副鸡禽杆菌aroA基因克隆及序列分析

旨在建立一种方便、高效的同源保守基因克隆方法,并对副鸡禽杆菌aroA基因进行克隆和结构分析。本试验以副鸡禽杆菌国际标准株145(C-3)基因组DNA为模板,用CODEHOP软件设计针对aroA基因的兼并引物,并运用改进的染色体步移方法扩增aroA基因序列;对该核酸序列及其编码蛋白进行结构分析并与该菌其他血清型及相关细菌进行序列比对分析。结果显示,获得了完整的aroA基因,全长1 293 bp。该基因编码由430个氨基酸组成的多肽,具有2个功能位点和5个抗原表位位点。不同血清型间氨基酸序列同源性为88.1%-100%,与其他相关细菌核酸同源性为75%以上。首次将兼并PCR和改进的染色体步移技术结合起来对副鸡禽杆菌aroA基因全长进行扩增研究,得到了预期的结果。     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

高效柴油降解菌Acinetobacter sp.W3分离鉴定及降解酶基因扩增分析

从柴油污染的海水样品中分离高效柴油降解细菌,分析菌株对柴油的降解能力及降解酶基因,为海洋柴油污染的生物修复奠定基础。选取浙江定海港柴油污染的海水样品,进行降解菌的富集培养;采用常规方法分离筛选高效柴油降解菌。利用革兰氏染色、形态学观察、生理生化鉴定及16S rDNA分析等方法对降解菌株进行种属鉴定。采用紫外吸收法测定菌株对柴油的降解率。采用PCR方法、核酸序列测定和比对,对其降解酶基因进行扩增分析。筛选出一株高效降解菌,形态学观察及生理生化鉴定初步确定为不动杆菌。16S rDNA序列分析及比对结果表明,其16S rDNA序列与威尼斯不动杆菌(Acinetobacter venetianus)属的序列同源性达到99.7%,命名为不动杆菌W3(Acinetobactersp.W3),该菌对柴油的7 d降解率达到84.7%。PCR方法从Acinetobactersp.W3菌株中的基因组DNA和质粒DNA上扩增到了大小为540 bp的烷烃羟化酶基因alkB和864 bp的CYP153A部分DNA片段,分别与Acinetobacter venetianus1-D-2的alkB和Acinetobactersp.OC4、Acinetobactersp.EB104的CYP153具有99%和98%的同源性。从定海港口柴油污染海水分离得到一株高效柴油降解菌Acinetobactersp.W3,该菌属于不动杆菌属,含有烷烃降解酶基因,能高效降解柴油污染物,有望应用于海水柴油污染的生物修复。     

新疆分离的0507JS60鉴定为辽宁病毒

0507JS60是从中国新疆喀什地区采集的库蚊标本分离的病毒株,可以在C6/36细胞上稳定传代.电镜观察显示完整病毒颗粒呈球形,直径55nm(n=10),无包膜,衣壳表面壳粒结构非常清晰.基因组核酸电泳显示基因组包括12条双链RNA(Double stranded RNA,dsRNA)片段.病毒第12基因片段序列测定结果显示该片段全长760bp(GenBank ID:FJ157354),具有长度为525bp的单一开放读码框(Single open reading frame,ORF),编码长度为174个氨基酸的蛋白,分子量约18.9kD.病毒第12基因片段序列比对显示与辽宁病毒(Liaoning virus,LNV)核酸序列同源性大于89%,氨基酸序列同源性大于91%.病毒第12基因片段系统进化分析显示该病毒与LNV病毒位于同一进化分枝内,与LNV-NE9712病毒株进化关系更接近.0507JS60经系统鉴定为LNV,这是首次在东北以外的地区分离到该病毒.     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9 Kb的基因片段.核酸序列分析结果表明,该片段全长1 984 bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9Kb的基因片段.核酸序列分析结果表明,该片段全长1984bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

