猪伪狂犬病病毒JSSQ2013株的分离鉴定及重要功能基因序列分析

为了解江苏省猪伪狂犬病病毒(Pseudorabies virus,PRV)野毒株的特点,本研究从2013年采自江苏省宿迁市的疑似PRV感染病料中分离纯化了一株PRV病毒,对其进行了PCR和间接免疫荧光法(IFA)鉴定,并进一步在Vero细胞上测定该分离株的病毒滴度TCID50和一步生长曲线,扩增其gB、gC、gD和gE基因进行序列比对及分子遗传进化分析,并将该分离株分别接种新西兰白兔和15日龄仔猪研究其致病性。结果显示,该病毒为一株PRV,命名为PRV JSSQ2013株,纯化后的病毒滴度为10^7.8 TCID50/ml;生长曲线测定显示在感染20h后病毒滴度即达到最高,为10^8.6 TCID50/ml。与我国近几年分离的PRV变异株序列相比,PRV JSSQ2013株的gB、gC、gD和gE基因核苷酸序列同源性分别为99.5~99.6%、99.5~99.6%、99.5~99.6%和98.7~99.7%,氨基酸序列同源性分别为98.9~99.0%、99.5~99.7%、99.0~99.2%和98.1~99.3%,均高于其与经典毒株(Ea、Fa和SC株)和欧美毒株(Becker、Kaplan、Bartha、Kolchis和NIA3)的同源性;基于gB、gC、gD和gE基因的遗传进化树分析均显示PRV JSSQ2013株与国内近几年分离的PRV变异株属同一分支。该病毒接种新西兰白兔后均出现典型的PR症状,如厌食、兴奋、啃咬或用爪挠接种部位等典型症状,且在48h内全部死亡;接种仔猪后第1天开始出现典型的PR症状,第5天全部死亡。以上结果证实,从江苏省宿迁市采集的疑似PRV感染病料中分离到一株强毒力的PRV变异株。本研究为了解江苏PRV分子流行特征、丰富我国PRV分子流行病学资料及新型疫苗的研制奠定了基础。     

亚洲棉bZIP蛋白家族的鉴定及GaFDs基因的组织表达分析

碱性亮氨酸拉链(basic leucine zipper, bZIP)是真核生物中数量最多并且最具多样性的转录因子之一,参与植物生长发育及响应生物和非生物胁迫。本研究利用亚洲棉(Gossypium arboreum)全基因组数据库,通过生物信息学分析分布在13条染色体上的159个bZIPs家族基因的全序列。系统进化、基因结构和保守基序分析表明这些基因分成13个亚家族。其中,A亚家族有3个GaFD基因GaFD1、GaFD2和GaFD3,通过实时荧光定量PCR分析3个GaFDs基因在不同组织中的表达,结果表明GaFD1和GaFD2在SAM中的表达量最高,GaFD3在茎中表达量最高。研究表明棉花基因组中具有数量众多的bZIP家族成员,不同基因结构及FD基因不同的表达特征表明bZIP基因在棉花生长发育中可能具有不同的功能,这些结果为进一步解析棉花bZIP家族基因的功能和作用机理积累了有价值的资料。     

海南省象耳豆根结线虫的鉴定研究

象耳豆根结线虫寄主范围广、致病力强以及能在携带Mi抗性基因的作物上寄生繁殖,是热区最具危害性的病原根结线虫种类之一。应用比较形态学,结合mt DNA序列分析、IGS序列分析和进化发育树等分子生物学方法对海南省18个市县的根结线虫进行鉴定,确定象耳豆根结线虫在蔬菜、番石榴、哈密瓜和枣树等作物上广泛发生为害,其中枣树为首次发现的象耳豆根结线虫新寄主。研究结果表明,象耳豆根结线虫已成为海南省蔬菜作物上重要的病原根结线虫种,并有进一步扩散加重为害的趋势。     

Diet-related composition of the gut microbiota of Lumbricus rubellus as revealed by a molecular fingerprinting technique and cloning

Interactions between earthworms and microorganisms are essential for the functioning of soil ecosystems as they affect organic matter degradation and nutrient cycling. This is also true for the alpine region, where socio-economic changes lead to the increasing abandonment of pastures, which in turn, causes a considerable shift in the diet of saprotrophic invertebrates and thus impacts food web and decomposition processes. To enhance our understanding of how this diet shift influences earthworms and associated microorganisms, we studied the gut content and cast microbiota of Lumbricus rubellus (Lumbricidae, Oligochaeta), a key macrodecomposer on alpine pastureland in the Central Alps. A feeding experiment with L. rubellus and three different food sources that represent the vegetation shift from an alpine pasture to an abandoned site was set up. Earthworms were collected in the field, transferred to a climate chamber and fed with cow manure, dwarf shrub or grass litter for six weeks. PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) analysis of the DNA extracted from the substrates, the earthworms' gut contents and casts revealed that the gut and cast microbiota was strongly influenced by the food source ingested. Cloning of bacterial 16S rRNA gene fragments demonstrated that the intestinal content was dominated by Proteobacteria, especially from the Gamma-subclass, followed by members of the phyla Bacteroidetes, Actinobacteria and Firmicutes. In contrast, Actinobacteria were detected abundantly in all samples types when a cultivation approach was used. In conclusion, the gut microbiota of L. rubellus was shown to be substantially affected by the food source ingested, suggesting that this essential macrodecomposer is exposed to the diet shift resulting from a land-use change in the alpine area.     

