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phi29 DNA聚合酶结合随机引物(Random Primer)进行扩增,获得了丰富且质量较高的PCR产物.以全基因组扩增产物(稀释10倍)对四个常用的基因位点(TEF1、SSU rRNA、18S-ITS1-5.8S、a-tubulin)进行扩增,均成功获得相应的基因片断.     

海洋单细胞藻对紫外吸收的比较研究

用日本岛津UV-260分光光度计,对9种海洋单细胞藻的紫外吸收变化进行了研究.结果表明:(1)9种海洋单细胞藻的吸收峰的波长均在280nm以下,它们相应的波长范围在252.8～266.4nm之间,高低相差仅为13.6nm;(2)按吸收峰值从大到小排列依次为:盐藻＞扁藻＞小球藻＞塔胞藻＞角毛藻＞三角褐指藻＞新月菱形藻＞金藻3011＞金藻8701,呈现明显的规律性变化,即:绿藻门＞硅藻门＞金藻门;(3)在UV-B(280～320nm)波段,9种海洋单细胞藻的吸收度均随波长的增加呈现逐渐下降的趋势,而吸收度的大小排列次序同吸收峰值完全一致,同样表现出绿藻＞硅藻＞金藻的规律.     

Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council     

用于单一藻细胞PCR扩增反应的样品保存方法比较

用室内培养的塔玛亚历山大藻(Alexandrium tamarense ATHK藻株)作为实验对象,分别选择了5%福尔马林固定后常温保存、95%乙醇固定后常温保存、-20℃冷冻保存、鲁哥氏液(Lugols solution)固定后常温保存等方法,在5,15,30,60 d后对保存的样品进行单细胞PCR反应,并与新鲜样品进行比较。实验结果表明,用95%乙醇保存、-20℃冷冻保存、鲁哥氏液保存的样品在60 d后仍可扩增出目标条带,与新鲜样品没有差别,但是样品中的藻细胞形态有一些改变;而以5%福尔马林固定保存的样品只能在5 d后扩增出目标条带,但藻细胞形态比较完好。因此,对用于单细胞PCR实验的微藻样品,短期内(不多于5 d)可以采用福尔马林保存,而需要长期保存的样品,应当采用乙醇或鲁哥氏液固定,或者采用-20℃冷冻保存的方法。     

聚苯乙烯微塑料与铜离子单一和复合污染对THP-1细胞的DNA损伤效应研究

采用单细胞凝胶电泳实验研究了不同浓度的Cu²⁺(10、50、100、500、1000、2500、5000μg/L)和不同粒径(0.1、0.2、0.3、0.5、0.7、1.0、2.0μm)、不同浓度(1、10、15、20、25 mg/mL)的聚苯乙烯微塑料对人单核细胞白血病(Human Acute Monocytic Leukemia,THP-1)细胞的DNA损伤效应,结果表明:单一污染时,Cu²⁺(≥50μg/L)对THP-1细胞的DNA损伤极显著(p<0.01)。而且,DNA损伤值随Cu²⁺浓度的增大而增加。2.0μm粒径的聚苯乙烯微塑料(10 mg/mL)对THP-1细胞产生明显的DNA损伤效应。复合污染作用下对THP-1细胞的DNA损伤效应,相比单一污染明显增强,而且DNA损伤值比单一污染的理论加和值高。本研究表明Cu²⁺和聚苯乙烯微塑料均对DNA产生损伤效应,显示一定的基因毒性,二者复合对损伤具有交互增强的作用。     

危险的“荧光海滩”

<正>近年来,"荧光海滩"现象成为热议,全世界有7个地方先后报道出现这种奇特现象,其中3个在波多黎各、2个在澳大利亚、1个在马尔代夫、另外1个就是我国大连的大黑石村浴场。海洋生物学者普遍认为"荧光海滩"其实是由无数散发着幽蓝光芒的浮游生物随着浪花冲集在海滩上形成的壮观场景。目前的"荧光海     

Effects of virus infection on expression of cell cycle regulatory proteins in the unicellular marine algae Emiliania huxleyi

The authors have investigated the biochemical events by which marine algal virus infection induces cell cycle arrest. The key G₂/M-phase regulatory proteins are analyzed by immunobloting in unicellular Emiliania huxleyi, suggesting that virus induced cell cycle arrest is related with virus''s effect on cyclins and cyclin dependent kinases. E. huxleyi virus (EhV) represses Cdc2/cyclinB complex activity by inhibiting the activity of Cdc2 kinase in a phosphorylation-related manner, blocking host cells G₂/M checkpoint. Dephosphorylated/inactive Cdc25C combined with up-regulation of Wee1 expression at early infect period appears to be important mechanisms by which EhV represses Cdc2/cyclinB complex activity that is required for entry into M phase. This study has allowed us to confirm that algal virus infection leads to selective activation or inhibition of certain cell-cycle factors, which may play a significant role in establishing a more efficient environment for viral gene expression and DNA replication.     

蓟县高于庄组燧石中原位保存的单细胞微体化石新类型

本文描述的化石产自天津市蓟县中、新元古界剖面长城系高于庄组（小红峪沟）一段，即官地亚组燧石条带中。其基本外形为两端浑圆的橡皮艇状，沿长轴方向具有一个贯通化石体的纵向开口，开口的大小约占标本宽度的1／3，以开口为界，化石体具有两侧对称特征。该类形态化石在蓟县剖面的报道尚属首次。文中就化石体的基本形态特征及产出特点与国内外相关化石进行了详细对比。另据标本所显示的规则的开口结构，推测其可能具有释放内生孢子的功能，因此视其为可能的真核生物。此外，笔者还就该层位化石种类单调，个体微小（8～17μm）等特征，结合官地亚组的沉积特点，探讨了化石的形成环境。     

单细胞凝胶电泳用于检测低温保存的真鲷(Pagrosomus major)精子DNA损伤

应用单细胞凝胶电泳(SCGE)方法,对超低温冷冻保存的真鲷精子DNA的损伤状况进行了检测研究。针对研究对象,在实验过程中对传统的碱性单细胞凝胶电泳在铺胶方法、电泳条件等进行了改进。对精子细胞进行预处理,在碱性电泳液中使核DNA双链解链变性后电泳,EB染色10min后,在荧光显微镜下观察,每次随机观察50个左右的核DNA。结果表明,对荧光显微镜下观察到的精子核按彗尾长度及荧光强度划分等级,出现损伤的精子核DNA的损伤程度主要为轻度损伤和中度损伤,很少见有完全损伤的真鲷精子核。对比真鲷冷冻精液与新鲜精液的精子DNA的损伤状况,表明仅用30%DMSO冷冻精子DNA损伤状况与鲜精差异显著(P>95%),其他冷冻方法保存的精子与鲜精差异不显著(P>95%)。     

纤毛虫单细胞基因组DNA微量提取方法的比较及优化

在纤毛虫的分子生物学研究中,单细胞基因组DNA的微量提取是后续基因分析的关键。本研究以海洋纤毛虫红色伪角毛虫为材料,采用直接取完整虫体、裂解法以及3种改良后的试剂盒微量提取DNA方法,分别提取了新鲜样品与4种不同方法保存样品的基因组DNA,比较了9种组合在PCR产物质量、提取所需时间、成本及便捷性方面的优劣,并进一步比较、讨论和探索了4种样品保存方法和5种DNA微量提取方法在纤毛虫单细胞基因组PCR扩增中的可行性。本工作旨在为后续的纤毛虫分子生物学研究提供可资借鉴的技术路线和方法。