基于荧光方法的大肠杆菌单管定量检测技术研究

通过研究培养温度、培养基pH值对大肠杆菌生长和培养液荧光强度的影响,确定44℃,培养液pH值7.0～7.5为大肠杆菌MUG酶荧光检测的最佳条件。通过研究培养时间和接种菌浓度与培养液荧光强度的关系,建立起基于荧光方法检测大肠杆菌的单管定量检测技术。通过与平板菌落计数法和最大可能数法比较发现单管定量检测法的相对标准偏差小于这两种常用的方法,而且更为快速、经济,适合用于大肠杆菌的定量检测。     

珊瑚和海葵来源红荧光蛋白的研究和应用

绿色荧光蛋白作为标记蛋白和报告蛋白在生物学研究中应用越来越广。但在荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET)等技术中存在一些缺陷,需要更大波长范围的荧光蛋白。最近研究发现了多种来源于珊瑚和海葵的红荧光蛋白,这些长波长的荧光蛋白对绿色荧光蛋白是一种很好的代替和补充,可以实现细胞内多荧光标记,提供更理想的FRET荧光对。经随机突变和定点突变等方法改建获得的红荧光蛋白变种显示出更高的荧光强度,成熟时间也更短。目前应用较多的是来源于香菇珊瑚(Discosomasp.)的红荧光蛋白DsRed。     

镁离子对柱孢鱼腥藻(Anabaena cylindrica)类囊体膜吸收光谱和荧光光谱的影响

研究了不同浓度(5和10mM)的镁离子对柱孢鱼腥藻类囊体膜吸收光谱和荧光光谱的影响。5mM镁离子浓度降低柱孢鱼腥藻类囊体膜的叶绿素α在红区和蓝区的吸收峰,表现相似于镁离子对叶绿体的叶绿素α吸收峰的变平效应。在室温下,加入5mM镁离子浓度使膜的光系统Ⅱ684nm发射荧光强度降低。在液氮低温下,镁离子降低膜的光系统Ⅱ684nm和光系统Ⅰ728nm发射荧光强度的比值。可能表明镁离子促进光系统问激发能的传递。同时,镁离子增大叶绿素α所吸收光的激发能对光系统Ⅰ发射荧光的贡献,促进叶绿素α至光系统Ⅰ的激发能传递。     

γ辐射对红细胞膜流动性的影响

我们实验以红细胞膜作为材料,使用ANS和DPH荧光探针测量红细胞膜的荧光强度和偏振度。发现γ辐照红细胞膜剂量从4.6×10~3拉德增至148×10~3拉德,而ANS和DPH的荧光强度随剂量的增加而降低,荧光偏振度随剂量的增加而增加,这些结果表明γ辐照引起红细胞膜流动性降低。但荧光强度的减少并不伴随荧光光谱的变化。ANS与γ辐照的膜结合量也没有改变,因此荧光强度的减少可能是由于在辐照的膜中ANS荧光量子产率的降低。在我们的实验中γ辐射对ANS探针所引起的荧光变化较DPH灵敏,看来γ辐射首先影响膜蛋白。     

基于荧光强度快速判断蛋白可溶性方法的建立

目的:建立一种通过观察荧光强度,快速、直观地判断蛋白可溶性的方法。方法:选择抗辐射蛋白CBL502-AA及其突变体CBL502-ΔAA′作为目的蛋白,增强型绿色荧光蛋白作为报告分子,分别构建融合蛋白表达载体;通过SDS-PAGE和荧光观察两种手段检测和比较融合蛋白的可溶性。结果:荧光观察融合蛋白的可溶性与SDS-PAGE检测结果一致。结论:建立了一种基于荧光强度,快速、简单、直观的比较蛋白可溶性的方法。     

