暴发性流行病病原对中国对虾亲虾人工感染及对子代影响的PCR检测

１９９７年４月在山东海阳市近海捕获１０尾中国对虾,采用患暴发性流行病的虾池中的病虾为毒种,进行人工感染试验采用两次了聚合酶链反应（ＰＣＲ）检测方法,对感染亲虾的胃、腮、卵巢,以及卵和各期幼体进行跟踪检测,对经人工感染的１０尾亲虾组织的ＰＣＲ检测结果表明,６尾虾的胃样呈阳性,其中４尾为第１次检测出阳性;１尾虾的产呈阳性;２尾虾的卵巢样呈阳性,且其所产的孵子也呈阳性;每尾亲虾产卵所孵化出来的各期纪体,     

Detection of prawn white spot baculovirus by polymerase chain reaction

INTRODUCTIONAprawnbaculovirushasbeenresponsibleformostoftheseriousshrimpdiseaseinChinasince1992.Studiesonthepathology,Pathogenesisandmorphologyofthevirusshowedthatitwasanon-occlUSiontheybaculoviruswhichcouldinfectPenaeusjaponicus,P.nzonham,P.chinests...     

甘糖酯对大鼠尿激酶型纤溶酶原激活物(uPA)mRNA水平的影响

从分子水平上探讨海洋新药甘糖酯的抗栓作用机理。实验以大鼠为材料,采用定量ＲＴＰＣＲ方法定量检测口服甘糖酯后大鼠尿激酶型纤溶酶原激活物（ｕＰＡ）基因ｍ ＲＮＡ水平变化。研究表明大鼠口服甘糖酯后ｕＰＡ基因ｍ ＲＮＡ 水平高于对照组,差异显著。结论认为甘糖酯可提高体内ｕＰＡ基因的ｍ ＲＮＡ 水平,从而提高ｕＰＡ蛋白的活性,激活机体的纤溶系统,达到抗栓作用。     

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.     

近海水域病原菌多样性研究进展

水产动物病原菌广泛分布于近海,不但影响海水养殖业的发展、海产品的质量安全,甚至还威胁到人类公众健康,因此,开展近海水域病原菌多样性的研究,对于海水养殖业的可持续发展及人类健康都具有重要意义。文章综述了近海水域水产动物病原菌多样性研究主要方法的原理、特点以及局限性,介绍了16S rRNA序列分析、分子杂交、指纹图谱等分子生物学技术,阐述了实时荧光定量PCR、竞争定量PCR等定量研究技术的应用,展望了病原菌多样性研究的发展趋势是原位、快速、高通量、灵敏、多技术联合和准确定量。     

Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences

Vibrios are widespread in the marine environment and a few pathogenic species are known to be commonly associated with outbreaks of diarrheal diseases in humans due to the consumption of raw or improperly cooked seafood. However, there are also many Vibrio species which are potentially pathogenic to vertebrate and invertebrate aquatic animals, and of which little is known. In an attempt to develop rapid PCR detection methods for these latter class of vibrios, we have examined the 16S-23S intergenic spacers (IGSs) of 10 lesser-known Vibrio species and successfully developed species-specific primers for eight of them--Vibrio costicola, V. diazotrophicus, V. fluvialis, V. nigripulchritudo, V. proteolyticus, V. salmonicida, V. splendidus and V. tubiashii. The IGS amplicons were amplified using primers complementary to conserved regions of the 16S and 23S rRNA genes, and cloned into plasmid vectors and sequenced. Analysis of the IGS sequences showed that 37 ribosomal RNA (rrn) operons representing seven different IGS types have been cloned from the 10 vibrios. The three IGS types--IGS(0), IGS(IA) and IGS(Glu)--were the most prevalent forms detected. Multiple alignment of representative sequences of these three IGS types from different Vibrio species revealed several domains of high sequence variability, which were used to design species-specific primers for PCR. The specificity of the primers were evaluated using total DNA prepared from different Vibrio species and bacterial genera. The results showed that the PCR method can be used to reliably detect eight of the 10 Vibrio species in marine waters in this study.     

Regulation of Tilapia metallothionein gene expression by heavy metal ions

Tilapia is a common fish species inhabiting inland waters and estuarine regions in Hong Kong and Southeast Asia, and useful for bio-monitoring of metal pollution. Metallothionein (MT) gene expression in fish tissues has been useful to sub-lethal risk assessment as biomarker of exposure to metal ions in fishes inhabiting metal contaminated area. To investigate metal inductions of Tilapia MT gene expression in vivo, Tilapias were injected with different concentrations of heavy metals and tissues were then removed for quantitative PCR assay using mimic PCR methods. All of the metal ions tested (Cu²⁺, Cd²⁺, Hg²⁺, Ni²⁺, Pb²⁺ and Zn²⁺) were able to induce hepatic MT mRNA levels. Renal MT mRNA levels of Cd²⁺ and Zn²⁺ treated fish was not induced with significant fold induction, however MT mRNA levels in gills were sensitive to the administrations of these metal ions. These data indicated that Tilapia MT mRNA levels in gills and liver are sensitive biomarker of exposure to various metal ions.     

银鲳(Pampus argenteus)胰岛素样生长因子-I基因的克隆和表达分析

胰岛素样生长因子-I(Insulin-like growth factor-I,IGF-I)是影响脊椎动物生长、发育及代谢的重要调控因子。本研究采用RT-PCR和RACE技术,克隆了银鲳(Pampus argenteus)肝脏IGF-IcDNA序列,应用半定量RT-PCR、Real-time q PCR和原位杂交的方法检测了IGF-I的组织表达特性、在肝脏中的生长表达特性和IGF-I基因在肝脏中的定位。序列分析表明,IGF-I cDNA序列全长836bp,其5′非编码区128bp、3′非编码区92bp,开放阅读框(open reading frame,ORF)605bp,由此推导IGF-I前体蛋白由201个氨基酸组成;前体肽由信号肽、成熟肽、E肽三部分组成,其中信号肽59个氨基酸,成熟肽68个氨基酸,E肽74个氨基酸;成熟肽由B、C、A、D四个区域组成,E肽分析表明,银鲳IGF-I属Ea-4型。同源性比较结果表明,银鲳与同目鲈形目鱼类的IGF-I编码序列同源性较高,为83.52%—91.40%;与哺乳类、鸟类和爬行类的同源性较低。半定量RT-PCR和Real-time q PCR组织特异性表达结果显示,IGF-I m RNA在肝脏组织中的表达量最高,显著高于其它组织,肾脏、心脏、肌肉、脑、鳃、小肠、卵巢次之,嗅球、脾脏和胃中表达较低;半定量RT-PCR和Real-time q PCR不同生长阶段表达结果显示,IGF-I m RNA在30—50g肝脏组织中表达量最高(P0.05);IGF-I m RNA在肝脏中的原位杂交定位结果显示,在肝脏细胞中均有表达,阳性信号主要位于细胞质中,靠近细胞边缘处信号较强。