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。     

应用单细胞rDNA序列测定方法鉴定不同生活史阶段的亚历山大藻

通过实验室内诱导产生了重要的赤潮原因种链状亚历山大藻(Alexandrium catenella)不同生活史阶段的藻细胞,建立了低渗(TE缓冲液)溶胀和蛋白酶K处理相结合的细胞裂解方法和单细胞PCR扩增方法.应用所建立的方法,测定了不同生活史阶段的亚历山大藻细胞核糖体DNA(rDNA)两个高变区域(5.8S rDNA及其两侧的内转录间隔区和28S rDNA 5'端D1-D2区)的序列信息.结果显示,各生活史阶段的藻细胞rDNA序列在所测的两个高变区域完全相同,依据序列信息可以对处于不同生活史阶段的亚历山大藻进行鉴定.同时发现,在单个藻细胞中,两个所测片段均存有若干个多态位点,表明藻细胞内rDNA序列的多个拷贝之间可能存有差异.实验表明,对处于不同生活史阶段的赤潮原因种,通过单细胞rDNA序列测定的方法也可以进行准确鉴定.     

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.     

茭白黑粉菌的分离鉴定研究

以"余茭4号"灰茭为材料,进行了茭白黑粉菌的分离,并进行了ITS-5.8S rDNA的分子序列测定。结果表明:ITS-5.8S rDNA扩增片段大小775bp,通过DNA Star软件进行比较分析,结果表明:分离出的黑粉菌(Ustilago esculenta)是黑粉菌属的边缘物种。     

海带栽培品系和长海带ITS区的PCR扩增及序列分析

对遗传背景清晰且具有显著表型差异的海带6个栽培品系(CUL002,CUL860,CUL1170,CUE017,CUE018,CUL901)和长海带(L．longissia)的ITS区进行了PCR扩增和序列测定,并分析了8个种17个品系(其中7种共10株从GenBank中获得)海带之间的系统进化关系。结果表明：表型各异的6个海带栽培品系ITS区差别很小,其中ITS1 5．8S rDNA序列完全相同,部分品系间ITS2序列有个别碱基差异;进化树显示与日本海带(L．japonica,AF319018)聚在一起,相似性大于98％,其中CUL901、CUL860和CULll70的ITS序列完全相同。长海带(L．longissia)的ITS 5．8S rDNA和日本海带(L．japonica,AF319018)的完全相同。以Undaria peterseniana为外类群,根据ITS 5．8S rDNA序列构建进化树,6栽培品系及长海带与日本海带聚在一起;17株海带基本构成两个大的进化枝。研究结果揭示了我国海域栽培的长海带可能与日本海带(L．iaponica,AF319018)为同一个种。     

菰黑粉菌孢子萌发过程形态学观察及系统发育研究

通过显微观察法研究菰黑粉菌厚垣孢子在28℃、PDA液体培养基中静止萌发各时期的菌体形态,克隆了菰黑粉菌18S rDNA序列并测序,结合已有ITS-5.8S rDNA序列和18S rDNA序列分析其系统发育地位.结果表明,从厚垣孢子到形成稳定的担孢子需要62~72 h,经历厚垣孢子萌发、芽管形成、先菌丝形成、担孢子和小孢子等几个阶段,在体外培养时多以棒状担孢子形态存在,培养30 d后,在培养基表面或边缘形成白色丝状菌丝;系统发育分析结果显示菰黑粉菌与Ustilago属和Sporisorium属亲缘关系较近,与黑粉菌属中的Ustilago triodiae亲缘关系最近.     

基于DNA序列LZ复杂性距离的系统进化树重构

根据LZ复杂性的原理,提出了序列间条件LZ复杂性的概念.基于条件LZ复杂性,定义了非空序列间的LZ复杂性距离.选取20种有胎盘哺乳动物,以它们的全线粒体基因组作为分子数据,计算两两之间的LZ复杂性距离,得出距离矩阵.根据计算出的LZ复杂性距离矩阵重构了20种有胎盘哺乳动物的系统进化树.基于LZ复杂性距离所得的系统进化树支持有胎盘哺乳动物中的灵长动物(Primates)、原蹄动物(Ferungulate)和啮齿动物(Rodents)三者之间灵长动物与原蹄动物之间亲缘关系更近一些的观点.     