抗砷细胞荧光染色后的激光共聚焦显微镜检测

目的:应用激光共聚焦显微镜检测活细胞内荧光物质含量.方法:传代培养长期低剂量砷诱导的抗砷细胞,用荧光染料Rhodamine-123对细胞染色30min,实验组与维拉帕米(Verapamil)共同孵育,对照组为单加Rhodamine-123的抗砷细胞.应用激光共聚焦显微镜采集Rhodamine-123的荧光图像动态序列,并且记录不同时间段的细胞内荧光强度.结果:实验组细胞染色12h,24h,36h,48h,60h后,荧光强度依次为(51.567±0.7572)、(46.533±0.7095)、(39.557±0.601)、(38.6±0.6245)和(38.505±0.718),明显高于同时间段对照组的荧光强度,差异均有显著性(P＜0.01).结论:应用激光共聚焦显微成像技术能进行活细胞水平荧光物质实时定量检测.     

一种基于EdU的体外癌细胞与小鼠体内组织细胞标记方法的研究

摘要 目的：比较EdU标记对三种癌细胞和小鼠对细胞增殖的影响,为EdU作为标记开展相关细胞增殖实验和临床研究提供依据。方法：本研究使用不同剂量EdU对人非小细胞肺癌A549细胞、人宫颈癌Hela细胞、人肝癌Huh7进行标记2 h,然后使用荧光显微镜观测EdU在细胞中的标记效率,并使用多波长荧光酶标仪检测这三种癌细胞系标记后的荧光强度;使用流式细胞仪检测小鼠经不同剂量的EdU干预12 h后,体内肺、肝、肾组织标记的荧光强度。结果：与对照组相比,经EdU处理后,A549和Hela细胞系的荧光强度,三个剂量组均有显著性差异（P＜0.01）,Huh7细胞系的荧光强度,50 μmol/L有显著性差异（P＜0.05）;EdU在小鼠体内组织肺、肝、肾组织中均有分布,且在肝组织中分布比肺组织和肾组织高。结论：EdU的体外癌细胞与小鼠体内组织细胞的标记效率各不相同,建立的EdU体外标记癌细胞和小鼠体内组织的方法简单,易操作。     

Dworf序列增强GFP荧光强度

荧光蛋白质是重要且常用的标签蛋白质,但是在没有强启动子的情况下表达量低,会影响检测的灵敏度。DWORF（dwarf open reading frame）是由NONMMUG026737 编码的长度为34个氨基酸的短肽,其mRNA 5′端能够启动下游基因表达。Kozak序列是现今唯一一个位于成熟mRNA 5′端的增强子,能够提高基因在蛋白质水平的表达,但对某些基因在蛋白质水平的表达增强效果不理想。为了检测DWORF mRNA 5′端（命名为DW）是否能够通过提高荧光蛋白质基因在蛋白质水平的表达,从而增强荧光强度,我们设计了如下实验：将DWORF mRNA 5′端与绿色荧光蛋白 (green fluorescent protein, GFP)构建到表达载体中（命名为DW-GFP）,转染细胞后检测荧光强度,发现与对照组相比,转染DW-GFP质粒的细胞荧光强度显著提高（56.82 ± 1.77 vs. 45.97 ± 0.44, P<0.05）。为了便于后续应用,将DW截短到18个碱基（命名为DW18）,仍能显著提高GFP的荧光强度（33.57 ± 1.11 vs.25.34 ± 0.98, P<0.05）,且比Kozak序列提高6.80%。同时,我们发现DW18与Kozak序列同时存在能够进一步提高GFP的荧光强度,比Kozak序列单独存在时提高13.58%。本研究证明DWORF序列及其截短形式（DW18）能够有效提高表达效率,为研究低丰度蛋白质的生物学功能提供了可能的方法和工具。     

拟南芥叶细胞游离钙离子的测定

低温(4℃)条件下将钙离子荧光探针Fluo-3／AM导入拟南芥叶细胞,利用激光共聚焦显微技术检测了胞内钙离子荧光强度的分布。实验证明,低温导入Fluo-3／AM法测定拟南芥叶细胞中钙离子荧光强度的变化切实可行。茉莉酸(JA)处理能够诱导胞内游离钙离子浓度的升高。     