A diazotrophic, indole-3-acetic acid-producing endophyte from wild cottonwood

An endophytic bacterium, wild poplar strain B (WPB), isolated from stems of wild cottonwood (Populus trichocarpa) was identified to Burkholderia vietnamiensis by analyzing the recA and rDNA genes. Phylogenetic analysis of the nifHDK cluster indicates that the WPB isolate shares high sequence similarity with known B. vietnamiensis strains. The nitrogenase activity of WPB was determined by a ¹⁵N₂ incorporation assay and an acetylene reduction assay. WPB was also monitored for production of indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. In addition, its plant growth promotion capacity was assessed by inoculating the WPB strain onto Kentucky bluegrass in nitrogen-free medium. Compared to uninoculated control plants, the plants inoculated with WPB gained more dry weight (42%, p = 0.01) and more nitrogen content (37%, p = 0.04) in 50 days.     

High HEV presence in four different wild boar populations in East and West Germany

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969 nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.     

黄河中游粗山羊草三种y-型高分子量谷蛋白亚基的鉴定、克隆及系统进化分析

粗山羊草(Aegilops tauschii, 2n = 2x = 14, DD)是六倍体普通小麦的祖先之一,其高分子量谷蛋白亚基(HMW-GS)变异类型丰富,是小麦品质改良的重要基因资源。利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析了黄河中游地区161份粗山羊草的HMW-GS,发现3种编码序列未知的y-型亚基,即Dy10.5^t、Dy10.4^t和Dy10.5^*t亚基。通过AS-PCR扩增、克隆、测序和氨基酸序列推导,发现3种未知序列均具有典型HMW-GS的序列结构特征且较为相似,仅Dy10.4^t与Dy10.5^t亚基存在一个氨基酸重复单元的缺失,Dy10.5^t与Dy10.5^*t亚基在信号肽部位有一个氨基酸的替换(L-F)。通过对这3种HMW-GS与32个已知氨基酸序列的HMW-GS多序列比对和系统进化关系分析,证实Dy10.5^t、Dy10.4^t和Dy10.5^*t 3个亚基是D基因组编码的高分子量谷蛋白y-型亚基家族的新成员。     

利用ITS核酸序列分析鉴定海洋青霉菌

[目的]利用ITS核酸序列对6株根据形态学特征初步鉴定为青霉菌的海洋真菌进行鉴定。[方法]采用分子生物学技术,对6株样品的ITS序列进行克隆,并利用ClustalX1．83软件对结果进行系统发育分析。[结果]通过PCR克隆获得了6株样品的ITS序列,将结果与GenBank中已登陆的其他序列进行系统发育分析,确定了6株菌株均属于青霉菌属。[结论]该6株菌株均属于青霉菌属。     

新疆小家鼠控制区序列分析

[目的]研究新疆地区小家鼠控制区的序列多样性。[方法]对14只来自新疆于田、和丰和乌鲁木齐的小家鼠个体进行基因组DNA提取,控制区PCR扩增和序列测定,并分析其遗传多样性,构建系统进化树。[结果]14只小家鼠基因组大小均在20000bp以上;小家鼠控制区序列中A、T、G、C的平均含量分别为：T：29．0％;C：25．2％;A：33．9％;G：11．9％,个体间碱基组成几乎无差别;14只小家鼠个体与M.m.musculus的亲缘关系较近,在系统进化树中共享1个分支,且该分支中2225、2228和2226具有相同的支长,2242和2243具有相同的支长,2249、2247和2251具有相同的支长。[结论]供试14只新疆小家鼠个体属于M.m.musculus亚种。     

缠绕植物手性的系统发育分析(英文)

利用matK、rbcL与trnL基因,分析了中国薯蓣属植物缠绕手性的系统发生关系;利用matK基因,从大分类单元角度进一步分析了缠绕植物的系统特征。结果显示左、右旋缠绕植物物种存在显著的系统发育特征:matK数据中的右旋薯蓣聚成独立的进化枝与rbcL基因的分析共同支持右旋物种的单系性;rbcL和trnL的数据均较好地显示了右旋薯蓣的单系特征[除多毛叶薯蓣(D.decipiens)和甘薯(D.esculenta)外];在大分类单元上,缠绕植物手性性状则呈现出明显的多系特征。结果表明在中国区薯蓣属的分类中,手性特征是较可靠的性状特征,《中国植物志》薯蓣属章节直接将右旋物种归入周生翅组(sect.Enantiophyllum)较合理。但在世界范围上看,在小分类单元的薯蓣属内和薯蓣科及其他大分类单元内,手性特征都具有单独多次起源的性质,因此在执行分类时应当慎用这个性状。对于手性特征的起源问题,最新的观点倾向于否定南北半球物理差异相关的假说,但其结论的正确性有待商榷。结合地质事件研究薯蓣属等具有两种手性方向的小分类单元,以及构建突变体的方法,有助于最终解决植物手性的起源问题。