应用单细胞PCR方法研究胶州湾一株鳍藻的遗传特征

近年来，分子生物学技术与方法被越来越多地应用于有害赤潮研究。其中，单细胞PCR方法是对难以室内培养的有害赤潮藻种进行遗传特征研究的一项重要技术。本实验尝试建立微藻的单细胞PCR方法，并将其应用于鳍藻研究。 应用室内培养的亚历山大藻，研究了微藻的单细胞PCR方法，并对藻细胞的固定和保存方法进行了比较。结果表明，浮游植物研究中常用的甲醛固定方法只能用于短期保存样品（少于5天），而乙醇固定、鲁格氏液固定、或者在-20℃下冷冻保存的样品，在较长的时间后（60天）仍可以得到比较理想的PCR扩增结果。 应用单细胞PCR方法，对青岛近海采集的鳍藻进行了研究，扩增并测定了包括核糖体大亚基（LSU）rDNA的5’端D1-D2区序列，以及5.8S rDNA和ITS区的部分序列信息。通过分析软件对所得到的鳍藻序列信息与基因库中已知的鳍藻序列信息进行了分析与对比。根据ITS和LSU序列信息构建的系统进化树都显示，本文采集的鳍藻藻种与国外报道的圆形鳍藻聚为一支，初步确定采集的鳍藻应为圆形鳍藻，对该藻种形态学特征的观察也支持这一结果。该藻种部分LSU rDNA序列与欧洲同种鳍藻相似度达到99%,与其它等鳍藻遗传距离在18%-20%之间。测定的ITS区序列与同种圆形鳍藻有62个碱基的差异，与LSU rDNA序列相比，ITS序列变异更大。应用LC-MS方法对该鳍藻的进行了DSP毒素分析，结果未检测到OA或DTX1毒素。 这是我国首次应用单细胞PCR方法对鳍藻开展的研究工作，首次报道了我国鳍藻的核糖体部分序列信息。圆形鳍藻在青岛近海海域是初次报道，显示了单细胞PCR方法在鳍藻研究中的重要意义。

亚历山大藻分子鉴定和荧光原位杂交检测方法研究

近年来，世界沿海国家有害赤潮发生的频率、规模及危害都有上升趋势，有害赤潮已经成为重要的近海环境问题之一。要有效防范有害赤潮带来的危害效应，建立和发展可靠、有效的赤潮监测手段非常重要。目前，对于赤潮藻种的监测主要依靠显微观察的方法，在实际应用中经常遇到困难。首先，亲缘关系相近的物种在形态上差异很小，如甲藻门亚历山大藻属的一些种类，仅细胞壁上个别甲片的结构有细微差别，并且这些形态学指标还容易受环境条件及生长阶段的影响。另外，这种以形态学为基础的分析方法，分析速度慢、耗时长，对操作人员的要求较高，难以满足浮游植物种群动力学监测“量大、连续”的要求。因此，本研究将分子生物学的技术和方法应用于赤潮监测，力求提高赤潮藻种鉴定的准确性和检测工作的效率。 亚历山大藻是一类重要的有害赤潮藻，该藻属中一些产毒特性差别很大的藻种，单从表形特征难以明确区分，从而限制了基于形态观察的监测技术的应用。本研究中，我们尝试应用分子生物学技术与方法，开展了该藻属藻种分子鉴定和荧光原位杂交检测方法的研究。在亚历山大藻的分子鉴定方面，我们采用了核糖体RNA基因（rDNA）序列分析的方法，首次测定了9株分离自中国沿海的（以及实验室保有的其它两株）亚历山大藻的rDNA序列全长，其中包括核糖体小亚基（SSU）rDNA、大亚基（LSU）rDNA、5.8S rDNA及内转录间隔区（ITS）区序列。序列分析结果显示，这些藻株包含了5种核糖体类型，分别是塔玛复合种亚洲温带（Temperate Asian）核糖体类型（TSC-TA），塔玛复合种西欧（West European）核糖体类型（TSC-WE），相关亚历山大藻（A. affine）核糖体类型（AF），微小亚历山大藻（A. minutum）葡萄牙（Portugal）核糖体类型（M-PO）和微小亚历山大藻新西兰（New Zealand）核糖体类型（M-NZ）。将测获的rDNA序列划分为若干保守性不同的区段，分别进行系统发育分析（结合GenBank数据库中保存的其它亚历山大藻相关序列）。结果显示，LSU rDNA D1-D2区是对该藻属藻种进行分子鉴定和系统发育研究的较好区段。同时，为解决建立亚历山大藻克隆培养的困难，我们应用单细胞rDNA序列分析方法，对亚历山大藻单个细胞直接进行了种类鉴定。结果表明，该方法适用于不同生活史阶段的亚历山大藻。 在亚历山大藻的检测技术方面，我们进一步扩展和完善了针对完整细胞的荧光原位杂交检测方法。首先，通过对不同核糖体类型藻株rDNA序列信息的对比分析，针对各自特异的序列位点，设计了特异性rRNA标记探针。经荧光原位杂交实验检验，实现了对5种核糖体类型亚历山大藻的特异性标记。其中，针对WE、M-PO及M-NZ核糖体型的特异性探针为首次获得，另外两个探针是针对TA和AF核糖体类型rRNA新的位点所设计。同时，对影响探针标记效果的诸多因素进行了分析和探讨。此外，在2007年春季长江口海域赤潮调查中，首次应用特异性核酸探针和荧光原位杂交检测方法，调查了该海域亚历山大藻的丰度。结果表明，在4月4日-4月10日的样品中，亚历山大藻达到了较高的密度，最高密度达到103cells/L。同时发现，实验中样品的保存方法有待改进。随后的研究表明，盐醇固定方法及多聚甲醛/甲醇固定方法，可以较好的保持rRNA不被降解并适宜杂交（至少3个月时间）。 总之，本研究首次测定并分析了11株亚历山大藻（9株分离自中国沿海）的rDNA全序列信息。在此基础上，获得了5种核糖体类型亚历山大藻的特异性rRNA标记探针，其中3种为首次获得。另外，实验证明，单细胞rDNA分析技术和荧光原位杂交检测方法，在自然水体中亚历山大藻的直接鉴定及丰度调查中，均具有良好的应用前景。这一工作为我国近海亚历山大藻的鉴定和检测提供了理论依据和方法学基础，希望对该藻赤潮的监测工作有推动作用。 关键词：亚历山大藻 遗传探针 rRNA rDNA 荧光原位杂交 系统发育

