赤潮生物原甲藻Prorocentrum分子识别和系统发育学研究

对东海原甲藻Prorocentrum donghaiense、海洋原甲藻P.micans、微小原甲藻P.minimum、立玛原甲藻P.lima(CCMP1966)四种赤潮生物的18S rDNA进行了PCR扩增和全序列测定及分析,比较了它们的遗传距离和相似系数,获得其18S rDNA序列中的三处高变异区,为设计快速识别原甲藻种间分子探针提供分子依据.同时结合12种具有代表性的重要赤潮甲藻的18S rDNA序列,采用邻接法(Neighbour-Joining,NJ)和最大似然法(Maximum Likelihood,ML)构建系统发育树,研究原甲藻的种间关系和进化地位,结果表明,营底栖生活的P.lima与营浮游生活的P.micans、P.minimum、P.donghaiense为多系起源,浮游型原甲藻与凯伦藻Karenia有更近的起源关系.     

用荧光原位杂交(FISH)技术鉴定赤潮甲藻的研究

探索了用荧光原位杂交（Fluorescence In Situ Hybridization,FISH）技术检测赤潮藻的可行性。用FISH技术鉴定了2株已知的赤潮甲藻——东海原甲藻（Prorocentrum donghaiense）和塔马亚历山大藻（Alexandrium tamarense）。荧光标记的DNA探针是东海原甲藻的核糖体转录单元内间隔区（ITS）序列。研究结果如下：（1）在荧光显微镜下观察与荧光探针杂交后的东海原甲藻细胞,在细胞顶端可见绿色的荧光,而作为对照的塔马亚历山大藻在与标记的探针杂交后没有产生绿色荧光,说明探针只与目标株东海原甲藻反应,不与对照组反应,表明根据ITS序列设计的探针是高度特异的;（2）材料固定后,经蓝光激发,均未产生细胞内源的叶绿素和甲藻素等色素荧光干扰杂交信号的现象,说明固定材料的方法正确。以上的结果表明,FISH技术在鉴定赤潮微藻方面具有其它技术无法比拟的优越性：准确、快速、简单,将其应用到现场定性、定量检测赤潮微藻方面将具有很大的潜力。     

坛紫菜5.8S rDNA和ITS区片段的序列分析及应用

对坛紫菜(Porphyra haitanensis)野生(GL)和栽培(PXV)品系的5.8S rDNA-ITS兀区进行了PCR扩增和序列分析,扩增的GL和PXV的DNA片段长度分别为1213 bp和1221bp,包含完整的ITS1-5.8S-ITS2区.然后对紫菜7个种9个品系(其中6种7个品系从GenBank数据库中获得)的rDNA相应序列进行了排序和系统进化分析,结果表明:9个紫菜品系rDNA中5.8S区的长度和序列非常保守,而ITS区的长度和序列则变异较大;根据它们的序列差异,计算出这9个紫菜品系的遗传距离在0.010～0.551之间,遗传相似性在44.9%～99%之间;并且采用邻接法构建了这9个紫菜品系的系统发育树,发现可以明显分为4个进化枝,由此讨论了分子分类方法同传统分类方法的分歧.实验结果表明,5.8S rDNA.ITS区序列可以成为紫菜种质鉴定和系统进化研究的强有力工具.     

两种快速细菌菌种鉴定方法的比较

采用Riboprinter全自动核糖体RNA指纹分析系统,进行了两株送测细菌的鉴定,同时与16S rDNA序列测定结合生理生化实验的方法进行比较.结果表明,两种方法均成功、快速地完成了送测菌株的鉴定,其分别为大肠杆菌和铜绿假单胞菌.相比而言,Riboprinter系统更为简洁,可以在8h内完成未知菌株的鉴定,最大化避免了其他因素对实验的影响,且在鉴定到种的基础上可以进一步分类,进行溯源分析.     

运用荧光定量PCR技术检测中肋骨条藻的研究

以rDNA序列为寻找种特异性引物的靶区域,通过分析中肋骨条藻rDNA序列,设计出7套适合用于实时荧光定量PCR方法(RFQ-PCR)的引物与探针,经引物验证,选择Primer6(F/R)进一步分析,对比扩增序列及核酸杂交验证,表明该探针可作为中肋骨条藻特异性探针.最后以Primer6(F/R)和TaqMan6建立了定量检测中肋骨条藻的RFQ-PCR方法,并绘制出了定量检测中肋骨条藻的标准曲线,测定数据用镜检计数进行了验证,证明该方法是准确可靠的.     