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。     

厦门海域产蛋白酶浮游细菌的多样性研究

从厦门海域分离筛选到33株产蛋白酶浮游细菌,并通过对其16S核糖体RNA(16S rRNA)基因进行聚合酶链式反应(PCR)扩增和限制性片段长度多态性(RFLP)分析,初步研究了该海域产蛋白酶浮游细菌的多样性.研究发现,这33株菌可分为12种操作分类单元(OTU),其中OTU-Ⅰ和OTU-Ⅱ分别包含8株和12株菌株,分别占总菌数的24.2%和36.4%,为优势分离类群.分别从12种OTU中随机选取1株细菌作为代表进行16S rRNA基因测序.序列分析结果显示,属于OTU-Ⅶ的细菌为假交替单胞菌属(Pseudoalteromonas sp.),而包括OTU-Ⅰ和OTU-Ⅱ在内的9种OTU 都属于弧菌属(Vibrio sp.),表明弧菌在厦门海域产蛋白酶浮游细菌中占优势.初步的生理检测显示,这些细菌的适宜生长温度为16℃左右,适宜生长盐度范围为3%～5% NaCl.     

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     

Assessing phytoplankton using a two-rank database based on excitation-emission fluorescence spectra

The feasibility of using a two-rank database of reference spectra based on in vivo fluorescence excitation-emission matrix (EEMs) spectra to assess dominant groups of phytoplankton was explored. Twenty-six species belonging to 20 genera of seven divisions were studied. First, fluorescent characteristics of these EEMs were extracted using Daubechies-7 wavelet analysis. Second, the optimal characteristic spectra of scale vectors (SOCS) and time-series vectors (TOCS) were selected; phytoplankton of different genera were classified using Fisher linear discriminant analysis. Third, SOCS and TOCS reference spectra databases were obtained by hierarchical cluster analysis. Using non-negative least squares as the method to assess the phytoplankton, a two-rank reference spectra database was established according to their respective ability to identify the 2818 single-species samples. Using this fluorimetric technique, the correct identification rates (CIRs) for single-species samples were 88.8-100% at the genus level and 98.8-100% at the division level. Dominant species in the 465 laboratory mixtures had corresponding CIRs of 85.6% and 96.1%. Moreover, 15 of the 19 species used as dominants were correctly identified at the genus level. In 27 natural seawater samples, Prorocentrum donghaiense, Thalassiosira nordenskioldi, and Chaetoceros socialis (bloom-forming species with a density of about 10(7) cell L(-1)), and Alexandrium sp. (dominant species with abundance of about 10(6) cell L(-1)) were qualitatively identified at the genus level; other dominant species, with densities of 10(5) to 10(6) cell L(-1), were identified at the division level. The quantitative identification was relatively poor in the natural water samples, and several potential resolutions, including trying both new measuring methods and calculating methods, for future study are given.     

16SrRNA基因测序确定根瘤菌的系统发育

采用聚合酶链反应和ＤＮＡ测序技术分析了两个新根瘤菌群的代表菌株的部分１６ＳｒＲＮＡ基因序列，并对所得序列进行了聚类分析。在系统发育树状图中，菌株ｓｈ２２６２３所代表的根瘤菌群与菜豆根瘤菌、豌豆根瘤菌及发根土壤杆菌等构成一个亚群，另一个菌群的代表菌株ｓｈ１９３１２与土壤杆菌的３个种构成一亚群。这些结果初步确定了两菌群的系统发育地位，并提示了根瘤与土壤杆菌两属间进化上的密切关系。     

Cryptic species of planktonic foraminifera: their effect on palaeoceanographic reconstructions

Shells of planktonic foraminifera recovered from marine sediments provide a multitude of important palaeoproxies. Most of these proxies are based on the assumption that each morphospecies of planktonic foraminifera represents a genetically continuous species with a unique habitat. Recent discovery of hitherto hidden genetic diversity among modern planktonic foraminifera has significant repercussions on palaeoproxies derived from their fossil shells. We have compiled all available data on this genetic diversity. To date, 33 cryptic genetic types were found in 9 out of the 22 sequenced morphospecies of modern planktonic foraminifera. An examination of this database suggests that cryptic genetic diversity may be a prevalent pattern among modern planktonic foraminifera, but that the total number of cryptic genetic types per morphospecies is not large and that most genetic types show a non-random pattern of distribution in the oceans. Using modern distribution data from the Atlantic Ocean as constraints, the relationship between abundances of three genetic types of Globigerina bulloides and sea-surface temperature has been modelled and this model has been applied to a database of species counts in Atlantic coretops (761 samples). Trials with artificial neural networks (ANNs), the modern analogue technique and Imbrie-Kipp transfer functions showed that the splitting of G. bulloides into three genetic types resulted in substantial reduction in the prediction error rate (by 5 to 34%) and that this improvement was by far greatest in ANN trials (on average more than 20%). We conclude that such a large reduction in error rate occurred because the models resonated with a real pattern in the original data. This study indicates that genetic diversity among planktonic foraminifera may become more of a gift than malaise to palaeoproxies. If it becomes possible to distinguish these genetic types in the fossil record, the accuracy of proxies based on planktonic foraminifera will indeed substantially increase.     