一种提高质粒DNA凝胶电泳检测灵敏度的方法

比较了凝胶电泳示检测质粒ＤＮＡ时不同激发波长对ＤＮＡ－ＥＢ荧光强度的影响,发现短波长激发光可增加ＤＮＡ的探测灵敏度。采用２６０ｎｍ作为激光光时可探测到少至０．７ｎｇ的线性ＤＮＡ。且在很广的ＤＮＡ的质量范围内,ＤＮＡ－ＥＢ荧光强度 与ＤＮＡ量或正比。以此改进方法检测电离辐射诱导的ＤＮＡ单、双链断裂岢得到与其它研究结果相一致的Ｇ（ＳＳＢ）和Ｇ（ＤＳＢ）值。     

论山西中元古代晚期汝阳群微体化石组合

我国北方18-19亿年前的串岭沟组中获得的大球形疑源类,迄今,被认为是可信的最古老真核化石记录。自上世纪80年代以来,东秦岭北坡的中元古代晚期汝阳群陆续发现了丰富的球形光面疑源类,具刺疑源类,丝状微体化石和一些有疑问的动物遗迹。很显然,在中元古代晚期真核生物曾有重大辐射。     

Fluorescence intensity studies of Triassic acritarchs from the Yanchang Formation in Ordos basin,northwestern China

Fluorescence properties of Early Cambrian acritarchs were investigated using Leica das Mikroskop (DM)microscopy with a mercury lamp.Well-preserved autoflurescence properties show a correlation between acritarchs morphology and the intensity of emitted fluorescence.In accordance with the fluorescence intensity of organic cell walls,two groups ofmicrofossils were distinguished.Results of observation in this study,which are consistent with those of the previous foreign studies,are in good agreement with regular difference in autofluorescence intensity among palynomorphs reported by McPhilemy (1998).Spores and algae,including Botryococcus,have very bright fluorescence while acritarchs often show less intense fluorescence.Dark brown microfossils have been reworked,and have little or no fluorescence.     

湖北峡东地区九龙湾剖面震旦系陡山沱组微体化石的新发现

中国南方扬子地台震旦系陡山沱组产出丰富的微体化石,它们主要保存在磷块岩以及燧石结核和条带中。文章详细报道湖北峡东地区九龙湾剖面震旦系陡山沱组微体化石,描述以前未曾在峡东地区碳酸盐相燧石中发现的8属8种微体化石。研究九龙湾剖面陡山沱组大型带刺疑源类、微体多细胞藻类和动物胚胎化石的分布特征,发现Tianzhushania spinosa是最早出现的大型带刺疑源类分子,大冰期后微体生物的辐射是一个阶段性渐进的过程。同时．本项研究进一步证实华南扬子区陡山沱组碳酸盐相燧石和磷块岩地层中保存的微体化石面貌基本一致。     

山西永济白草坪组具刺疑源类的发现及其地质意义

该文着重报道了山西永济白草坪组(一般认为系中元古界上部相当于蓟县系的层位),以具刺疑源类为主的微化石群,计有4类6属15种,其中包括1新属,4新种,1新组合,1亲近种,2未定种,新属是具细网状纹饰的复杂具刺疑源类Shuiyousphaeridium Yan gen.nov.,4新种是Shuiyousphaeridium membraniferum Yan sp.nov.,Dictyosphaera incrassate Yan sp.nov.,Foliomorpha stipuliferaYan sp.nov.,Manicosiphoninema shuiyouense Yan sp.nov.。本微化石群以球形疑源类为主体,以具刺疑源类的大量发育为特征,并伴生有推测是绿藻和褐藻的化石,其组合面貌与蓟县上前寒武系各组皆有明显区别,具有更高的进化程度,从生物地层标志推测白草坪组所在的汝阳群应归入震旦系。白草坪组具刺疑源类可能是横裂甲藻的原始分子,它在地层对比和藻类进化方面皆具有重要意义。     