一株Ⅱ型甲烷氧化菌中甲烷单加氧酶基因和16S rDNA的分析

[目的]利用分子生物学方法对-株甲烷氧化菌Methylosinus trichosporium IMV 3011中的16S rDNA和溶解性甲烷单加氧酶基因序列进行分析并探索其进化分类地位.[方法]利用基因数据库已有的基因序列信息,设计PCR扩增引物和基因测序引物,对溶解性甲烷单加氧酶基因和16s rDNA进行扩增和测序,并进行溶解性甲烷单加氧酶的6个基因和氨基酸序列与同类菌株的相应序列进行联配分析.[结果]获得了全长为5319 bp甲烷单加氧酶基因序列和长度为1290 bp的16S rDNA序列.该菌株与Methylosinus trichosporium OB3b中相对应基因的同一性是99.0%～82.7%,MMOX氨基酸序列的同一性为99.4%～81.8%,相似性为99.8%～89.2%.基于以上分析表明MMOX组分有很高的序列保守性,特别是在活性中心区域.[结论]菌株IMV3011属于甲基弯菌属,最近似的菌株是Methylosinus trichosporium OB3b.     

两株真养产碱杆菌部分基因的克隆与序列分析

采用随机引物PCR技术从新建细胞培养室空气中获得两段长度414 bp及450 bp的片段.通过克隆测序及序列分析,结果表明,所测序列与真养产碱杆菌主要参考菌株的同源性分别高迭79%-83%及79%-84%,由其推导的氨基酸序列与真养产碱杆菌主要参考菌株的同源性高达86.4%-89.1%,由两分离菌株所获得基因片段推导的氨基酸序列之间的同源性高达98.1%,从而确定所分离两菌株为真养产碱杆菌不同亚型的菌株.     

中国农大小型猪胰淀素基因部分未知序列扩增与分析

目的扩增中国农大小型猪IAPP基因序列,分析其序列结构特点。方法提取中国农大小型猪血液基因组DNA,设计3对特异性引物进行PCR扩增,产物鉴定、测序和同源性比对分析。结果成功扩增出中国农大小型猪IAPP基因的1028bp的序列。结论经同源性比对分析显示,中国农大小型猪与人的IAPP基因的同源性为77%。     

松材线虫伴生菌中产纤维素酶细菌的筛选、鉴定及其相关基因的克隆

[目的]从松材线虫伴生菌中筛选出高效降解纤维素的细菌菌株,初步鉴定后,对其相应的纤维素酶基因尝试克隆.[方法]首先从河南南阳松材线虫病疫区采集到的木材样本中,分离获得松材线虫.采用刚果红平板初筛法,从松材线虫伴生菌中获得具有分泌较高活性纤维素酶的细菌菌株.基于该菌株的形态学、生理学及16 s rDNA序列特征等对高活性菌株进行分类鉴定.设计兼并引物,从高活性菌株中克隆该菌株的纤维素酶基因,并进行序列分析.[结果]获得7株具有分泌较高活性纤维素酶的细菌菌株,其中编号为C8的菌株酶活最高.经鉴定该菌株归为肠杆菌属,命名为Enterobacter sp.C8.进一步从C8菌株中成功克隆出该菌株的一个全长1104 bp的纤维素酶基因(GenBank JQ845065),在NCBI比对后发现该基因分别与产气肠杆菌( Enterobacter aerogenes) KCTC 2190的纤维素合成酶亚单位BcsC的核苷酸序列有97％的同源性,氨基酸序列有92％的同源性 ;与克雷白氏肺炎菌(Klebsiella pneumoniae)的endo-1,4-D-glucanase基因有82％的同源性,与不可培养的细菌内切纤维素酶基因有82％的同源性.[结论]本文首次从松材线虫伴生菌中筛选到了一株简单的产纤维素酶的细菌菌株并从中克隆出了一个新型纤维素酶基因,为下一步进行新能源的利用奠定了理论基础.     

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.     

Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.     

