TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

微生物法生产对苯二甲酸进展

对苯二甲酸(terephthalic acid,TPA)是一种重要的有机工业化学品,广泛应用于聚酯、纤维、涂料、粘合剂和薄膜等行业的生产,目前主要采用对二甲苯的化学氧化法进行生产。本文综述了微生物法生产TPA的方法和国内外的生产现状以及研究进展。     

酸性矿山废水污染的水稻田土壤中重金属的微生物学效应

采样调查了广东大宝山地区受酸性采矿废水长期污染的亚热带水稻田的土壤理化性质 ,重金属 Cu、Pb、Zn、Cd的全量及其 DTPA浸提量 ,以及微生物生物量及其呼吸活性等指标。利用主成分和逐步回归分析了影响土壤重金属的有效性及其微生物学效应的因素。结果表明 :土壤高含硫 ,强酸性 ,有机碳、全氮较低 ,4种金属的全量普遍超标。DTPA可提取态金属含量较高 ,不仅与其全量呈显著正相关 ,而且与土壤酸度和粘粒含量正相关 ,和 Mn含量负相关。过量的金属显著降低了土壤微生物生物量 C、N、微生物商、生物量 N/全 N比 ,并抑制了微生物呼吸强度和对有机碳的矿化率 ,导致了土壤 C/N比的升高。同时 ,金属对微生物群落及生理代谢指标 ,如微生物生物量 C/N比和代谢商的影响不显著。 DTPA可提取态金属 ,特别是 DTPA- Cu是导致微生物生物量和活性指标变化的主要因素。以有机碳 (或全氮 )为基数的复合微生物指标降低了土壤性质差异造成的干扰 ,较单一指标更能准确指示微生物对金属胁迫的反应。土壤硫没有对金属有效性和微生物指标产生明显影响 ,但其氧化过程可能引起酸化和金属离子的释放     

草丁膦对转bar基因水稻GS酶活性和光合功能的影响

喷施草丁膦后,对草丁膦无抗性水稻Cypress(未转bar基因)叶片的谷氨酰胺合成酶(GS)活性先受到抑制,随后叶片内NH 4积累上升,叶绿素含量、PSⅡ原初光化学效率(Fv/Fm)、光能转化效率(ΦPSⅡ)和叶片叶绿素荧光的光化学猝灭系数(qp)下降,光合速率显著降低;最后引起植株死亡.另一方面,Cypress PB-6(转bar基因抗草丁膦水稻)的GS酶活性在喷施草丁膦后虽然先被抑制,但随后能恢复至正常水平,接着NH 4积累下降,草丁膦对叶绿素含量、荧光参数Fv/Fm、ΦPSⅡ、qp的影响被解除,光合速率恢复到正常水平,整个植株生长正常.     

转Bt基因抗虫棉叶面可培养微生物多样性的变化

在大田栽培条件下,以转Bt基因抗虫棉GK-12和常规棉泗棉3号为材料,在棉花的苗期、现蕾期、花铃期和吐絮期分别测定棉花叶面细菌、真菌和放线菌的数量变化,并在花铃期和吐絮期对叶面细菌生理群的数量和多样性进行分析.结果表明：棉花叶面可培养微生物数量与其生长发育呈正相关,叶面可培养微生物的数量一般由苗期开始增多,到花铃期达最高峰,吐絮期明显减少;花铃期转Bt基因抗虫棉叶面细菌生理群Simpson指数、Shannon-Wiener指数和均匀度比常规棉升高,吐絮期比常规棉下降.     

Genetic differentiation between populations of swimming crab <Emphasis Type="Italic">Portunus trituberculatus</Emphasis> along the coastal waters of the East China Sea

Portunus trituberculatus is a commercially important species widely spread in the East China Sea. Intraspecific variation of the mitochondrial DNA cytochrome oxidase subunit I (mtDNA COI) gene was investigated in 213 individuals from six localities (Changjiang Estuary, Shengsi Islands, Zhoushan Islands, Dongtou Islands, Dinghai Bay, and Quanzhou Bay) ranging from north (31°21′N) to south (24°55′N) coastal waters of the East China Sea. Overall, a total of 27 mtDNA haplotypes and 21 variable sites were detected in the 787 bp segment of COI gene. Analysis of mtDNA COI sequence data revealed that crabs from the six localities were characterized by moderately high haplotypic diversity (h = 0.787 ± 0.026), while sequence divergence values between haplotypes were relatively low (π = 0.00241 ± 0.00098). Each population was characterized by a single most frequent haplotype, shared among all six localities, and a small number of rare ones, typically present in only one or two individuals and representative of a specific population. However, neither the neighbor-joining tree nor the minimum spanning network (MSN) based on the haplotype data exhibited geographical patterns of the six populations. Mismatch distribution analysis of P. trituberculatus individuals sampled from the six localities suggested that sudden population expansion might have occurred in CJ and SS population that might be consistent with over-exploitation of the swimming crab. Analysis of molecular variance (AMOVA) and F _ST statistics showed that significant genetic differentiation existed among the SS, ZS, DT, DH, and QZ populations, suggesting that gene flow might be reduced, even between the geographically close sites, despite the high potential of dispersal. The possible causes of the observed genetic heterogeneity among the P. trituberculatus populations and the potential applications of the mtDNA COI marker in the artificial breeding and fisheries management are discussed. Handling editor: C. Sturmbauer     

半干旱区采煤沉陷对地表植被组成及多样性的影响

对神府-东胜矿区6个矿3种不同地貌下2个不同沉陷年份及对照区地表植被进行了调查,计算并分析了各样地植物种的重要值、物种多样性指数和均匀度等.结果表明:(1)神府-东胜矿区植物种类总体上来看较为稀少,生态较为脆弱;在调查区内共出现植物30科 82属 114种.(2)6个矿中以上湾、大柳塔和活鸡兔3矿区植物种类较丰富,补连塔和乌兰木伦矿区物种相对单一.(3)经沉陷干扰后,植物群落主要组成成分没有发生明显变化,群落的建群种没有改变;除生态十分脆弱的乌兰木伦矿区外,其余矿区地表沉陷后物种多样性与对照相比没有显著性差异;(4)沉陷后群落组成成分较沉陷前增多,且沉陷1年区比沉陷当年区物种数多,它们之间具有明显的相关性.     

Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh 2 gene, one of the two tdh genes ( tdh 1 and tdh 2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh 1– and tdh 2– lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh 2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh 2 and tdh 1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh 2 promoter; they differ at position −34 but have the same base at position −24 as the tdh 2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.