Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

荧光实时定量PCR技术及其在水产养殖研究中的应用

荧光实时定量PCR技术是近年来快速发展起来的一门新技术,它是核酸探针技术、荧光共振能量传递技术(Fluorescence Resonance Energy Transfer,FRET)和PCR技术的有机结合。与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高、能较准确定量等特点。本文综述了荧光实时定量PCR技术的基本原理及几种广泛应用的荧光化学方法,并概述了荧光实时定量PCR技术在水产养殖研究领域的应用现状,并对荧光实时定量PCR技术的应用前景进行了展望。     

吐鲁番黑羊MSTN/Smad信号通路基因表达的发育性变化

为探讨MSTN/Smad信号通路基因对吐鲁番黑羊肌肉生长发育的影响,采用实时定量PCR法,分别对1～6月龄吐鲁番黑羊的腿肌和尾脂MSTN/Smad信号通路基因进行检测.结果表明：MSTN/Smad信号通路基因在腿肌和尾脂组织中均有表达,MSTN/Smad信号通路基因在吐鲁番黑羊不同生长阶段的腿肌和尾脂中的表达没有出现随月龄的增加而一直增加或下降的趋势.     

甘蔗SPS基因家族成员表达与糖分积累关系解析

以甘蔗高糖品种福农95-1702和低糖品种ROC-5为材料,分析SPS I、Ⅱ、Ⅲ、Ⅳ、Ⅴ在高、低糖这两个品种的幼叶、第三功能叶和不同蔗茎的表达,测定甘蔗糖分积累早期、中期和后期的糖分含量。结果显示,在功能叶和茎第二节中,三个阶段SPS家族成员在高糖品种的含量整体趋势高于低糖品种,结合蔗糖积累特点分析表明其可能在蔗糖合成中扮演着重要的角色。与正三叶相反,在幼叶中,三个阶段SPS家族成员在高糖品种的表达量整体趋势低于低糖品种,表明其可能与嫩叶的生长等其他生理性状相关,在成熟茎中,随着蔗糖积累时间的增加,高糖品种中的优势表达基因减少,而低糖品种中的优势表达基因增加,这可能与低糖品种中、后期大量积累蔗糖相关。     

马银花愈伤组织RoWUS基因及其启动子的克隆与表达分析

WUSCHEL(WUS)基因是植物干细胞的标志基因。采用同源克隆法结合RACE技术从马银花愈伤组织中克隆得到Ro WUS c DNA全长序列,采用染色体步移法克隆得到该基因的启动子序列,登录号分别为KF365488、KF861578。马银花Ro WUS c DNA全长1 123 bp,编码302个氨基酸;DNA序列2 001 bp,包含2个内含子和3个外显子;启动子序列3 122 bp。生物信息学分析表明:Ro WUS亚细胞定位于细胞核,是不稳定的亲水蛋白,无信号肽,具有跨膜结构,包含homeodomain功能位点。系统进化树分析结果表明:Ro WUS单独形成一个分支,与葡萄、大豆和苜蓿的亲缘关系最近。对Ro WUS的启动子进行分析表明,该启动子除了含有丰富的TATA-box和CAAT-box等基本元件以外,还含有多个光响应元件、逆境胁迫响应元件、激素应答元件和其他功能元件。q PCR结果表明:Ro WUS基因在马银花愈伤组织不同生长时期中呈先上升后下降的趋势,在第5个继代周期时表达量最高。外源赤霉素和脱落酸浓度为15 mg/L时表达量最高,说明Ro WUS基因在该浓度时对赤霉素和脱落酸的响应最强。     

花生中UDP-glucosyltransferase基因的克隆及在非生物胁迫下的表达研究

通过RACE技术从花生叶片cDNA中克隆到了UDP-glucosyltransferase基因,命名为AhUGT83A1-like （Genbank注册号为KF411463）。该基因全长为1 530bp,ORF为1 380bp,编码460个氨基酸。序列比对与进化分析结果表明,该序列有保守的UDPGT结构域,并且与其它植物的UGT蛋白同源性较高。荧光定量PCR结果表明,AhUGT83A1-like基因在高盐和低温处理的花生根和叶片中均上调表达;在干旱处理时,根中表达也受到显著上调,但叶片中表达量则有明显下降。以上结果表明AhUGT83A1-like可能参与了花生对干旱、低温和高盐的抗性调控。ABA处理结果表明,AhUGT83A1-like在花生根中对ABA响应明显,但在叶片中对ABA没有明显响应,表明该基因在花生根中对非生物胁迫的调控可能是以依赖ABA的方式发挥作用的。?     

基于SMP的总线式软运动控制引擎设计

提出了基于SMP实时架构的EtherMAC实时以太网主站的设计方法,开发了一种运行于内核空间的运动控制引擎,并给出了一种通过在共享内存建立内存堆栈方式的跨平台编程接口设计方法。通过设计总线抽象层将总线控制资源抽象,降低了运动控制引擎与总线之间耦合性,增强了总线系统或从站设备的可替换性。原型机进行功能验证表明,该引擎实现了较好的易用性和可移植性,并间接增强了EtherMAC的实时性能。     

Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout, Oncorhynchus mykiss (Walbaum)

Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD.