鳗弧菌(Vibrio anguillarum)侵染对青蛤(Cyclina sinensis)髓样分化因子88基因表达的影响

经转录组测序后筛选并克隆得到青蛤(Cyclina sinensis)髓样分化因子88(myeloid differenttiation factor 88,My D88)的c DNA序列。在鳗弧菌(Vibrio anguillarum)胁迫下,采用荧光定量PCR法分析了My D88基因在青蛤体内的表达过程。结果显示,青蛤My D88基因的开放阅读框为1521bp,编码506个氨基酸,分子量约为57.14k Da,氨基酸N段存在DEATH结构域,C段存在TIR结构域(Toll/Interleukin-1 receptor domain)。My D88基因在青蛤血淋巴、肝脏、外套膜、鳃和闭壳肌等组织中普遍表达,但在血淋巴中表达量最高,与其它组织出现显著性差异(P0.05)。通过检测鳗弧菌刺激下My D88基因在青蛤血淋巴中的表达值,发现My D88基因在24h开始升高,48h达到最大值,约为对照组的10倍,实验组与对照组及空白组均出现了极显著性差异(P0.01);研究结果表明,该基因在软体动物的免疫应答反应中对革兰氏阴性菌有识别作用。     

牙鲆(Paralichthys olivaceus)TLR21 基因在迟缓爱德华氏菌(Edwardsiella tarda)感染后的表达特征

应用荧光定量PCR技术, 检测了TLR21 基因在牙鲆(Paralichthys olivaceus)感染迟缓爱德华 氏菌(Edwardsiella tarda)后, 在0 h、1 h、3 h、6 h、12 h、1 d、3 d、6 d 后, 在心脏、肝脏、脾脏、 头肾、鳃、小肠、肌肉和血的时空表达特征, 并探讨了它们与牙鲆先天免疫反应的关系。结果表明, 大 多组织在感染病原6 h 后TLR21 基因表达明显上调, 尤其是头肾和小肠。头肾6 h 的表达量达到了 对照组的59.3 倍, 小肠6 h 的表达量为对照组的38.6 倍。迟缓爱德华氏菌感染引起牙鲆体内各组织 中TLR21 的上调表达和变化, 为研究牙鲆对迟缓爱德华氏菌的防御机制提供了理论依据。     

长牡蛎(Crassostrea gigas)微卫星多重PCR 体系构建及其在家系鉴定中的应用

筛选多态性高、特异性好、片段大小适中的10 个长牡蛎微卫星位点进行优化组合, 根据扩 增片段大小及同一荧光不重叠原则构建了两组五重PCR 体系。运用CERVUS 3.0 软件对0527 组27 个长牡蛎全同胞家系的643 个子代和0612 组27 个全同胞家系382 个子代分别进行亲权鉴定。结果 发现, 用两组微卫星五重PCR 鉴别时, 在0527 组和0612 组的鉴定成功率均为100%; 只用第一组微 卫星五重PCR, 可以将0527 组96%的子代和0612 组96%的子代鉴定到亲本; 只用第二组微卫星五 重PCR, 可以将0527 组97%的子代和0612 组95%的子代鉴定到亲本。本研究中筛选出的两组微卫 星五重PCR 体系在两组家系中的鉴定效率均较高, 可以快速有效地将子代个体鉴定至所属父母本, 在长牡蛎家系鉴定中具有很好的应用价值。     

Depuration dynamics of oysters (Crassostrea gigas) artificially contaminated by Salmonella enterica serovar Typhimurium

The State of Santa Catarina produces the greatest quantity of edible mollusks in Brazil. To guarantee sanitary qualify, mollusk cultures should be monitored for contamination by pathogenic microorganisms. A self-purification or "depuration" system that eliminates Salmonella enterica serovar Typhimurium contamination from oysters has been developed and evaluated. The depuration process occurred within a closed system, in which 1000 L of water was recirculated for 24 h. The water was sterilized with ultraviolet (UV) light, chlorine, or both together. Oysters (Crassostrea gigas) artificially contaminated with S. typhimurium were harvested every 6 h. Samples of oyster tissue were excised and both the presence and numbers of bacteria were determined. Combined UV light and chlorine treatments resulted in total elimination of bacteria within 12 h. Polymerase chain reaction detected bacteria in water exposed to the three treatments. This pioneering study is the first of its kind in Brazil and represents a major contribution to commercial mollusk culture in this country.     

Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.     

A Method To Detect Viable Helicobacter pylori Bacteria in Groundwater

The inability to detect the presence of viable Helicobacter pylori bacteria in environmental waters has hindered the public health community in assessing the role water may play in the transmission of this pathogen. This work describes a cultural enrichment method coupled with an H. pylori‐specific PCR to identify these bacteria in water. While far from perfected at the present time, this represents an exciting new approach to studying the significance of water as a transmission mechanism for H. pylori. Evidence is presented that indicates culturable H. pylori bacteria were found using this enrichment/PCR method in a local groundwater source.     

Identification of Microplankton in South China Sea with Image-Matching Individual PCR

In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.     

链状亚历山大藻组蛋白H3基因克隆与生长过程中的表达分析

链状亚历山大藻(Alexandrium catenella)是一种典型的产毒赤潮甲藻。甲藻曾被认为没有组蛋白,但近年来在多种甲藻中检测到全部四个核心组蛋白的活跃转录,而目前对甲藻中组蛋白表达模式与具体功能还缺乏深入的研究。本文报道了链状亚历山大藻组蛋白H3变体之一H3.c的全长ORF序列的克隆和分析;分析了链状亚历山大藻生长过程中组蛋白H3在基因和蛋白水平的表达。目前研究发现的链状亚历山大藻H3变体共三个,其中H3.b在N末端序列与其他物种差异最大,为链状亚历山大藻特有的H3变体。对数生长期的组蛋白H3在基因与蛋白水平上均活跃表达,但表达量并不会随着爆发性增长而显著变化,其中H3.b的基因表达在对数末期呈现上调;培养至衰亡期,各H3变体表达均下调,尤其在蛋白水平上呈现明显衰减。结合前期研究,本文认为链状亚历山大藻三个H3均为复制非依赖型(RI)变体,一些变体可响应伴随藻不断生长而逐渐增强的细胞密度等复杂胁迫,推测其参与某些表观遗传修饰,调控生长进程;而衰亡期基于H3的表观遗传调控受到抑制。     

基于线粒体cyt b基因序列差异的组合PCR技术快速鉴定五种鳗鲡养殖种类的方法研究

在自然或人为因素下容易出现鳗苗种质混杂的现象,由于鳗鲡苗种在形态上十分相似,难以区分。为了保障鳗农的合法利益和提高养殖效益,急需建立一种能够在现场快速、高效使用的鳗鲡种质鉴定方法。本研究通过比对找出5种常见鳗鲡养殖种类:日本鳗鲡(Anguilla japonica)、美洲鳗鲡(A.rostrata)、花鳗鲡(A.marmorata)、太平洋双色鳗鲡(A.bicolor pacifica)、欧洲鳗鲡(A.anguilla)线粒体细胞色素B(cytochrome b,cyt b)基因的差异序列,基于5个cyt b基因序列差异较大的片段,设计多对引物,分别经过PCR验证和条件优化,挑选出4对引物:aj S1/A1、r-a S1/A2、bp-m S5/A3和m-a S4/A4。将这4对引物组合在同一个反应体系中进行PCR扩增,进行条件优化,筛选出组合PCR的最优条件:退火温度为58oC;退火时间为45s;循环数为27。结果表明,通过扩增产物凝胶电泳中条带的有无及大小可快速准确的鉴定出五个鳗鲡种类。本研究建立的组合PCR方法在3小时内可完成整个种类鉴定过程,同时可使用便携式小型仪器完成操作,可满足现场快速、高效鉴定的要求。此外,通过MEGA5.0软件构建5种鳗鲡线粒体cyt b基因序列NJ进化树,发现花鳗鲡和太平洋双色鳗鲡先聚为一支,再与日本鳗鲡聚在一起,而欧洲鳗鲡和美洲鳗鲡聚在一起,进化树图显示的遗传距离和它们的地理分布的远近相似。