全细胞杂交在三种赤潮微藻检测中的应用

从核糖体DNA的转录间隔区ITS和18SrRNA的基因序列中设计特异性荧光核酸分子探针,采用全细胞杂交方法（即荧光原位杂交法）对锥状斯氏藻（Scrippsiella trochoidea（Stein）Loeblich Ⅲ）、赤潮异弯藻（Heterosigma akashiwo（Hada）Hada）和球状棕囊藻（Phaeocystis globosa Scherffel）三种单胞微藻进行了分子鉴定,用荧光显徽镜和流式细胞仪对杂交结果进行了观察。结果表明,实验所设计的特异性分子探针结合全细胞杂交方法和流式细胞仪检测手段能够有效地实现对上述三种赤潮微藻的检测。由于该三种藻细胞的细胞外被物存在差别,荧光显微镜检测效果存在差异,即在荧光显微镜下,赤潮异弯藻和球状棕囊藻的杂交细胞的荧光强度比锥状斯式藻杂交细胞的荧光强度弱,在一定程度上影响了观察效果。同时,用该方法还成功地从模拟自然海区混合样品中检测出球状棕囊藻。因此,该研究方法适用于对实验室和自然海区样品的赤湖微藻的检测。     

赤潮生物原甲藻Prorocentrum分子识别和系统发育学研究

对东海原甲藻Prorocentrum donghaiense、海洋原甲藻P.micans、微小原甲藻P.minimum、立玛原甲藻P.lima(CCMP1966)四种赤潮生物的18S rDNA进行了PCR扩增和全序列测定及分析,比较了它们的遗传距离和相似系数,获得其18S rDNA序列中的三处高变异区,为设计快速识别原甲藻种间分子探针提供分子依据.同时结合12种具有代表性的重要赤潮甲藻的18S rDNA序列,采用邻接法(Neighbour-Joining,NJ)和最大似然法(Maximum Likelihood,ML)构建系统发育树,研究原甲藻的种间关系和进化地位,结果表明,营底栖生活的P.lima与营浮游生活的P.micans、P.minimum、P.donghaiense为多系起源,浮游型原甲藻与凯伦藻Karenia有更近的起源关系.     

浮游植物活体三维荧光光谱特征提取

测量了6种东海常见的浮游植物在两个温度(20℃,15℃)、三个光照(7000Lux,4100Lux,1100Lux)下的不同生长期的三维激发／发射荧光光谱,研究了光谱特征提取方法。对去除散射干扰后的三维光谱进行了奇异值分解,得到的相应于激发光谱的第一主成分具有区分藻种的能力,可作为三维光谱的特征光谱。分析结果表明,实验条件下,等鞭金藻(Isochrysis galbana)、岛国大扁藻(Platymonas helgolanidica)和中肋骨条藻(Skeletonema costatuma)的特征光谱相似度高,塔玛亚历山大藻(Alexandrium tamarense)、东海原甲藻(Prorocentrum dentatum)和尖刺拟菱形藻(Pseudo—nitzschia pungens)光谱相似度稍差。     

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     

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.     

海洋微藻产生的不同粒级溶解有机物质的三维荧光光谱

通过过滤(0.7μm)和切向超滤(100kDa、10kDa、1kDa)技术将实验室内培养的赤潮异弯藻和中肋骨条藻藻液分离成100kDa-0.7μm、10～100kDa、1～10kDa的胶体浓缩液和＜1kDa的超滤液,利用激发-发射矩阵荧光光谱技术测定了不同粒级溶液中有机物的荧光性质,分析了三维荧光光谱图中的荧光峰位置、数量及荧光强度的变化情况.结果表明,两种微藻产生的类腐殖质荧光物质主要是＜1kDa的小分子物质.赤潮异湾藻产生的类蛋白荧光物质中,15%是100kDa-0.7μm的大胶体,8%是10～100kDa的中胶体,而大部分是＜1kDa的小分子物质.赤潮异弯藻各级超滤截留液的类蛋白荧光峰的发射波长与过滤液相比,发生蓝移,而超滤液的则发生红移,表明赤潮异湾藻产生的类蛋白荧光物质随着粒径的增大极性减弱.     

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。     

海洋浮游植物生长过程中溶解有机物质的三维荧光光谱研究

利用三维激发-发射矩阵荧光光谱技术,监测了赤潮异弯藻和中肋骨条藻培养过程中产生的溶解有机物,分析了三维荧光光谱图中的荧光峰位置、数量及荧光强度的变化情况.结果表明,微藻生长过程中会产生类蛋白和类腐殖质两类荧光有机物质,这两类有机物的荧光峰的位置及荧光强度有较大差异.在不同生长期,两类荧光有机物的产生机制不同.在指数生长期,两类有机物的荧光强度与藻密度成正相关,说明浮游植物释放了一定量的荧光物质;在平稳期和衰亡期,两类有机物的荧光强度迅速增加,这可能由于衰老、死亡藻细胞的破碎释放出大量的荧光有机物质所致,此外细菌对非荧光有机物进一步降解,也可能是产生该现象的一个原因.     

