Molecular Typing and Presence of Genetic Markers Among Strains of Banana Finger-Tip Rot Pathogen, Burkholderia cenocepacia, in Taiwan

ABSTRACT Burkholderia cenocepacia (genomovar III of B. cepacia complex), the causal agent of banana finger-tip rot, is a common plant-associated bacterium but also an important opportunistic pathogen of humans. To better understand the nature of B. cenocepacia from banana, the genetic variation among B. cenocepacia isolates from various banana-growing regions in southern Taiwan was examined. Forty-four serial isolates recovered from diseased banana stigmata from three banana-growing regions during the periods ranging from 2002 to 2004 were investigated. All B. cenocepacia isolates picked from quinate-yeast extract tetracycline-polymyxin semiselective medium could cause onion maceration and were polymerase chain reaction (PCR) positive for bcscV, which is a type III secretion gene present in all members of the B. cepacia complex except B. cepacia (formerly genomovar I). Genetic diversity was assessed using recA PCR restriction fragment length polymorphism, recA nucleotide sequence analysis, and pulsed-field gel electrophoresis assays. The assays revealed the genetic variability among the isolates and also allowed us to trace the relationship among isolates. The isolates all were assigned to genomovar III and consisted of two groups, A and B, which corresponded to recA lineage IIIA and IIIB. The group B strains were separated into B1 and B2 subgroups and the B1 strains were further divided into distinct lineages. The B1 strains were the most frequently detected and occurred in all regions tested. There was no significant difference between strains from each subgroup in the virulence on banana fingers of cv. Cavendish. PCR assays were further used to determine whether B. cenocepacia from banana contained the cable pilus subunit gene (cblA), IS1356, and B. cepacia epidemic strain marker (BCESM), which are DNA markers associated with epidemic B. cepacia clinic strains. The results indicated that cblA and IS1356 were absent but the BCESM was found in all isolates. The present study revealed that banana is a natural reservoir of genetically diversified B. cenocepacia strains.     

筇竹与黄皮树人工混交林中筇竹地上部分生物量模型构建

本研究以筇竹与黄皮树人工混交林中筇竹地上部分为研究对象,测定分析了1~4年生分株地上部分各构件生物量及含水率,建立人工筇竹分株地上部分各构件的生物量及总生物量模型,以期为人工筇竹林的经营管理及其碳汇项目的开发提供科学依据。结果表明：随着筇竹分株年龄的增加,各构件含水率和生物量均逐渐减少,筇竹1~4年生分株地上部分平均含水率分别为57.62%、53.40%、50.01%、42.66%,平均生物量分别为133.99、123.31、109.76、85.39 g/m²;各年龄分株地上部分生物量的分配均呈现出秆＞枝＞叶的变化规律。不同年龄分株的胸径与秆、枝、叶生物量及地上部分总生物量均有极显著相关性(P＜0.01)。以胸径为自变量建立的各年龄筇竹分株地上部分总生物量模型的决定系数(R²)均在0.93以上,具有较高的可信度,也有着较强的适用性,可用于类似立地条件下的筇竹分株生物量估测。     

青稞转录因子HvnANT1基因的克隆与表达分析

为探究HvnANT1基因在青稞粒色形成过程中的基因表达模式,以紫粒青稞‘涅如姆扎’和白粒青稞‘昆仑10号’为试材,利用简化基因组GBS(Genotyping-by-Sequencing)对青稞紫粒进行基因定位,克隆到HvnANT1基因,对其进行生物信息学分析。结果表明:1)在7H染色体的84.30—86.00 cM获得1个MYB类转录因子,命名为HvnANT1。2)该基因长1 033 bp,其完整开放阅读框为762 bp,编码253个氨基酸。HvnANT1蛋白分子量为27.14 kU,是亲水性的不稳定碱性蛋白且不存在跨膜结构,无信号肽。该蛋白的二级结构主要是由无规卷曲、α-螺旋、延伸链和β-转角组成,具有2个SANT结构域(分别位于第13—第63个;第66—第114个氨基酸)。3)同源比对与系统进化分析表明:青稞HvnANT1蛋白与大麦、乌拉尔图小麦、高梁、玉米、二型花、小米、水稻、土瓶草、车轴草和杨梅10种植物的ANT1蛋白序列相似性分别为100.00%、88.85%、54.64%、60.95%、60.82%、58.46%、57.61%、37.76%、36.05%和36.33%;这些序列都具有2个高度保守的2个SANT结构域;与大麦的亲缘关系最近,其次是与乌拉尔图小麦,与水稻最远。4)亚细胞定位结果表明,该基因定位在细胞核内。5)实时荧光定量PCR(qRT-PCR)结果显示:与籽粒颜色形成的乳熟早期和乳熟晚期相比,软面团期的HvnANT1基因在‘涅如姆扎’中表达量极显著上调,而在‘昆仑10号’中各时期的表达量较低且差异不显著;且软面团期的‘涅如姆扎’籽粒HvnANT1基因的表达量极显著高于‘昆仑10号’(P<0.01)。花青素合成相关的结构基因HvnCHI、HvnANS和HvnDFR与该基因表达量模式相似。综上,青稞HvnANT1蛋白结构中的SANT结构域在物种进化过程中比较保守;该基因定位在细胞核内,符合转录因子特性;HvnANT1基因表达模式与结构基因HvnCHI、HvnANS和HvnDFR相似,在籽粒颜色形成的软面团期表达量极显著升高。     