Characteristics of rDNA intergenic spacer region from a waterbloom cyanobacteria species Oscillatoria sp.

Sequence of rDNA intergenic spacer region (ISR) from a waterbloom cyanobacterial species Oscillatoria sp, was determined and analyzed. The results of sequence comparison showed that the spacer had a high level sequence divergence, suggesting the sequence may be a target sequence for developing cyanobacteria genus- and species-specific oligonucleotide probes. In addition, a 20bp sequence of rDNA ISR was found highly conserved in all species of cyanobacteria, which was not found in other eubacteria. This conserved sequence within a variable region indicates that it might be a functional oligonucleotide in the processing of the rRNA precursor.

Sequence analysis of the ITS region and 5.8S rDNA of Porphyra haitanensis

The sequences of the ITS (internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli (NB, PT and ST) were amplified, sequenced and analyzed. In addition, the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated. The results are as follows: the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains. The sequences of ITS l were 331 by to 334 bp, while those of the 5.8S rDNA were 158 by and the sequences of ITS2 ranged from 673 by to 681 bp. The sequences of the ITS had a high level of homology (up to 99.5%) with that of P. haitanensis (DQ662228) retrieved from GenBank, but were only approximately 50% homologous with those of other species of Porphyra. The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis. These results suggest that the three cultivated strains of P. haitanensis evolved conservatively and that the ITS showed evolutionary consistency. However, the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations. Therefore, the relationship of Porphyra interspecies phyletic evolution could be judged, which provides the proof for Porphyra identification study. However, proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.

Distribution of rDNA in the nucleus of Giardia lamblia - Detection by Ag-I silver stain

OBJECTIVE: To determine whether rDNA of Giardia lamblia forms a nucleolus organizer region (NOR)-like structure and is in a very primitive state. STUDY DESIGN: G lamblia was used as the experimental animal, with Euglena gracilis as the control. The distribution was demonstrated indirectly by the modified Ag-I silver technique, which can specifically indicate the NOR under both light and electron microscopes. RESULTS: In the ultrathin sections of silver-stained Euglena cells, all the silver grains were concentrated in the fibrosa of the nucleolus, while no grains found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver-stained Giardia cells, no nucleolus was found; a few silver grains were scattered in the nucleus but were not concentrated in any specific region. CONCLUSION: The distribution of silver grains in G lamblia showed that the transcription of rDNA occurs inside the nucleus, though no nucleolus is present. It is possible that chromosomes are in a very primitive state in diplomonad cells; as each chromosome has few prRNA genes, the transcription is independent of a nucleolus. These results imply that the rDNA of Giardia does not form a NOR-like structure and seems to represent a very primitive state in the evolution of the nucleolus.

