Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

牛凝乳酶原基因克隆及序列的进化分析

利用RT-PCR克隆获得牛凝乳酶原基因的cDNA序列, 测序后与GenBank中凝乳酶原基因进行序列比对和生物信息学分析。序列比对统计分析显示, 该基因为牛凝乳酶原B基因, 与已知牛和其他哺乳动物的凝乳酶原基因具有很高的同源性,18种哺乳动物凝乳酶原基因密码子一、二、三位点的碱基偏倚度分别为：6.227、1.042和1.456。这表明该基因具有整体保守性和突变位点偏倚性两个特征, 可以作为哺乳动物系统进化的研究对象。采用多种方法构建的该基因系统进化树一致表明, 偶蹄动物与灵长动物的亲缘关系比偶蹄动物与啮齿动物的亲缘关系更近, 比偶蹄动物与食肉动物的亲缘关系更远, 并且18种哺乳动物的亲缘关系与动物种系进化关系一致, 为哺乳动物系统进化关系研究提供了分子水平的佐证和依据。     

The fur gene from Klebsiella pneumoniae: characterization,genomic organization and phylogenetic analysis

The Fur (ferric uptake regulator) protein controls the expression of a number of bacterial virulence determinants including those involved in iron uptake. The fur gene was cloned and characterized from Klebsiella pneumoniae. The gene is preceded by a single autoregulated promoter whose −10 region overlaps the putative Fur binding site. The autoregulated nature of the K. pneumoniae fur gene and functionality of the encoded Fur repressor were tested in Fur titration and complementation assays. A partial open reading frame upstream from the fur gene was identified as a flavodoxin (fldA) gene. An open reading frame located 50 bases downstream from the fur stop codon appears to be a truncated citA gene that, if functional, would encode only the carboxy terminus of a citrate utilization protein. The fldA-fur arrangement is also present in Escherichia coli. However, the fur-citA arrangement found in K. pneumoniae is novel. It appears that the chromosomal region downstream from the fur gene is unstable and, thus, variable even in closely related bacterial lineages. To assess the ability of the Fur protein sequence to reflect organismal phylogeny, the Fur protein tree was compared to the tree of 16S rRNA (ribosomal RNA). The Fur dataset comprises almost an order of magnitude fewer characters than the 16S rRNA but is nonetheless able to track the phylogenetic signal reasonably well, suggesting that the fur gene, like the 16S rDNA, may not be subject to horizontal gene transfer in these bacteria.     

家蚕Piwi亚家族基因的克隆、表达及进化分析

为克隆家蚕Bombyx mori Piwi亚家族蛋白基因cDNA全长序列,分析其分子特征和表达模式,探究Piwi亚家族蛋白在家蚕中的生理功能,本研究利用已知物种的Piwi亚家族蛋白搜索家蚕基因组,预测获得家蚕Piwi亚家族蛋白基因siwi1和siwi2,采用RACE技术克隆siwi1和siwi2的全长cDNA序列,利用ORFfinder、Gene-Explorer、InterPro等分析其分子特征;其次,利用已知的所有物种Piwi蛋白及其类似物Piwil构建系统发育树;最后,通过荧光定量PCR技术检测了siwi1和siwi2在丝腺、马氏管、中肠、头部、卵巢和精巢以及不同发育时期(卵、1～5龄幼虫、蛹、成虫)的表达水平,结果显示,克隆获得了siwi1 cDNA全长3 277 bp,包含部分5′UTR、完整的开放阅读框ORF和3′UTR,获得了siwi2的部分序列,其中siwi1对应BmPiwi,siwi2对应BmAgo3。系统发育结果显示,家蚕Piwi亚家族蛋白与乳草长蝽Oncopeltus fasciatus、黑腹果蝇Drosophila melanogaster、橘小实蝇Bactro...     

拟态弧菌溶血素基因的克隆测序与生物信息学分析

目的对拟态弧菌安徽分离株HX4(V.mimicusHX4株)的全长溶血素基因(vmh)进行克隆测序和生物信息学分析,为表达溶血素蛋白(VMH)奠定基础。方法采用PCR法扩增V.mimicusHX4菌株全长vmh基因,将其克隆至pMD18-Tvector并进行测序,应用生物信息学软件分析vmh基因的同源性及其编码蛋白的分子特征。结果V.mimicusHX4菌株vmh基因全长序列2235 bp,编码由744个氨基酸组成的分子量约为82.85 kDa的VMH蛋白。V.mimicusHX4菌株vmh基因的核苷酸序列和氨基酸序列与参考株相应序列的同源性分别介于98.9%~99.1%和96.6%~97.3%。VMH蛋白N端前25个氨基酸组成信号肽,7~27位氨基酸之间存在一个跨膜区域,蛋白二级结构中无规卷曲含量最高,达39.52%,其次为α-螺旋和β-折叠,分别占25.81%和26.75%,β转角含量最低,仅占7.93%。VMH蛋白含有多个T细胞和B细胞抗原表位,同时存在T、B细胞抗原表位的区域最有可能位于肽链第86~95、193~211、419~440和459~501位区段。结论拟态弧菌VMH蛋白是一种高度保守的毒素蛋白,对HX4菌株vmh基因及其编码蛋白信息特征的了解,有助于进一步表达VMH蛋白。     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

家蚕CyPA基因的克隆、表达谱及进化分析

CyP蛋白家族在蛋白质折叠过程中起着重要作用。本研究克隆了家蚕Bombyx mori CyPA基因(BmCyPA),该基因由2个外显子和1个内含子组成,推导开放阅读框编码165个氨基酸,分子量为19.4 kD,等电点为8.79。序列分析表明BmCyPA在不同物种间具有高度的保守性,含有肽酰脯氨酸顺反异构酶活性位点及与CsA侧链结合的氨基酸,提示BmCyPA可能具有肽酰脯氨酸顺反异构酶活性和与CsA结合的特性。组织表达谱及EST数据分析显示,BmCyPA在丝腺中高丰度表达。通过对不同物种来源的CyP基因的进化分析,进一步预测了BmCyP基因的功能,BmCyP可能与丝蛋白的正确折叠相关。     