运用荧光定量PCR技术检测中肋骨条藻的研究

以rDNA序列为寻找种特异性引物的靶区域,通过分析中肋骨条藻rDNA序列,设计出7套适合用于实时荧光定量PCR方法(RFQ-PCR)的引物与探针,经引物验证,选择Primer6(F/R)进一步分析,对比扩增序列及核酸杂交验证,表明该探针可作为中肋骨条藻特异性探针.最后以Primer6(F/R)和TaqMan6建立了定量检测中肋骨条藻的RFQ-PCR方法,并绘制出了定量检测中肋骨条藻的标准曲线,测定数据用镜检计数进行了验证,证明该方法是准确可靠的.     

大豆根瘤菌8－1－4A色氨酸营养缺陷突变型的筛选及其原养型回复突变株共生效率的研究

采用转座子Ｔｎ５诱变快生型大豆根瘤菌（Ｒｈｉｚｏｂｉｕｍｆｒｅｄｉｉ）８－１－４Ａ筛选到１０个色氨酸营养缺陷型突变株，植物结瘤试验表明这些菌均失去了结瘤能力。经自发突变，从培养平板和振瘤中分离出２３个色氨酸原养回复突变株，盆栽试验结果表明有２２株的结瘤能力得到恢复，其中有５株的共生效率与出发菌相比有显著提高。对部分回复突变株进行了质粒检测和以标记Ｔｎ５为探针的ＳｏｕｔｈｅｒｎＤＮＡ杂交分析。     

In situ Visualization of Gene Expression Using Polymer‐Coated Quantum–Dot–DNA Conjugates

Imaging of specific mRNA targets in cells is of great importance in understanding gene expression and cell signaling processes. Subcellular localization of mRNA is known as a universal mechanism for cells to sequester specific mRNA for high production of required proteins. Various gene expressions in Drosophila cells are studied using quantum dots (QDs) and the fluorescence in situ hybridization (FISH) method. The excellent photostability and highly luminescent properties of QDs compared to conventional fluorophores allows reproducible obtainment of quantifiable mRNA gene expression imaging. Amine‐modified oligonucleotide probes are designed and covalently attached to the carboxyl‐terminated polymer‐coated QDs via EDC chemistry. The resulting QD–DNA conjugates show sequence‐specific hybridization with target mRNAs. Quantitative analysis of FISH on the Diptericin gene after lipopolysaccharide (LPS) treatment shows that the intensity and number of FISH signals per cell depends on the concentration of LPS and correlates well with quantitative real‐time PCR results. In addition, our QD–DNA probes exhibit excellent sensitivity to detect the low‐expressing Dorsal‐related immunity factor gene. Importantly, multiplex FISH of Ribosomal protein 49 and Actin 5C using green and red QD–DNA conjugates allows the observation of cellular distribution of the two independent genes simultaneously. These results demonstrate that highly fluorescent and stable QD–DNA probes can be a powerful tool for direct localization and quantification of gene expression in situ.     

Incomplete chromosome exchanges are not fingerprints of high LET neutrons

A new fluorescence in situ hybridisation (FISH) technique combining whole chromosome specific DNA libraries with pan-centromeric DNA and telomeric PNA probes was introduced to investigate the induction of chromosome exchanges in human lymphocytes after exposure to low (4 Gy X rays) and high (1 Gy neutrons) linear energy transfer radiation. This combination of probes allowed accurate detection of exchange aberrations involving the painted chromosomes and an unambiguous discrimination between complete and incomplete exchanges, as well as terminal and interstitial deletions. Data obtained in the present study using combined FISH assay with telomeres detection showed no differences between two types of radiation regarding the induction of incomplete exchanges.     

Nonbleaching fluorescence of gold nanoparticles and its applications in cancer cell imaging

In this paper, we investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half-maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at the single particle level. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. On the basis of this principle, we imaged living HeLa cells using GNPs as fluorescent probes and obtained good cell images by photobleaching of cellular autofluorescence. Furthermore, anti-EGFR/GNPs were successfully used as targeted probes for fluorescence imaging of cancer cells. Our preliminary results demonstrated that GNPs possessed excellent behaviors of antiphotobleaching and were good fluorescent probes in cell imaging. Our cellular imaging method described has potential applications in cancer diagnostics, studies, and immunoassays.     