Molecular characteristics of Camallanus spp. (Spirurida : Camallanidae) in fishes from China based on its rDNA sequences

In the,paper, we explored the intra- and interspecific evolutionary variation among species of Camallanus collected from different fish species in various regions of China. We determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) sequences of these nematodes. The divergence (uncorrected p-distance) of ITS 1, ITS2, and ITS rDNA data sets confirmed 2 valid species of Camallanus in China, i.e., C. cotti and C. hypophthalmichthys. The 2 species were distinguished not only by their different morphologies and host ranges but also by a letranucleotide microsatellite (TTGC)n present in the ITS I region of C cotti. Phylogenetic analyses of the nematodes disclosed 2 main clades, corresponding to different individuals of C cotti and C. hypophthalmichthys from different fish species in various geographical locations, although the interior nodes of each clade received poor support.

Phylogeny of diplozoids in five genera of the subfamily diplozoinae palombi, 1949 as inferred from ITS-2 rDNA sequences

The phylogenetic relationship of 5 genera, i.e. Diplozoon Nordmann, 1832, Paradiplozoon Achmerov, 1974, Inustiatus Khotenovsky, 1978, Sindiplozoon Khotenovsky, 1981, and Eudiplozoon Khotenovsky, 1985 in the subfamily Diplozoinae Palombi, 1949 (Monogenea, Polyopisthocotylea) was inferred from rDNA ITS-2 region using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian methods. The phylogenetic trees produced by using NJ, ML and Bayesian methods exhibit essentially the same topology. Surprisingly, freshwater species of Paradiplozoon from Europe clustered together with species of Diplozoon, but separated from Chinese Paradiplozoon species. The results of molecular phylogeny and lower level of divergence (4(.)1-15(.)7%) in ITS-2 rDNA among Paradiplozoon from Europe and Diplozoon and, on the other hand, high level of divergence (45(.)3-53(.)7%) among Paradiplozoon species from Europe and China might indicate the non-monophyletic origin of the genus Paradiplozoon. Also, the generic status of European Paradiplozoon needs to be revised. The species of Paradiplozoon in China is a basal group in Diplozoinae as revealed by NJ and Bayesian methods, and Sindiplozoon appears to be closely related to European Paradiplozoon and Diplozoon. with their relationship to Eudiplozoon and Inustiatus being unresolved.

Molecular variation of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda : Pseudophyllidea) in different fish host species based on ITS rDNA sequences

The molecular variation in Bothriocephalus acheilognathi Yamaguti, 1934 from 11 species of freshwater fish collected in Australia, China, the Czech Republic, England and Hawaii was investigated by determining the nucleotide sequences of the internal transcribed spacer region. The length of the first and second internal transcribed spacer sequences of multiple individuals ranged from 553 to 571 bp and 553 to 615 bp, and the G + C content from 53.1 to 53.5%. The percentage sequence divergence varied between 0 and 0.9% in the ITS1 and 0 and 6.6% in the ITS2, respectively, indicating the occurrence of intraspecific variation. It is demonstrated that the fragment length variation resulted primarily from microsatellite polymorphisms present in the ITS region, especially in the ITS2 region. Phylogenetic analyses revealed that B. acheilognathi examined in this study consisted of three closely related genotypes with certain degrees of host-specificity, and the genotype representing isolates from Cyprinus carpio L. was the most common and diverse form within the species B. acheilognathi.

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.

