原生动物单细胞PCR应用初探——以天蓝喇叭虫为例

以天蓝喇叭虫(Stentor coeruleus)为研究对象,探索了单细胞单基因PCR扩增及单细胞全基因组PCR扩增技术在原生动物中的应用.经过不断探索和优化条件后,试验取得了理想的结果.在40例单细胞单基因(SSU rDNA基因全序列)PCR的一次性扩增中,新鲜细胞和经过中性红染色的细胞都获得了100%的成功率,室温下酒精(95%)保存一周的细胞获得了82.5%的成功率.在单细胞全基因组PCR扩增中,采用高效高保真的phi29DNA聚合酶结合随机引物(Random Primer)进行扩增,获得了丰富且质

利用RAPD-PCR探讨天蓝喇叭虫生物群系分化

选择天蓝喇叭虫(Stentor coeruleus)作为研究对象,对武汉市南湖、月湖、关桥3个水体共5个样点天蓝喇叭虫(S.coeruleus)样本的总DNA进行随机扩增多态DNA聚类分析,以检测各个样本的遗传相似性和趋异程度,借以评估样本间的遗传变异度。结果如下:(1)从98条随机引物中筛选12条引物共扩增出89条大小为100～1500bp的清晰条带,平均每条引物扩增出7.4条片段。(2)用Rapdistance1.04分析显示,不同样点样本之间存在着一定的变异,其遗传距离在0.076～0.416之间。

全细胞PCR扩增用于鱼腥藻种类鉴定

传统鉴定藻种的方法主要是通过形态学观察的方法加以判断。蓝藻在自然条件和人工培养过程中 ,其形态、代谢能力等都可能发生变化 ,同时该过程需要的时间长 ,难以区分种或种以下的分类、单位 ,亦难以在水华暴发早期阶段准确鉴定。本文利用rDNA通用引物扩增 ,表明在 5 0 μL的反应体系中加入 2 0个鱼腥藻细胞能扩增出目的条带 ;对已知的鱼腥藻PC基因的分析设计引物 ,在BSA浓度为 0 2 %— 1% (w/v)下 ,全细胞扩增出实验室保存的四种鱼腥藻的部分PC以及PC IGS序列 ,序列分析结果表明PC I

鲤鱼3个亚种线粒体DNA ND5/6区段的PCR-RFLP分析及亚种间分子标记的确立

从鲤鱼3个亚种(Cyprinus carpio carpio,Cyprinus carpio haematopterus和Cyprinus carpiorubrofuscus)中选出具代表性的品系共10个,运用PCR方法,扩增出2.4 kb的mtDNA ND5/6区段.共用9种限制性内切酶进行酶切分析,结果表明,每个亚种拥有一种单倍型,有4种限制性内切酶(Dde I,Hae III,Taq I和Mbo I)酶切后产生了能将3个亚种区分开来的诊断性限制性酶切位点.可利用其作为鉴定3个亚种的遗传标记和遗传育种

RT-PCR法检测污染物甲基汞对调钙质基因表达的抑制

应用RT -PCR方法 ,通过用不同浓度的甲基汞对Hep2B细胞株进行不同时间长短的处理 ,检测其调钙质的mRNA表达水平 ,发现微量的甲基汞处理就可以引起细胞株细胞损伤 ,同时抑制调钙质mRNA的表达 ,进一步证实了在肝脏损伤的病理状态下调钙质基因的表达减少 ,从而为检测环境中甲基汞的含量提供了一个可能的分子生物学手段 .

原位杂交和原位PCR技术在鱼类基因定位中的应用

国家自然科学基金（项目编号：39730290）

水华蓝藻产毒特性的PCR检测法

特异引物对（ＴＯＸ　１Ｐ／１Ｆ；ＴＯＸ　２Ｐ／２Ｆ）用于检测微囊藻毒素合成酶基因ｍｃｙＢ片段在３８种水华蓝藻中的分布情况。结果显示，所有能产生微囊藻毒素的微囊藻都有特异扩增条带，非产毒株则没有。几种常规的毒性检测方法验证了ＰＣＲ方法所获结果的准确性。本研究发展了以全细胞ＰＣＲ法检测ｍｃｙＢ片断，说明全细胞ＰＣＲ检测法适用于不同来源的蓝藻材料。结果证明以ＤＮＡ为基础鉴别产毒和非产毒微囊藻及其他水华蓝藻的方法是可行和实用的。

集胞藻ORFs侧翼序列的PCR扩增和定向基因插入失活策略

针对集胞藻ＰＣＣ６８０３的１９２７个待定编码基因进行了两侧序列的ＰＣＲ扩增。４个亚株基因组在。ｓｌｌ０２６７－ｓｌｌ０２６８－ｓｌｌ０２６９区域的 ＰＣＲ扩增产物与 Ｋａｚｕｓａ ＤＮＡ数据存在差异。以叶绿素合成基因ｃｈｌＨ和ｃｈｌＬ为例，显示三片段连接ＰＣＲ产物可有效用于集胞藻６８０３基因组定向插入失活。