新疆哈密地区晚泥盆世疑源类化石的发现及地质意义

从新疆哈密地区石城子北剖面7件样品获得分异度较高、有机质壁显著炭化保存的疑源类化石,根据其形态特征,共鉴定出14个形态属和15个形态种(其中8个未定种,3个比较种)。结合国内外古生代晚泥盆世已知疑源类组合进行比较,清楚表明当前获得的疑源类组合代表了晚泥盆世海洋微体浮游植物群面貌。我国涉及晚古生代疑源类生物地层的调查研究相对薄弱,而有关晚泥盆世疑源类化石的发现和报道更为匮乏,当前疑源类化石的发现,填补和丰富了新疆乃至国内晚泥盆世疑源类化石研究资料。该发现佐证了关于卡拉麦里洋在早石炭世闭合的认识;作为基础食物链的海洋微体浮游植物是重要成烃生物,晚泥盆世疑源类的保存预示研究区域具有石油、天然气勘察的前景。     

Fluorescence intensity studies of Triassic acritarchs from the Yanchang Formation in Ordos basin, northwestern China

Fluorescence properties of Early Cambrian acritarchs were investigated using Leica das Mikroskop (DM) microscopy with a mercury lamp. Well-preserved autoflurescence properties show a correlation between acritarchs morphology and the intensity of emitted fluorescence. In accordance with the fluorescence intensity of organic cell walls, two groups of microfossils were distinguished. Results of observation in this study, which are consistent with those of the previous foreign studies, are in good agreement with regular difference in autofluorescence intensity among palynomorphs reported by McPhilemy (1998). Spores and algae, including Botryococcus, have very bright fluorescence while acritarchs often show less intense fluorescence. Dark brown microfossils have been reworked, and have little or no fluorescence. __________ Translated from Acta Micropalaeontologica Sinica, 2006, 23(3): 309–312 [译自: 微体古生物学报]     

Microplankton associations, biofacies and palaeoenvironment of the type lower Ludlow Series, Silurian

The acritarchs and prasinophyte algae from the type lower Ludlow Series of the Goggin Road section, Ludlow, England, are resolved into seven recurrent associations comprising taxa with similar environmental preferences. Endemic and environmentally sensitive associations of acritarchs and prasinophytes are identified and high-resolution fluctuations in the early Ludlow palaeoenvironment are established. An early Ludlow crisis in the acritarchs is recognized in the lower part of the Middle Elton Formation, when an abrupt palaeoenvironmental change in the Ludlow area resulted in a large decline in the abundance of the acritarchs, but allowed Tasmanites and retiolitid graptolites to flourish briefly. Cymbosphaeridium sp. A, Pulvinosphaeridium ludlowense and Multiplicisphaeridium arbusculum forma A are taxa possibly specialized, or produced as a response, to a stressed palaeoenvironment, as they are most abundant when other acritarchs and prasinophytes are uncommon. The low abundance of acritarchs and prasinophytes in the Upper Elton Formation may be related to high sedimentation rates and to the slumping of sediments caused by instability on the shelf of the Welsh Basin, or to lower plankton productivity.     

BASAL CAMBRIAN MICROFOSSILS FROM THE YURTUS AND XISHANBLAQ FORMATIONS (TARIM, NORTH-WEST CHINA): SYSTEMATIC REVISION AND BIOSTRATIGRAPHIC CORRELATION OF MICRHYSTRIDIUM-LIKE ACRITARCHS