Load-Unload Response Ratio and Accelerating Moment/Energy Release Critical Region Scaling and Earthquake Prediction

The main idea of the Load-Unload Response Ratio (LURR) is that when a system is stable, its response to loading corresponds to its response to unloading, whereas when the system is approaching an unstable state, the response to loading and unloading becomes quite different. High LURR values and observations of Accelerating Moment/Energy Release (AMR/AER) prior to large earthquakes have led different research groups to suggest intermediate-term earthquake prediction is possible and imply that the LURR and AMR/AER observations may have a similar physical origin. To study this possibility, we conducted a retrospective examination of several Australian and Chinese earthquakes with magnitudes ranging from 5.0 to 7.9, including Australia's deadly Newcastle earthquake and the devastating Tangshan earthquake. Both LURR values and best-fit power-law time-to-failure functions were computed using data within a range of distances from the epicenter. Like the best-fit power-law fits in AMR/AER, the LURR value was optimal using data within a certain epicentral distance implying a critical region for LURR. Furthermore, LURR critical region size scales with mainshock magnitude and is similar to the AMR/AER critical region size. These results suggest a common physical origin for both the AMR/AER and LURR observations. Further research may provide clues that yield an understanding of this mechanism and help lead to a solid foundation for intermediate-term earthquake prediction.

A generalized wave action conservative equation for the dissipative dynamical system in the nearshore region

To describe the various complex mechanisms of the dissipative dynamical system between waves, currents, and bottoms in the nearshore region that induce typically the wave motion on large-scale variation of ambient currents, a generalized wave action equation for the dissipative dynamical system in the nearshore region is developed by using the mean-flow equations based on the Navier-Stokes equations of viscous fluid, thus raising two new concepts: the vertical velocity wave action and the dissipative wave action, extending the classical concept, wave action, from the ideal averaged flow conservative system into the real averaged flow dissipative system (that is, the generalized conservative system). It will have more applications.

Computation of stress intensity factors by the sub-region mixed finite element method of lines

Based on the sub-region generalized variational principle, a sub-region mixed version of the newly-developed semi-analytical 'finite element method of lines' (FEMOL) is proposed in this paper for accurate and efficient computation of stress intensity factors (SIFs) of two-dimensional notches/cracks. The circular regions surrounding notch/crack tips are taken as the complementary energy region in which a number of leading terms of singular solutions for stresses are used, with the sought SIFs being among the unknown coefficients. The rest of the arbitrary domain is taken as the potential energy region in which FEMOL is applied to obtain approximate displacements. A mixed system of ordinary differential equations (ODEs) and algebraic equations is derived via the sub-region generalized variational principle. A singularity removal technique that eliminates the stress parameters from the mixed equation system eventually yields a standard FEMOL ODE system, the solution of which is no longer singular and is simply and efficiently obtained using a standard general-purpose ODE solver. A number of numerical examples, including bi-material notches/cracks in anti-plane and plane elasticity, are given to show the generally excellent performance of the proposed method.

An improvement on the theoretical model for the large-scale coherent structures in the outer region of a turbulent boundary layer

According to the experimental results, there exist large-scale coherent structures in the outer region of a turbulent boundary layer, which have been studied by many authors.As experimental results, Antonia (1990) showed the phase- aver aged streamlines and isovorticity lines of the large-scale coherent structures in a turbulent boundary layer for different Reynolds numbers. Based on the hydrodynamic stability theory, the 2-D theoretical model for the large-scale structures was proposed by Luo and Zhou, in which the eddy viscosity was defined as a complex function of the position in the normal direction. The theoretical results showed in ref. were in agreement with those in ref. However, there were two problems in the results. One is that in the experimental results, there were divergent focuses between two saddle points in the streamlines, but in the theoretical results, there were centers. The other is that the stretched parts of the isovorticity lines appear at the location of centers in the theoretical results, while in the experimental results they located somewhere between the focuses and saddle points. The reason is that the computations were based on a 2-D model.

Triple-region structure for turbulent-flow in a square duct - a finite-element approach

The Reynolds-averaged Navier-Stokes equations for describing the turbulent flow in a straight square duct are formulated with two different turbulence models. The governing equations are then expanded as a multi-deck structure in a plane perpendicular to the streamwise direction, with each deck characterized by its dominant physical forces as commonly carried out in analytical work using triple-deck expansion. The resulting equations are numerically integrated using higher polynomial (H-P) finite element technique for each cross-sectional plane to be followed by finite difference representation in the streamwise direction until a fully developed state is reached. The computed results using the two different turbulence models show fair agreement with each other, and concur with the vast body of available experimental data. There is also general agreement between our results and the recent numerical works anisotropic k-epsilon turbulence model.