七株微囊藻系统进化关系的RAPD-PCR分析(英文)

应用RAPD－PCR的方法，选用24个随机引物，分析来自不同地区的7株微囊藻的基因组多态性．结果显示，Microcystis．viridis及M.wesenbergii明显与M．aeruginosa区分开．M．aeruginosa分为两个可视为不同种的异源分类单位．作为对照的Anabaenasp．7120与其他微囊藻株表现出完全不同的基因型及更远的遗传距离．此项研究表明，以基因型而不是表现型为基础，分析蓝藻种内及种间区别是可能的．因此，为解决蓝藻分类问题，特别是在种和属的水平上，提供了重要的线索．结合正在

七株微囊藻系统进化关系的RAPD-PCR分析

应用RAPD-PCR的方法,选用24个随机引物,分析来自不同地区的7株微囊藻的基因组多态性。结果显示,Microcystis.viridis及M.wesenbergii明显与M.aeruginosa区分开。M.aeruginosa分为两个可视为不同种的异源分类单位。作为对照的Anabaena sp.7120与其他微囊藻株表现出完全不同的基因型及更远的遗传距离。 此项研究表明,以基因型而不是表现型为基础,分析蓝藻种内及种间区别是可能的。因此,为解决蓝藻分类问题,特别是在种和属的水平上,提供了重要的线索。结合

PCR技术在环境生物学上的应用

ＰＣＲ技术在环境生物学上的应用邱东茹，吴振斌（中国科学院水生生物研究所武汉４３００７２）水是许多疾病的传播途径之一，为监测水环境、水处理系统和供水系统的卫生学质量，需要对水中的病原菌和病毒作检测，由于直接检查水中各种病原微生物方法复杂，如有些细菌很难...

PCR-DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were characterized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communities, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eukaryotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE-patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the concentration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR-DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations.

Plankton community composition in the Three Gorges Reservoir Region revealed by PCR-DGGE and its relationships with environmental factors

To explore the relationships between community composition and the environment in a reservoir ecosystem, plankton communities from the Three Gorges Reservoir Region were studied by PCR-denaturing gradient gel electrophoresis fingerprinting. Bacterial and eukaryotic operational taxonomic units (OTUs), generated by DGGE analysis of the PCR-amplified 16S and 18S rRNA genes, were used as surrogates for the dominant "biodiversity units". OTU composition among the sites was heterogeneous; 46.7% of the total bacteria] OTUs (45) and 64.1% of the eukaryotic OTUs (39) were identified in less than half of the sampling sites. Unweighted pair group method with arithmetic averages (UPGMA) clustering of the OTUs suggested that the plankton communities in the Xiangxi Rive sites were not always significantly different from those from the Yangtze River sites, despite clear differences in their environmental characterizations. Canonical correspondence analysis (CCA) was applied to further investigate the relationships between OTU composition and the environmental factors. The first two CCA ordination axes suggested that the bacterial community composition was primarily correlated with the variables of NO3--N, dissolved oxygen (DO), and SiO32--Si, whereas, the eukaryotic community was mainly correlated with the concentrations of DO, PO43--P, and SiO32--Si.

Spatiotemporal heterogeneity of plankton communities in Lake Donghu, China, as revealed by PCR-denaturing gradient gel electrophoresis and its relation to biotic and abiotic factors

The 16S and 18S rRNA genes of planktonic organisms derived from five stations with nutrient gradients in Lake Donghu, China, were studied by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, and the relationships between the genetic diversity of the plankton community and biotic/abiotic factors are discussed. The concentrations of total nitrogen (TN), total phosphorus (TP), NH4-N and As were found to be significantly related (P < 0.05) to morphological composition of the plankton community. Both chemical and morphological analyses suggested that temporal heterogeneity was comparatively higher than spatial heterogeneity in Lake Donghu. Although the morphological composition was not identical to the DGGE fingerprints in characterizing habitat similarity, the two strongest eutrophic stations (I and II) were always initially grouped into one cluster. Canonical correspondence analysis suggested that the factors strongly correlated with the first two ordination axes were seasonally different. The concentrations of TN and TP and the densities of rotifers and crustaceans were generally the main factors related to the DGGE patterns of the plankton communities. The study suggested that genetic diversity as depicted by metagenomic techniques (such as PCR-DGGE fingerprinting) is a promising tool for ecological study of plankton communities and that such techniques are likely to play an increasingly important role in assessing the environmental conditions of aquatic habitats.

Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.