高活性几丁质酶产生菌的筛选及鉴定

从四川、陕西、浙江等地食用菌栽培的土壤样品中,筛选到一株性能优良、产几丁质酶活力较强的菌株BLC08609,并结合菌落形态、生理生化指标和16S rDNA序列分析对其进行鉴定.结果显示,菌株BLC08609能以食用菌细胞壁(以几丁质为主)为唯一碳源生长,在菌株BLC08609个体形态和生理生化特性鉴定的基础上,通过16S rDNA测序鉴定,确定BLC08609为地衣芽孢杆菌(Bacillus licheniformis).     

基于高光谱反射率的藻类水体基线荧光峰高度与叶绿素a浓度关系研究

采用现场实测和室内培养两种方式测定了甲藻、赤潮异弯藻、叉角藻赤潮和新月菱形藻、海洋蓝绿藻、叉鞭金藻、塔胞藻、扁藻和小球藻等非赤潮藻类光谱曲线。采用度量太阳激发的叶绿素荧光峰高度的基线荧光高度法 ,建立了不同藻类基线荧光高度与叶绿素浓度的关系。基线荧光高度法所用的 3个荧光高度波段分别为 6 6 5nm、6 80nm和86 5nm。在采用线性方程对不同藻类水体基线荧光高度与叶绿素浓度进行回归分析时 ,不同藻类产生了明显不同的结果。其中赤潮异弯藻、海洋蓝绿藻和甲藻为负相关 ,其余为正相关。在正相关的藻类中 ,小球藻最低 ,为 0 .4 6 92。结果偏差主要来自于两个方面 :一是藻类荧光峰位置变化影响 ;二是浮游植物红光和近红外波段高反射率的影响     

纤维脱胶菌RJ6的筛选及脱胶性能

针对植物纤维因其纤长且坚韧在造纸和防治工业中应用受限的问题,从腐烂菜堆中筛选到分离得到纤维脱胶菌株RJ6,将该菌接种到以苎麻汁为唯一有机营养成分的培养基中对苎麻进行脱胶实验,对其进行了形态鉴定、生理生化特性实验以及16S rDNA序列的比对,并对菌株的发酵条件进行优化.测得接种RJ6菌株实验组相对于对照组的脱胶率达到91％,说明RJ6对植物纤维原料有高效的脱胶作用.对菌株的发酵条件进行优化,显示RJ6菌株在转速160 r/min、接种量5％、温度37℃和起始pH 7.0条件下保持一个高水平的发酵.形态鉴定、生理生化特性实验以及16S rDNA序列比对结果表明,RJ6为短杆状,两端钝圆,大小约为(0.4～0.5)μm×(1.1～1.8)μm,革兰氏染色结果呈阴性,存在端生鞭毛.其16S rDNA序列与GenBank中的果胶杆菌属(Pectobacterium.sp)同源性大于99％,RJ6属于果胶杆菌属(Pectobacterium.sp).     

基于表面反射率的赤潮卫星荧光线高度算法比较

采用现场实测和室内培养两种方式测定了甲藻、赤潮异弯藻和叉角藻等赤潮藻以及新月菱形藻、海洋蓝绿藻、叉鞭金藻、塔胞藻、扁藻和小球藻等非赤潮藻类光谱曲线。采用的各卫星(MERIS,GLI,MODIS)的荧光波段数据按照其中心波长,从实际测定的高光谱反射率曲线提取而来,并按照荧光高度的计算公式得到其荧光高度。同时,采用统计分析方法建立荧光高度与叶绿素浓度的关系。10种藻类水体的荧光线高度与叶绿素α的回归分析结果显示了良好的线性关系,但部分藻种出现了负相关的结果。因为在高叶绿素浓度即赤潮条件下,浮游植物在荧光波段(685nm附近)和近红外波段(700～750nm)复杂的光谱行为,使得采用星载遥感器的叶绿素荧光波段探测某些藻类的赤潮时会出现偏差。同时,由于不同藻类的荧光高度与叶绿素浓度的关系也不一致,本文建议针对单独的赤潮种类应建立特定的荧光算法。相关问题还需要在实测的基础上进行更深入的研究。     

