环境因子对南极菌Pseudoalteromonas sp.S-15-13多糖合成关键酶UGD基因表达的影响

以南极菌Pseudoalteromonas sp.S-15-13为材料,采用实时定量PCR的方法研究了温度、冻融循环及培养基中NaCl、葡萄糖含量和pH对多糖合成关键酶基因ugd表达水平的影响。结果表明,低温有利于ugd的表达,在2℃和10℃培养温度下,培养24h后ugd的表达量约为20℃时的4倍;冻融循环后,ugd的表达量上调,第2个冻融循环后ugd的表达量较对照提高了6.85倍;NaCl对ugd的影响表现为低促高抑,即NaCl含量为6.0%时ugd的表达量是对照(3.0%)的3.97倍,但含量达9.0%时ugd表达量仅为对照的2/5;葡萄糖能够提高ugd的表达量,当含量为2.0%时ugd表达量为对照的13.68倍;在一定范围内(pH5.0—8.0),pH的改变会促进ugd的表达,当pH为6.0时ugd表达量约为pH7.0时的2.15倍。     

辽宁沿海巨蛎属牡蛎的分布

作者采用多重种特异性PCR(multiplex species-specific PCR)技术,研究了巨蛎属(Crassostrea)牡蛎在辽宁沿海的分布。从辽宁沿海的共11个采样点共采集802个牡蛎样本,通过对COI基因的扩增,随机检测了其中的531个牡蛎样本,结果517个个体为长牡蛎(Crassostrea gigas),14个为近江牡蛎(Crassostrea ariakensis),未发现其他巨蛎属牡蛎。结果表明,辽宁沿海有长牡蛎和近江牡蛎等2种巨蛎属牡蛎分布,其中长牡蛎为优势种,分布于潮间带和潮下带,近江牡蛎为稀有种,分布于潮下带,而且在黄海和渤海海域均有分布。     

转rhG-CSF基因鱼腥藻的重组基因及蛋白检测

提取质粒进行PCR和酶切鉴定人粒细胞集落刺激因子(rhG-CSF)基因成功转入鱼腥藻7120中,并用酶联免疫方法测得转基因鱼腥藻7120-G-CSF中G-CSF蛋白在第21d表达量达到最大。对转基因蓝藻的生长曲线、叶绿素a含量测定结果表明,rhG-CSF基因转化蓝藻后,对蓝藻前中期的生长影响不大,但21d后生长速度下降,同时外源蛋白表达也达到最大值,这有利于转基因藻的大规模培养。     

PCR检测对虾白斑综合征病毒(WSSV)中假阴性的探讨

采用PCR、核酸探针斑点杂交、组织切片等方法研究了PCR检测白斑综合征病毒(WSSV)中的假阴性问题。设计了一对引物用于PCR检测WSSV,PCR扩增的产物长度为235bp,检出极限为0.1pg WSSV DNA。结果表明,PCR检测15例攻毒螯虾鳃样品时出现一例阴性,而经10—106倍稀释后的样品却呈现阳性,推断PCR出现了假阴性。核酸探针斑点杂交及组织切片结果表明注射WSSV的螯虾鳃组织确已被严重感染,进一步证实了PCR假阴性。根据PCR的检出极限及模板稀释梯度,推算出该PCR能成功扩增的引物与模板浓度的比例范围约为2.4×105—2.4×1010。     

口虾蛄高血糖激素基因全长cDNA的克隆及表达分析

本文首次采用RACE技术获得口虾蛄(Oratosquilla oratoria)眼柄部高血糖激素(crustacean hyperglycemic hormone,CHH)基因cDNA全长序列,共2 421 bp,5'UTR长度为180 bp,3'UTR 为1 821 bp,开放阅读框(ORF)长420 bp,编码139个氨基酸。预测蛋白二级结构α-螺旋23.74%,延伸链占23.02%,无规则卷曲占53.24%,预测分子量为15.47 kD,等电点(pI)为7.049。CHH成熟肽包含6个CHH家族中位置保守的Cys残基,C端为GK,推断为ES型CHH基因。聚类分析表明:口虾蛄CHH和十足目CHH聚为同一个分支的两亚群,具有较近的亲缘关系。荧光定量PCR分析结果表明,CHH基因在肠中表达量最高,在眼柄、触角中有较高的表达量,在肌肉中表达量相对较低,在卵巢中表达最低,而在精巢中不表达。     

半滑舌鳎病原鳗利斯顿氏菌双重PCR检测方法的研究

对引起江苏连云港工厂化养殖半滑舌鳎大量死亡的病原鳗利斯顿氏菌,进行了基于溶血素和金属蛋白酶两种基因的双重PCR检测.根据鳗利斯顿氏菌溶血素基因和金属蛋白酶基因序列设计2对特异性引物,在同一PCR反应体系中,鳗利斯顿氏菌可同时扩增出上述2种基因片段,扩增片段大小分别为493 bp和248bp,两对引物对5种其他水产动物病...     