机载激光雷达和高光谱组合系统的亚热带森林估测遥感试验

为了提高森林的类型识别及生物物理参数反演精度,采用国产机载激光雷达和高光谱组合系统(ALHIS),选择湖北典型亚热带森林开展了航空遥感试验,获取了试验区激光雷达点云、高光谱和CCD影像数据,提取了森林高度和优势树种类别信息。对数据的分析表明,激光雷达林分平均高的估测精度达到90.67%,激光雷达估测平均高与地面实测胸径加权平均高之间显著相关(R2=0.73,RMSE=1.29m)。按照优势树种分类结果进行统计,发现马尾松、栓皮栎和其它树种的林分平均高分别为9.62m、9.30m、8.79m,不同树种之间的林分平均高相差不大。高光谱优势树种识别总体精度达到82.00%(Kappa=0.70),试验区森林和非森林面积所占比例分别为60.01%和39.99%,马尾松、栓皮栎和其它树种面积在森林中所占比例分别为59.77%、24.99%和15.23%。试验证明,ALHIS能够同时获取高分辨率的植被遥感特征数据,以用于森林制图、优势树种/树种组识别、碳储量估算及生态环境建模等研究。     

Isolation of the epsilon-caprolactam denitrifying bacteria from a wastewater treatment system manufactured with acrylonitrile-butadiene-styrene resin

epsilon-Caprolactam has high COD and toxicity, so its discharge to natural water and soil systems may lead to an adverse environmental effect on water quality, endangering public health and welfare. This investigation attempts to isolate epsilon-caprolactam denitrifying bacteria from a wastewater treatment system manufactured with acrylonitrile-butadiene-styrene (ABS) resin. The goal is to elucidate the effectiveness of isolated pure strain and ABS mixed strains in treating varepsilon-caprolactam from synthetic wastewater. The results reveal that Paracoccus versutus MDC-3 was isolated from the wastewater treatment system manufactured with ABS resin. The ABS mixed strains and P. versutus MDC-3 can consume up to 1539mg/l epsilon-caprolactam to denitrify from synthetic wastewater. Complete epsilon-caprolactam removal depended on the supply of sufficient electron acceptors (nitrate). Strain P. versutus MDC-3, Hyphomicrobium sp. HM, Methylosinus pucelana and Magnetospirillum sp. CC-26 are related closely, according to the phylogenetic analyses of 16S rDNA sequences.     

Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes

Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of approximately 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.     

Formation of persisters in <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilms induced by antibacterial dental monomer

Antibacterial monomers can combat oral biofilm acids and caries; however, little is known on whether quaternary ammonium monomers (QAMs) would induce drug persistence in oral bacteria. The objectives of this study were to investigate the interactions of Streptococcus mutans (S. mutans) with dimethylaminohexadecyl methacrylate (DMAHDM), and determine for the first time whether DMAHDM could induce persisters in S. mutans. DMAHDM was synthesized using a modified Menschutkin reaction. Dose-dependent killing curves and time-dependent killing curves of planktonic S. mutans and biofilms were determined to evaluate drug persistence, using chlorhexidine (CHX) as control. The inheritability assay, minimum inhibitory concentration (MIC) and live/dead biofilm assay were determined to investigate persister characteristics. DMAHDM matched the killing potency of the gold standard CHX against S. mutans biofilms. DMAHDM and CHX induced drug persistence in S. mutans biofilms but not in planktonic bacteria. S. mutans biofilm persistence was not inheritable in that the tolerance to DMAHDM or CHX of the surviving persisters in the initial population was not transferred to subsequent generations, as displayed by the inheritability assay. The MIC of S. mutans parental strain and induced persisters remained the same. The induced persisters in S. mutans biofilms could be eliminated via higher doses of 300?μg/mL of DMAHDM and CHX. In conclusion, this study showed for the first time that (1) DMAHDM induced persisters only in biofilms, but not in planktonic bacteria; and (2) both DMAHDM-induced and CHX-induced S. mutans persister biofilms could be completely eradicated by even higher concentrations of DMAHDM and CHX. More studies are needed on the induction of persisters in oral biofilms for the development and use of a new generation of antibacterial dental monomers and resins.