16S rRNA基因与16S-23S rRNA转录单元内间隔区序列分析及其在节旋藻和螺旋藻分类鉴定中的应用

测定了节旋藻属3个品系和螺旋藻属1个品系的全长16SrRNA基因基因和16S rRNA转录单元内间隔区序列（ITS）,分析了已知的节旋藻、螺旋藻和相关品系的相应序列的同源性,构建了系统发生树,并评价了这两段DNA序列在节旋藻、螺旋藻种属分类和种质鉴定中的意义。结果表明：（1）16SrRNA基因序列和ITS序列均可用于节旋藻属和螺旋藻属的属间分类,以两序列为基础的系统学分析结果一致;（2）ITS序列变异程度高于16SrRNA序列,适用于节旋藻和螺旋藻属内品系或种质鉴定;（3）节旋藻属可明确界定,16SrRNA基因序列相似性大于98％,ITS序列相 似性大于88％;（4）螺旋藻属某些品系间16SrRNA序列和ITS序列相似性较低,与不同属间的序列相似性程度为同一水平。     

Two-photon excited fluorescence of a conjugated polyelectrolyte and its application in cell imaging

The two-photon excited fluorescence of a conjugated polyelectrolyte (CPE), PPESO3, was studied in methanol and in water. The photophysical and amplified quenching properties of the CPE observed under two-photon excitation were comparable to the results obtained under one-photon excited conditions. Two-photon fluorescence microscopy performed with PPESO3-coated silica nanoparticles in HeLa cells provided images with significantly improved resolution compared to one-photon microscopy, demonstrating the utility of the CPE as a fluorescent probe in two-photon fluorescence cell imaging.     

Detection of Epstein-Barr virus infection in gastric carcinomas using quantum dot-based fluorescence in-situ hybridization

Quantum dots were proposed as new fluorochromes for use in fluorescence in-situ hybridization. EBV-encoded small RNA, the most abundant viral product in latently infected cells, was detected by quantum dot fluorescence in-situ hybridization in paraffin-embedded tissue sections of gastric carcinoma. An indirect FISH approach using quantum dots streptavidin conjugates as secondary reporters and digoxigenin labeled EBV-encoded small RNA oligonucleotide probes as detectable molecules was employed. Quantum dot fluorescence in-situ hybridization offered a slightly higher sensitivity in detecting EBV-encoded small RNA in gastric carcinoma than chromogenic in-situ hybridization. Statistical analyses showed that the detected EBV-associated gastric carcinoma was not associated with any clinicopathological parameters of the Chinese gastric carcinoma patients investigated in this study.     

Potential for using isotopically altered metalloproteins in species-specific isotope dilution analysis of proteins by HPLC coupled to inductively coupled plasma mass spectrometry

The production and evaluation of an isotopically enriched metalloprotein standard for use as a calibrant in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is described. Using a model system involving the copper-containing protein rusticyanin (Rc) from the bacterium Acido-thiobacillus ferrooxidans, it was possible to demonstrate the analytical conditions that could be used for the measurement of metalloproteins by on-line IDMS analysis. Rc was chosen because it is a well-characterized protein with an established amino acid sequence and can be produced in suitable quantities using a bacterial recombinant system. Three different forms of the protein were studied by organic and inorganic mass spectrometry: the native form of the protein containing a natural isotopic profile for copper, an isotopically enriched species containing virtually all of its copper as the 65Cu isotope, and the nonmetalated apo form. Incorporation of the copper isotopes into the apo form of the protein was determined using a UV-vis spectrophotometric assay and shown to be complete for each of the copper-containing species. The experimental conditions required to maintain the conformational form of the protein with a nonexchangeable copper center were established using +ve electrospray mass spectrometry. A pH 7.0 buffer was found to afford the most appropriate conditions, and this was then used with HPLC-ICP-MS to verify the stability of the copper center by analysis of mixtures of different isotopic solutions. No exchange of the enriched copper isotope from Rc with an added naturally abundant inorganic copper cation was observed under a neutral pH environment, indicating that species-specific ID-MS analysis of metalloproteins is possible.     

Binding-induced fluorescence turn-on assay using aptamer-functionalized silver nanocluster DNA probes

We present here a binding-induced fluorescence turn-on assay for protein detection. Key features of this assay include affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters (Ag NCs) using guanine-rich DNA sequences. In an example of an assay for human α-thrombin, two aptamers (Apt15 and Apt29) were used and were modified by including additional sequence elements. A 12-nucleotide (nt) sequence was used to link the first aptamer with a nanocluster nucleation sequence at the 5'-end. The second aptamer was linked through a complementary sequence (12-nt) to a G-rich overhang at the 3'-end. Binding of the two aptamer probes to the target protein initiates hybridization between the complementary linker sequences attached to each aptamer and thereby bring the end of the G-rich overhang to close proximity to Ag NCs, resulting in a significant fluorescence enhancement. With this approach, a detection limit of 1 nM and a linear dynamic range of 5 nM-2 μM were achieved for human α-thrombin. This fluorescence assay is performed in a single tube, and it does not require washing or separation steps. The principle of the binding-induced DNA hybridization and fluorescence enhancement of Ag NCs can be extended to other homogeneous assay applications provided that two appropriate probes are available to bind with the same target molecule.