浮游植物活体三维荧光光谱特征提取

测量了6种东海常见的浮游植物在两个温度(20℃,15℃)、三个光照(7000Lux,4100Lux,1100Lux)下的不同生长期的三维激发／发射荧光光谱,研究了光谱特征提取方法。对去除散射干扰后的三维光谱进行了奇异值分解,得到的相应于激发光谱的第一主成分具有区分藻种的能力,可作为三维光谱的特征光谱。分析结果表明,实验条件下,等鞭金藻(Isochrysis galbana)、岛国大扁藻(Platymonas helgolanidica)和中肋骨条藻(Skeletonema costatuma)的特征光谱相似度高,塔玛亚历山大藻(Alexandrium tamarense)、东海原甲藻(Prorocentrum dentatum)和尖刺拟菱形藻(Pseudo—nitzschia pungens)光谱相似度稍差。     

Mechanical characterization of cellulosic thecal plates in dinoflagellates by nanoindentation

Dinoflagellates constitute an important group of microorganisms. Symbiotic dinoflagellates are responsible for the primary production of coral reef ecosystems and the phenomenon of their demise is known as "coral bleaching." Blooming of the planktonic dinoflagellates is the major cause of "red tides." Many dinoflagellates have prominent membrane-bound thecal plates at their cell cortices. These thecal plates have high cellulose content and are biologically fabricated into various shapes. However, the mechanical properties of theca have not previously been characterized; understanding these properties, including hardness and elastic modulus, will give insights into the ecological significance and biotechnological potential of bio-fabricated structures. A series of nanoindentation tests were performed on various locations of cellulosic thecal plates isolated from the dinoflagellates Alexandrium catenella and Lingulodinium polyedrum. Despite having transparent properties, thecal plates possess mechanical properties comparable to softwood cell walls, implicating their role as a protective cell covering. Consistent measurements were obtained when indentation was performed at various locations, which contrasts with the high variability of cellulose microfibers from plant sources. The present study demonstrated the novel properties of this potential new source of cellulose.     

SERS microscopic imaging as novel tool for assessing viability and enumerating yeast cells at various stages of cell cycle in lag,log, exponential and stationary phases of growth in culture

Surface enhanced Raman scattering (SERS) microscopic imaging was employed to enumerate the yeast cells in culture. We found this imaging method as an efficient tool for easily differentiating and quantitatively enumerating yeast cell at different stages of cell-division cycle (G1, S, G2 and M phase) at various stages of growth phases namely lag, log, exponential and stationary phases in culture. Apart from enumerating the cells at different stages of cell cycle under lag, log, exponential and stationary phases, it was possible using SERS microscopy to differentiate the live cells from dead ones. The dead cells were SERS inactive and gave enhanced autofluorescence compared with the live cells, which were SERS active. The results from the present investigation suggest that SERS microscopic imaging, using silver nanoparticles (AgNPs) as a sensitive tool to enumerate the yeast cells in culture.     

The effect of cyclic prestraining on the fatigue life and the microstructural evolution of fatigued copper polycrystals

Almost all engineering materials undergo a mechanical pre-treatment during forming process. For this reason, the effect of prestraining on fatigue life and microstructure evolution was investigated at different stages of cycling up to failure. In this context, several specimens were tested under various plastic strain amplitudes in air and vacuum environments. The obtained results showed that the fatigue life is not affected by prestraining. In addition, microscopic analysis revealed that the characteristic dislocation microstructure of a virgin specimen fatigued at constant plastic strain amplitude is never observed after prestraining. Furthermore, the equiaxed dislocation cell structure tended to form very quickly with cumulative plastic deformation.     

Hygrothermal degradation in glass/epoxy — evaluation via stress wave factors

The paper documents an experimental study on nondestructive evaluation of hygrothermally degraded glass fiber/epoxy matrix composite laminates. Specimens were subjected to accelerated life tests, i.e. high temperature and high pressure steam. The efficiency of the stress (ultrasonic) wave propagation through the specimens was evaluated quantitatively, at various stages of degradation, via the so-called stress wave factors (SWFs). Scanning electron micrographs were taken at some stages of degradation. In addition, the specimens were tested in bending mode. All measurements are correlated with the SWFs.     