异色瓢虫过氧化氢酶(Catalase)的基因克隆及序列分析

【目的】克隆异色瓢虫Harmonia axyridis保护酶系中过氧化氢酶(Catalase,CAT)的全长cDNA序列,并分析该基因的基本特性。【方法】采用同源克隆和锚定PCR技术,从异色瓢虫中克隆到HaraxCAT基因的cDNA全序列(GenBank登录号KC991026),并采用生物信息学的相关方法进行了分析。【结果】HaraxCAT的cDNA序列全长1 781 bp,其包含110 bp的3′非编码区域和45 bp的5′非编码区域,可读框长1 626 bp,编码541个氨基酸。预测该基因编码蛋白的分子量为61.55 ku,理论等电点为8.33,包含3个糖基化位点,无信号肽序列和跨膜结构。并且该基因包含了一个长达18个氨基酸的潜在的活性位点序列FDRERIPERVVHAKGAGA和血红素配体信号序列RIFSYGDTH。同源比对不同昆虫的CAT蛋白序列,发现昆虫CAT非常保守,HaraxCAT与其他昆虫的同源性高达65%及以上,与赤拟谷盗Tribolium castaneum同源性最高,达75.25%;系统发育分析表明其与鞘翅目赤拟谷盗和白星金花龟Protaetia brevitarsis亲缘关系最近。【结论】获得异色瓢虫catalase基因的cDNA全长序列,证实昆虫CAT蛋白非常保守。     

Recombination of Salmonella phase 1 flagellin genes generates new serovars.

To determine the evolutionary mechanisms generating serotypic diversity in Salmonella strains, we sequenced the central, antigen-determining part of the phase 1 flagellin gene (fliC) in strains of several serovars for which estimates of chromosomal genomic relatedness had been obtained by multilocus enzyme electrophoresis. The nucleotide sequence of this region was identical in several chromosomally divergent strains of Salmonella heidelberg (phase 1 antigen r) but differed by 19% from the corresponding and similarly invariant sequence in strains of the closely related serovar Salmonella typhimurium (phase 1 antigen i). Mutational drift of the sequence present in the common ancestor is unlikely to have generated the difference between the phase 1 flagellins of these two serovars, which we attribute instead to a recombination event. This interpretation is supported by evidence that Salmonella strains of very diverse chromosomal backgrounds but similar phase 1 antigens may have closely similar nucleotide sequences for this highly polymorphic region. We suggest that lateral transfer and recombination of phase 1 flagellin genes is a major evolutionary mechanism generating new Salmonella serovars.     

Characterization of antimicrobial resistance in Salmonella enterica food and animal isolates from Colombia: identification of a qnrB19-mediated quinolone resistance marker in two novel serovars

Ninety-three Salmonella isolates recovered from commercial foods and exotic animals in Colombia were studied. The serotypes, resistance profiles and where applicable the quinolone resistance genes were determined. Salmonella Anatum (n=14), Uganda (19), Braenderup (10) and Newport (10) were the most prevalent serovars, and resistance to tetracycline (18.3%), ampicillin (17.2%) and nalidixic acid (14%) was most common. Nalidixic acid-resistant isolates displayed minimum inhibitory concentrations ranging from 32 to 1024 μg mL(-1) . A Thr57→Ser substitution in ParC was the most frequent (12 of the 13 isolates). Six isolates possessed an Asp87→Tyr substitution in GyrA. No alterations in GyrA in a further seven nalidixic acid-resistant isolates were observed. Of these, four serovars including two Uganda, one Infantis and a serovar designated 6,7:d:-, all carried qnrB19 genes associated with 2.7 kb plasmids, two of which were completely sequenced. These exhibited 97% (serovar 6,7:d:- isolate) and 100% (serovar Infantis isolate) nucleotide sequence identity with previously identified ColE-like plasmids. This study demonstrates the occurrence of the qnrB19 gene associated with small ColE plasmids hitherto unrecognized in various Salmonella serovars in Colombia. We also report unusual high-level quinolone resistance in the absence of any DNA gyrase mutations in serovars S. Carrau, Muenchen and Uganda.     