应用新一代测序技术测定大室别藻苔虫线粒体基因组全序列

线粒体基因组的获得传统上通常是采取长PCR结合鸟枪法或步移法测序。近年来新一代测序技术蓬勃发展,以454,Solexa和SOLiD测序平台为代表。应用新一代高通量测序技术(Solexa)并结合巢式PCR扩增,完成了大室别藻苔虫(Membranipora grandicella)的线粒体基因组全序列。大室别藻苔虫线粒体基因组是目前国际上获得的第一条苔藓动物软壁亚目线粒体基因组全序列,基因组全长为15861 bp,比已完成的4种苔藓动物线粒体基因组略大。大室别藻苔虫线粒体基因组包含13个蛋白质编码基因、2个核糖体RNA基因和20个转运RNA基因。通过与已测得的苔藓动物进行比较,发现目前已完成的5个苔藓动物线粒体基因组的基因排列顺序显著不同。苔藓动物线粒体基因组基因排列的比较,今后可能会成为探讨该类群系统演化关系的重要信息来源。     

Monitoring toxic microalgae Ostreopsis (dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR-based assay combined with light microscopy

A molecular PCR-based assay was developed and applied to macrophyte and seawater samples containing mixed microphytobenthic and phytoplanktonic assemblages, respectively, in order to detect toxic Ostreopsis species in Mediterranean Sea. The specificity and sensitivity of the molecular PCR assay were assessed with both plasmidic and genomic DNA of the target genus or species using taxon-specific primers in the presence of background macrophyte DNA. The PCR molecular technique allowed rapid detection of the Ostreopsis cells, even at abundances undetectable within the resolution limit of the microscopy technique. Species-specific identification of Ostreopsis was determined only by PCR-based assay, due to the inherent difficulty of morphological identification in field samples. In the monitoring of the toxic Ostreopsis blooms PCR-based methods proved to be effective tools complementary to microscopy for rapid and specific detection of Ostreopsis and other toxic dinoflagellates in marine coastal environments.     

Darstellung der mikrobiellen Besiedlungsstruktur verschiedener Grundwasserhabitate durch Anwendung molekularbiologischer Methoden

Community Structures of Different Groundwater Habitats Investigated Using Methods of Molecular Biology The degradation of pollutants in groundwater and aquifers depends on microbiological and hydrogeochemical processes. To understand the transport and fate of anthropogenic compounds during bank filtration and artificial recharge of groundwater it is necessary to gain more information about the structure of microbial populations in these systems. The population structure of aerobic, anaerobic groundwater habitats and of water samples during artificial groundwater recharge was examined by 16S rDNA based analysis. Water and sediment samples were collected from a groundwater catchment area with artificial groundwater recharge near the river Ruhr in NW-Germany. 16S rRNA genes of mixed bacterial DNA from different samples were amplified by PCR (polymerase chain reaction) with eubacterial primer sequences. To reveal eubacterial population structure amplified PCR-products were separated by DGGE (denaturing gradient gel electrophoresis) on the basis of melting domain structure and nucleotide composition. DGGE patterns of groundwater enrichment cultures and groundwater samples were compared to demonstrate differences between the use of cultivation dependent and molecularbiological approaches. The DGGE pattern of groundwater is very complex and differs significantly from DGGE patterns of groundwater enrichment cultures characterized by a small number of distinct bands. This shows the small quantity of culturable microorganisms in groundwater eco-systems. Aerobic and anaerobic groundwater and sediment samples differ markedly in their DGGE profiles. Different hydrogeochemical zones of this groundwater catchment area are mirrowed by distinct DGGE patterns indicating changes in microbial community structure.Analysis of bacterial population structure in the course of artificial groundwater recharge shows identical DGGE patterns comparing surface water samples to samples taken be-fore gravel prefiltration and before sand filtration. In contrast the DGGE pattern of artificial recharged groundwater differs markedly, indicating significant changes in microbial population during underground passage.