Abstract:  Micrhystridium -like acritarchs are widely distributed in basal Cambrian cherts and phosphorites in South China. This paper describes similar acritarchs from the basal Cambrian Yurtus and Xishanblaq formations in Tarim, north-west China. The taxonomy of these acritarchs is revised. The basal Cambrian acritarch assemblage in Tarim and South China is characterized by three genera: Asteridium Moczydłowska, Heliosphaeridium Moczydłowska and Comasphaeridium Staplin, Jansonius and Pocock. This assemblage is named the Asteridium - Heliosphaeridium - Comasphaeridium (AHC) acritarch assemblage. In both South China and Tarim, the AHC acritarch assemblage is associated with the tubular microfossil Megathrix longus Yin L. and the small shelly fossil Kaiyangites novilis Qian and Yin G. This assemblage also occurs in the Lower Tal Formation in the Lesser Himalaya. Correlation with small shelly fossil (SSF) assemblages indicates that the AHC assemblage is restricted to the Meishucunian Stage, and possibly to the lower Meishucunian ( Anabarites trisulcatus - Protohertzina anabarica and Siphogonuchites triangularis - Paragloborilus subglobosus SSF assemblages). The AHC assemblage is broadly similar to the Asteridium tornatum - Comasphaeridium velvetum (acritarch) Zone in the East European Platform, which is considered to be Nemakit-Daldynian (and possibly Tommotian) in age.     

Diverse small spinose acritarchs from the Ediacaran Doushantuo Formation, South China

The Ediacaran Doushantuo Formation in South China is underlain by the Cryogenian Nantuo Formation (glacial rocks) and overlain by the late Ediacaran Dengying Formation. It is characterized by well-preserved, large (normally >100 μm in size) spinose acritarchs (LSAs), which have been shown to be probably the only useful biostratigraphic tool for the global correlation of the early- and middle-Ediacaran. Acritarchs are organic microfossils normally known as single-celled eukaryotic organisms (protists). Although recent research suggests that some large spinose acritarchs may represent diapause egg cysts of metazoans, the biological affinities of the Ediacaran spinose acritarchs, especially for those displaying remarkable size ranges, are still debatable.Recently, smaller specimens of the Ediacaran spinose acritarchs have been found in cherts and phosphorites of the Doushantuo Formation in South China. Many described Ediacaran spinose acritarch taxa display large size variation (from tens to hundreds of microns in vesicle diameter), but some taxa only have smaller (<70 μm) specimens. The morphological comparison with Paleozoic counterparts indicates that some Ediacaran spinose acritarchs may have phylogenetic affinity to eukaryotic algae. More evidence, including wall ultra-structure, geochemical analysis and comparison with modern analogs, is needed to understand the biological affinity of the Ediacaran spinose acritarchs. The remarkable radiation of planktonic protists, characterized by abundant, diverse spinose acritarchs, occurred as early as in the late Neoproterozoic, i.e., 40–60 million years earlier than previously thought.     

Population dynamics of galeate acritarchs at the Cambrian–Ordovician transition in the Algerian Sahara

Galeate acritarchs are a major component of Late Cambrian and Early Ordovician palynological assemblages. The populations of galeate acritarchs from the Cambrian–Ordovician transition section in borehole Nl-2 of the Algerian Sahara show a gradual increase in process length and in complexity of the process structures through the succession. While the number of striate elements at the process base, the number of distal ramifications, and the number of membranes between the processes increases progressively, the central body diameter shows only minor variation within the succession. The galeate acritarchs are here interpreted as probably being resting cysts of a microorganism similar to modern dinoflagellates. Published laboratory culture experiments on living dinoflagellates document a considerable morphological variation with respect to the process morphology of the resting cysts that can be produced by a single biologically defined species. Based on these experiments, it is possible to interpret the morphological changes in the galeate acritarchs as being possibly related to changing ecological conditions. By analogy to the cyst distribution of modern dinoflagellates, changing salinity may have played an important role. Depending on environmental parameters, and perhaps on the maturity attained before rupture of the outer membrane during cyst formation, the galeate acritarchs may show a wide variety of process morphologies that have to date been used to describe four genera (morphogenera) and 84 species (morphospecies).