分析BP神经网络自身局限性的基础上,研究了建模过程中输入输出数据预处理、网络结构设计、训练模式选择等问题,给出了水华预测中确定环境因子和建模方案的具体方法.该方法容易移植到其它湖库,提高了模型的实用性和通用性.     

2-甲基乙酰乙酸乙酯（EMA）抑制藻类生长的研究进展

水体富营养化及其引起的有害藻类水华是重要的环境问题,严重威胁着水体景观、生态功能以及人类健康.如何有效防治水华是目前环境领域的研究热点和前沿.通过植物的化感作用来抑制藻类生长作为一种新型的生物抑藻技术而备受关注.而从水生植物中提取、分离出的化感物质更是对控制水华爆发具有专一、高效的特点.综述了从自然环境里较易获得的芦苇中得到的化感物质EMA对常见水华和赤潮藻类的影响,对抑藻效果和抑制机理进行了分析阐述,并提出了目前仍存在的问题和该领域内的发展前景.     

不同湖泊水华发生机制研究进展

目前,世界各地湖泊水华的暴发越来越严重,对人类健康和生态系统有重大危害.本文调研了国内外典型湖泊水华的相关研究成果,从湖泊所属地理位置、气候带、受人类活动干扰强度、主要水华发生藻种和主控环境因子等不同角度进行了总结和分析,揭示了不同类型的湖泊水华特征和发生机制.最后以我国太湖为例,总结已有的研究成果,结合近几年太湖水华和水环境实测数据的分析结果,归纳了太湖水华的主要影响因子,包括Chl-a、TN、TP、COD、温度、风速六个指标,其中Chl-a、COD能指示说明水华的程度,TN、TP是营养盐状况的代表,温度和风速是水华发生的主要气象因子,并定量分析了水华程度和影响因子之间的关系,为水华的预警、预测和评价提供基础和支持.     

香溪河库湾春季水华纵向分布对水层结构的响应

为研究三峡水库支流库湾春季水华暴发空间差异及成因,本研究于2010年4月18日对三峡水库干流和香溪河库湾的水动力、营养盐、浮游植物等因子进行了现场监测.结果显示：三峡水库支流春季水华浮游植物生物量及群落结构空间差异较大,库湾中上游暴发了严重的硅藻水华,而下游及三峡水库干流没有水华;库湾中复杂的分层异重流对水温结构产生显著影响,使其呈独特的垂向分层;水温分层纵向差异导致的光混比（Zeu/Zmix）纵向分布差异是引起水华情势和浮游植物群落结构纵向差异的主要原因.研究结果将为三峡水库支流水华机理和防控措施研究提供支撑.     

Proteomic analysis of a toxic dinoflagellate Alexandrium catenella under different growth phases and conditions

Alexandrium is a widely spread dinoflagellate genus throughout many regions of the world,which not only causes the harmful algal blooms(HABs) but also results in the paralytic shellfish poisoning(PSP) throughout the world.This study compared protein profiles of A.catenella grown under different growth phases and conditions using a proteomic approach,and identified the differentially expressed proteins.The results showed that the expressions of proteins identified in three different regions of the gels,the groups 1,2 and 3 proteins,varied significantly with the growth phases and conditions.Group 1 proteins and six Group 2 proteins were highly expressed at the initial,exponential and stationary growth phases,eight Group 2 proteins were highly expressed only at the initial phase,and Group 3 proteins were highly expressed at the exponential and/or stationary phases.However,all these proteins were expressed at low levels or were barely visible at the dissipation phase.The expressions of groups 1 and 2 proteins were low or barely visible in various growth conditions except in continuous darkness they were highly expressed.Group 3 proteins,on the other hand,were overexpressed in continuous illumination and expressed at low levels or barely visible in continuous darkness or under nitrate-starvation.The data from MALDI-TOF-TOF mass spectrometry demonstrated that these differentially expressed proteins were associated with macromolecular biosynthesis,photosynthesis,tRNA synthesis and DNA stability,stress response and cell division regulation.Synthetase was the major component of the altered proteins.This is one of the first comprehensive proteomic study of a dinoflagellate,A.catenella,that provides a fundamental understanding of the proteins involved in A.catenella growth and response to environmental stresses,and potential physiological indicator proteins related to growth and environmental stress have been identified.     

CLN8P相互作用蛋白靶基因的获取与初步鉴定

为了获得CLN8P相互作用蛋白靶基因并对其进行初步鉴定,实验经抽提酵母质粒DNA,电转化大肠杆菌,选择性培养基分离质粒,酶切鉴定、测序、生物信息学分析,结果获得了CLN8P相互作用蛋白阳性克隆的一个靶基因BAG5;利用酵母双杂交共转化法初步验证,表明BAG5蛋白与CLN8P具有相互作用,并可能因此影响到神经细胞的正常生长.这将有利于进一步深入研究CLN8的功能及阐明NCLs发病机制.     