Strategic evolution of eco-products: a product life cycle planning methodology

This paper presents a methodology and a software tool to establish an eco-design concept of a product and its life cycle by assigning appropriate life cycle options to the components of the product. The product life cycle planning (LCP) methodology provides the following systematic procedures. First, the medium- or long-term production and collection plan for the product family is clarified. Next, target values for the product and its life cycle are set in the process of determination of customer-oriented specification and eco-specification. Then, eco-solution ideas to realize reasonable resource circulation are generated by using various life cycle option analysis charts. Finally, an eco-design concept which involves eco-solution ideas is evaluated for decision-making at early stages of product development. A design support tool was made for efficiently planning product life cycles by using quality function deployment and life cycle assessment data. Based on case studies, it was verified that the proposed methodology and tool are useful for developing multi-generational eco-products.     

Single-molecule detection of surface-hybridized human papilloma virus DNA for quantitative clinical screening

We present an improved method to quantify viral DNA in human cells at the single- molecule level. Human papilloma virus (HPV)-16 DNA was hybridized to probes that were covalently bound to a glass surface and detected with a single-molecule imaging system. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization. In the dual-probe mode, a second probe was introduced that has a fluorescently labeled 1-kb DNA strand connected to the 50-nt probe sequence. With the single-probe method, the detection limit was 0.7 copy/cell, which was similar to that reported in a flow system earlier. With the dual-probe method, the linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. Both methods were applied to cell line samples with known HPV-16 infection, and the result showed a good match with the reported viral load. DNA from cervical cells, collected with the Pap smear sampling method, was spiked with HPV-16 DNA and submitted to this assay to show compatibility with conventional sampling methods. The dual-probe method was further tested with a crudely prepared sample. The cells were heat lyzed and spun down, and the supernatant was immediately submitted to hybridization. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells with 600 copies/cell from healthy cells.     

Application of fracture mechanics to heterogeneous systems — prediction of fatigue life of ceramics

A prediction of fatigue life and fracture stress of ceramics of a heterogeneous system was made by application of fracture mechanics based on slow crack growth. It was shown that the prediction of fatigue life and fracture stress in ceramics of heterogeneous system in general could be dealt with in a similar way to those of ceramics of a homogeneous system. Experimental examination of the validity of the prediction was made with LAS glass ceramics (Li₂O-Al₂O₃-SiO₂) at various stages of devitrification as well as foamed glass. These results proved the validity of the formulae derived.     

An investigation on the high stress sensitivity of fatigue life of rolled AZ31 magnesium alloy under constant amplitude fatigue loading

In the present work, an investigation on the high stress sensitivity of the fatigue life of the AZ31 rolled magnesium alloy under constant amplitude fatigue loading has been carried out. Different damage parameters were involved to quantify fatigue damage accumulation at the various scales of material volume corresponding to the changing fatigue damage mechanisms that prevail at the various stages of the fatigue life. The experimental work included mainly nano‐indentation measurements to evaluate hardness evolution at the nano‐scale due to cyclic plasticity, results of micro‐crack monitoring by using the replication technique and fractographic analysis to obtain the fracture characteristics of the fatigue specimens after failure. The hexagonal close‐packed structure of the alloy and the resulting difficulty for the activation of five independent slip systems required for homogeneous plastic deformation were considered to determine the high stress sensitivity of the fatigue life observed for the rolled AZ31 alloy under the investigated loading conditions.     

Incorporation of microdosimetric concepts into a biologically-based model of radiation carcinogenesis

The generalised state-vector model of radiation carcinogenesis (SVM) simulates radiation induced biological effects by expressing the transition rates between the various initiation and promotion stages in terms of dose rate for low and high linear energy transfer (LET) particles. In the present work, the SVM has been reformulated to incorporate single track characteristics of particles with varying LET. Transition rates of the initiation phase were expressed as functions of LET by describing the complexity and clustering of DNA double strand breaks (DSBs) and its effect on repair kinetics, while the promotion phase was reformulated based on a multi-target single-hit hypothesis. Such an approach allows the consideration of hit frequencies and the variability of the specific energy and LET spectra of radon progeny alpha particles in bronchial target cells for different exposure conditions.