茶树肌动蛋白基因（CsActin1）全长cDNA克隆与生物信息学分析

以茶树（Camellia sinensis）萌动芽为材料,根据茶树萌动芽芽抑制消减杂交文库中分离得到的肌动蛋白（actin）基因的5′-片段设计引物,利用3′-RACE技术克隆了其cDNA全长序列,该基因cDNA全长1 470 bp,命名为CsActin1（GenBank登录号HQ235647）。序列分析表明,CsActin1开放阅读框长1 134 bp,编码377个氨基酸,5′非编码区100 bp,3′非编码区236 bp。推测的蛋白质分子量为41.70 kD,等电点约为5.31,具有肌动蛋白家族的特征信号序列（YVGDEAQs.KRG和WIAKaEYDE）和肌动蛋白相关蛋白的特征信号序列（LLTEApLNPkaNR）。CsActin1与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性在80%以上,氨基酸序列相似性在95%以上。与其它植物肌动蛋白的进化树分析结果表明,茶树肌动蛋白与杨树的两个肌动蛋白间的亲缘关系最为密切。并对推导的蛋白结构进行了分析。     

进口水产品中单增李斯特菌的分子流行病学特点

摘要：[目的] 探明进口水产品中单增李斯特菌的污染状况、致病性和分子特征。[方法] 针对2007年7月至2008年11月间从29个国家进口的1275批水产品,进行单增李斯特菌鉴定、谱系与血清型分析、小鼠毒力试验与多位点序列分析。[结果] 检出单增李斯特菌33批次（2.6%）,其中以4b型为主（65.2%）,而1/2a型、1/2b型与1/2c型仅分别占13.0%、17.4%与4.4%。这些分离株对小鼠均具有与强毒参考株相当的毒力。基于actA-hisJ-ribC-sigB的多位点序列分析可将32个菌株分为23个序列型,分辨力达0.97。其中3个序列型包含3个以上分离株,其中序列型9属于流行性克隆I。[结论] 进口水产品中单增李斯特菌污染率与国内水产品相近,但血清型分布以4b型为主,且有流行性克隆I检出,因此要加强对进口水产品中单增李斯特菌的监测。     

Species of Heterobasidion host a diverse pool of partitiviruses with global distribution and interspecies transmission

We investigated the geographic occurrence and genetic diversity of partitiviruses among 247 Heterobasidion specimens representing seven species and originating from Europe, Asia, and North America. Based on sequence analysis, partitiviruses were relatively rare, and occurred only in about 5 % of the Heterobasidion isolates analyzed, constituting a minority (about 28 %) of all virus-infected [double-stranded RNA (dsRNA)-positive] isolates. Altogether ten virus strains were characterized in sequence: one complete genome sequence of 3893 bp, six complete RNA-dependent RNA polymerase sequences of 2000-2033 bp, and three partial polymerase sequences. Based on phylogenetic analysis, the virus strains were assigned into three putative partitivirus species: HetRV1 (Heterobasidion RNA virus 1), HetRV4, and HetRV5. Degenerate consensus primers were designed for RT-PCR detection of these virus species. HetRV1 occurred in five different Heterobasidion species, and resembled the previously described Heterobasidion annosum virus (HaV). Highly similar HetRV1 strains with 98 % nucleotide level similarity were found from H. parviporum (member of the H. annosum species complex) and H. australe (member of the H. insulare complex) growing in the same region in Bhutan. This observation suggests recent virus transmission between these taxonomically distant Heterobasidion species in nature. It was also shown that HetRV1 can be transmitted by mycelial contact between the H. annosum and H. insulare complexes. The two other virus species, HetRV4 and HetRV5, were closely related to the Amasya Cherry Disease-associated mycovirus, to Heterobasidion parviporum partitivirus Fr110B, and also to several plant-infecting alphacryptoviruses. These results are in accordance with the view of a close evolutionary relationship between partitiviruses of plants and fungi.     

Two δ-Endotoxin Genes, cry9Da and a Novel Related Gene, Commonly Occurring in Lepidoptera-Specific Bacillus thuringiensis Japanese Isolates That Produce Spherical Parasporal Inclusions

Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta-endotoxin genes. Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da. The data revealed that the delta-endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta-endotoxin gene. DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region. The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta-endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans. The results showed that Japanese isolates of B. thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta-endotoxin allied to the class Cry9. It also appeared that heterogeneous multiple H serovars are involved in each group.     

Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.