哺乳动物Gpx 基因家族进化选择和功能分歧研究 (动物科学)

研究哺乳动物谷胱甘肽过氧化物酶基因家族(Gpx)的亲缘关系和进化选择压力性质。从NCBI等网站获取哺乳动物Gpx基因编码区核酸序列和GPX蛋白氨基酸序列,并用MEGA、Clustal构建亲缘关系树,用SignalP和TargetP分析信号肽和亚细胞定位信息;用K estimator和PAML分析其Gpx基因家族进化先后顺序和选择压力;用Diverge分析GPX蛋白家族功能分歧。结果表明,GPX蛋白家族进化中形成两个亚类,其一包括GPX1和GPX2,其二包括GPX3、GPX5和GPX6;各GPX蛋白成员均有高度保守的GPX蛋白家族基序和高度保守的硒代半胱氨酸或半胱氨酸残基,氨基端信号肽分析显示,GPX1蛋白为胞浆线粒体型,其余家族成员均为胞外分泌型;哺乳动物Gpx1基因进化上受到严格的负选择作用,Gpx基因家族成员保守区也受到负选择作用,但用枝位点模型却检出了正选择作用,GPX蛋白家族内存在轻微的功能分歧。哺乳动物GPX蛋白家族的进化上的负选择作用表明其在哺乳动物清除自由基抗氧化方面的关键作用。     

鱼腥藻铁调蛋白基因(alr0957)表达和Fe~(3+)对藻生长的影响

根据CyanoBase提供的鱼腥藻PCC7120 FurC基因(alr0957)序列信息设计特异性引物,用降落PCR法从染色体DNA中扩增得约450 bp目的片段.运用TA克隆将其连接到pMD18-T载体上,经筛选测序获得阳性重组质粒.再经过双酶切、纯化FurC基因,连接原核表达载体pET-28a(+),并转化表达菌株BL21和IPTG诱导表达.经测序鉴定的阳性菌株中的FurC,运用SDS-PAGE检测重组蛋白,并利用镍柱层析法纯化目的蛋白.由藻细胞密度、叶绿素a含量、可溶性糖含量等改变探讨不同Fe3+浓度对藻类生长的影响.结果表明:在37℃经1 mmol/L IPTG诱导18 h,成功表达了分子量约为19000的融合蛋白.小于0.5mg/L Fe3+促进藻生长,大于0.9 mg/L Fe3+抑制藻生长,最适藻生长Fe3+浓度为0.5⁰.9 mg/L.     

典型性微量金属元素对藻华的激励趋势探讨

Fe、Mn、Zn等典型性微量营养元素对藻类增殖的影响是探讨藻华成因分析的重要内容.通过设计系列正交实验,研究代表性微量元素Fe、Mn、Zn对常见藻华生物(蓝藻、绿藻)的影响,结果表明:P、Fe、Mn、Zn的质量浓度变化对藻生长速率激励效果明显,显著性顺序依次为:ZnPFeMn.通过单独添加Fe、Mn、Zn的方法进一步进行了藻增长潜力实验.结果表明:在Fe、Mn、Zn质量浓度分别为0.20、0.13、0.033mg/L时,藻类生长速率达到最大值,低于该质量浓度时,金属离子质量浓度与藻类生长速率、生物量呈正相关;高于该质量浓度时,金属离子质量浓度与藻生长水平呈反相关;拟合Fe、Mn、Zn质量浓度与藻生长速率之间的数值关系,分别得到相关系数为0.93、0.87、0.90的回归方程;根据微量元素与藻类生长速率之间的相关性,可以对藻华的爆发时间或程度进行一定精度的预测.正交实验中,锰的激励作用受微量元素之间的竞争性抑制而被消弱,反映出锰的作用机理比较复杂,有待于深入研究.     

禾本科植物抑制三峡库区水华优势蓝藻的探讨

抑藻化感物质主要为植物次生代谢物质,通常在自然条件下可以分解,难以在生态系统中积累,生态安全性好,在防控水华方面具有良好的应用前景.文章探讨了三峡水库消落带禾本科植物牛鞭草对水华鱼腥藻的化感抑制作用,提出了具体的研究方案,为下一步具体研究化感抑藻技术有效防控三峡库区水华提供参考.     

New 'phantom' dinoflagellate is the causative agent of major estuarine fish kills.

A worldwide increase in toxic phytoplankton blooms over the past 20 years has coincided with increasing reports of fish diseases and deaths of unknown cause. Among estuaries that have been repeatedly associated with unexplained fish kills on the western Atlantic Coast are the Pamlico and Neuse Estuaries of the southeastern United States. Here we describe a new toxic dinoflagellate with 'phantom-like' behaviour that has been identified as the causative agent of a significant portion of the fish kills in these estuaries, and which may also be active in other geographic regions. The alga requires live finfish or their fresh excreta for excystment and release of a potent toxin. Low cell densities cause neurotoxic signs and fish death, followed by rapid algal encystment and dormancy unless live fish are added. This dinoflagellate was abundant in the water during major fish kills in local estuaries, but only while fish were dying; within several hours of death where carcasses were still present, the flagellated vegetative algal population had encysted and settled back to the sediments. Isolates from each event were highly lethal to finfish and shellfish in laboratory bioassays. Given its broad temperature and salinity tolerance, and its stimulation by phosphate enrichment, this toxic phytoplankter may be a widespread but undetected source of fish mortality in nutrient-enriched estuaries.     

广西沿海赤潮多发区高浓度氮磷营养元素来源探讨

【目的】探讨广西沿海赤潮多发区氮磷营养元素的来源,分析广西沿海赤潮发生的影响因素,为预防或减少赤潮灾害发生、保护海洋生态环境提供科学依据。【方法】现场调查以钦州湾海域为研究对象,测定表层海水的溶解态氮(DN)、溶解态磷(DP)含量并分析其分布特征,结合历史资料追溯高浓度DN、DP来源及污染物输运过程。【结果】钦州湾内高浓度DN、DP分布区与入海陆源工业污染排放区不一致,离工业污染区近的地方DN、DP浓度偏低,而离工业污染区远的地方DN、DP浓度反而偏高。【结论】广西主要入海河流各类污染物的增加以及不同的海域通过动力途径输送而来的海水可能是广西沿海赤潮多发区的高浓度氮磷营养元素的主要来源。