Cloning and initial characterization of the Bordetella pertussis fur gene

In several Gram-negative pathogens the fur (ferric uptake regulator) gene product controls the expression of many genes involved in iron uptake and virulence. To facilitate the study of iron-regulated gene expression in Bordetella pertussis, we cloned the fur gene from this organism. The B. pertussis fur gene product was 54% identical to the Escherichia coli Fur and complemented two E. coli fur mutants. As with the E. coli fur gene, sequences upstream of the B. pertussis fur were homologous to the consensus Fur-binding site and to the consensus catabolite activator protein binding site.     

Cloning and characterization of the fur gene from Moraxella bovis

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient-iron conditions compared with deficient-iron conditions. An electrophoretic mobility-shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur-box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.     

Cloning and characterization of the fur gene from Helicobacter pylori

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.     

鸡IL-18成熟蛋白基因的克隆及分子进化分析

根据已发表的鸡白介素18(IL-18)基因序列设计合成引物,以植物凝集素(PHA)和脂多糖(LPS)激活的AA肉鸡脾细胞mRNA为模板,通过RT-PCR扩增出编码鸡IL-18成熟蛋白的eDNA。将该eDNA克隆于pUCm-T载体,并对其进行测序,结果表明所克隆的核苷酸片段包含了全部成熟蛋白编码基因,成熟蛋白编码区507个核苷酸,编码169个氧基酸。把该基因编码的鸡成熟IL-18蛋白氨基酸序列与已公布的禽及哺乳动物IL-8成熟蛋白基因氧基酸序列进行比较,其同源性分别在96．5％～100％和20．1％～26．6％之间,分子系统进化树分析表明鸡IL-18与哺乳动物IL-18有共同的祖先,亲源关系较近,但在免疫系统选择性压力下,形成独特的种族特异性。鸡IL-18基因的克隆为体外表达鸡IL-18蛋白及作为免疫佐剂应用于预防接种的研究奠定了基础。     

Cloning and characterisation of the Neisseria gonorrhoeaearoB gene

The gene coding for the 3-dehydroquinate synthetase (aroB) of Neisseria gonorrhoeae has been cloned by functional complementation of an Escherichia coli aroB mutant. The aroB gene isolated from a gonococcal plasmid library encodes a 359 amino acid protein with a molecular mass of 38.6?kDa. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 33 to 55%. An open reading frame coding for an aroK homologue is located immediately upstream of aroB. Downstream of aroB a region of inverted repeats and a gene showing high homology to yafJ of E. coli has been identified. Disruption of aroB generates a gonococcal mutant that is unable to grow in the absence of aromatic compounds. Complementation of the mutant with the intact aroB gene intrans indicates that the gene is responsible for the auxotrophic phenotype. In infection assays with AroB-deficient gonococcal strains, binding, entry and short-term survival in epithelial cells is not affected. The aroB gene might be useful as a selectable marker and target for attenuation of a gonococcal live vaccine strain or as a biosafe laboratory strain.     

Cloning and phylogenetic analysis of an amphioxus myogenic bHLH gene AmphiMDF

In this study, a member of the MyoD gene family, AmphiMDF, was isolated from the embryos of amphioxus by degenerate PCR, followed by rapid amplification of cDNA ends (RACE). Southern blot analysis confirmed that only a single myogenic bHLH gene was present in the genome of amphioxus Branchiostoma belcheri tsingtauense. Sequence and phylogenetic analyses indicated that AmphiMDF falls at the base of its vertebrate homologs. The amino acid sequence of AmphiMDF was almost equally similar to those of the four clusters of the vertebrate MyoD family. This suggests that AmphiMDF is not only the sister but also the archetype of the vertebrate myogenic bHLH genes. The scenarios to explain the origin of the vertebrate MyoD gene family from the ancestral myogenic bHLH gene like AmphiMDF are also discussed.     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

Nematophagous fungi, one of the natural enemies of nematodes, have been employed in biological control. Extracellular enzymes secreted from nematophagous fungi, including protease, chitinase and collagenase serve as virulence factors of infection. In this study, we found Lecanicillium psalliotae can penetrate the eggs of the root-knot nematode Meloidogyne incognita and influence development of the eggs. A chitinase gene Lpchi1 was isolated from L. psalliotae using degenerate primers and DNA-walking technique. Comparison of the chitinase amino acid sequences from different pathogenic fungi revealed that the enzymes were highly similar. The phylogenetic analysis demonstrated that the chitinases derived from different fungi were clustered into three main clades corresponding to different molecular weight.     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

食线虫真菌是植物寄生线虫的重要天敌,它们所产生的胞外水解酶(蛋白酶、几丁质酶和胶原蛋白酶等)能够降解线虫体壁和卵壳中的蛋白质及几丁质等结构成分并在侵染过程中发挥着重要的作用。本文中,我们发现刀孢轮枝菌Lecanicillium psalliotae对南方根结线虫Meloidogyne incognita卵具有较强的侵染能力。为了进一步研究刀孢轮枝菌胞外几丁质酶的性质,我们通过简并引物设计和DNA walking方法从刀孢轮枝菌的基因组中成功地克隆得到一个内切几丁质酶基因Lpchi1,该几丁质酶编码基因含有3个内含子,编码423个氨基酸。同源性和系统发育分析表明,不同生防真菌来源的几丁质酶具有较高的同源性并根据分子量的大小形成三个不同的进化分枝。