A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10³ colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.     

The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.     

水牛RNA聚合酶Ⅲ启动子的克隆与鉴定

[目的]获得有效的水牛RNA聚合酶Ⅲ启动子序列,为开展水牛源细胞的基因特异沉默研究奠定基础.[方法]通过启动子上、下游保守序列对水牛源启动子7SK、U6进行克隆和启动子关键顺式作用元件识别,利用一段针对EGFP的shRNA片段（shEGFP）对水牛7SK、U6启动子进行功能性分析,然后分别在水牛源及鼠源细胞中与pEGFP-N1共转染,转染48h后用荧光显微镜检测EGFP的表达情况,并用流式细胞仪和荧光实时定量PCR检测EGFP沉默表达情况.[结果]克隆获得水牛7SK、U6启动子序列分别为430和357 bp,其OCT-1（或CACCC盒）及TATA盒高度保守.连接shEGFP后与pEGFP-N1共转染细胞,通过荧光显微镜可观察到细胞发生了明显的荧光表达沉默现象;通过流式细胞分析,发现水牛7SK和U6启动子引导的shEGFP在水牛源细胞中沉默效率高达93.82％和87.45％;荧光实时定量PCR检测结果显示,在转染bu7SK-shEGFP和buU6-shEGFP的水牛源BFF细胞中,EGFP表达水平均显著低于其他物种启动子的细胞转染组（P＜0.05）,而在鼠源PT67细胞中,水牛启动子的启动效率与其他物种启动子差异不显著（P＞0.05）.[结论]水牛7SK和U6启动子可高效启动shRNA表达,且具有一定的物种特异性.     

淀山湖鱼类群落结构多样性的年际变化

淀山湖是上海市内陆水域最大的淡水湖泊,主要的淡水渔业水域和水产品来源地,也是重要的水生生物保护基地。为了评估增殖放流和生态环境变化对淀山湖鱼类群落结构变化的影响,本研究以2010-2012年淀山湖的渔业调查资料为基础,对该湖泊的鱼类优势种组成和多样性指数进行年际变化分析,并应用丰度生物量曲线方法对该湖的鱼类群落状况进行分析。结果表明:2010-2012年淀山湖鱼种类数基本稳定,基本鱼种组成变化不显著,优势鱼种组成趋势渐以小型鱼类为主;群落结构多样性指数年间差异不显著(P0.05);ABC曲线显示2010-2012这三年淀山湖群落的数量优势度曲线均高于生物量的优势度曲线,鱼类群落结构仍处于严重干扰状态。因此,建议改善增殖放流鱼种和加强渔业管理,以维持淀山湖鱼类群落的稳定。     

Gene Expression in Black Tiger Prawns Following Intramuscular Injection of β-gal Plasmid

Gene expression in black tiger prawns (Penaeus monodon) was studied following intra-muscular injection of CMVGal plasmid into the second abdominal segment. We used an in situ staining technique to detect -gal expression in one- and three-month-old injected prawns. We found that only one of the three-month-old prawns expressed the marker gene (2 days after injection), and the site of expression was confined to the sixth abdominal segment away from the injection site. We repeated the experiment on a new batch of three-month-old prawns, but using fluorometric determination technique. This time we found that -gal expression was detected (6/42) at the site of injection after 2, 7, and 14 days. In two other test samples, transgene expression was detected in the sixth abdominal segment only, further confirming the possibility of injected DNA dispersal. The results of the study also suggest that direct gene transfer is a feasible technique in black tiger prawns.