耐辐射球菌转录因子DdrO的基因克隆与生物信息学分析

目的：克隆耐辐射球菌ddrO基因,并对其进行生物信息学分析,预测其功能。方法：根据耐辐射球菌ddrO基因序列,由Primer Premier 5设计一对引物,以提取的耐辐射球菌基因组为模板,PCR扩增获得耐辐射球菌ddrO基因,序列测定并利用生物信息学软件对ddrO基因的理化性质、高级结构及生物学功能等进行分析与预测。结果：成功获得了ddrO基因。生物信息学分析发现,ddrO基因核苷酸序列长度为396bp,编码一个131aa组成的相对分子质量为14.993kD的预测的DdrO转录因子。核酸同源性搜索及比较分析仅在与耐辐射球菌同属的Deinococcus geothermalis和Deinococcus deserti中发现高度相似的序列;蛋白同源性搜索发现一些与DdrO显著同源的蛋白,如Deide_20570（95%）,Dgeo_0336（90%）,Deide_3p02170（82%）等;结构域分析发现DdrO含有HTH（helix-turn-helix）DNA结合结构域。结论：根据生物信息学结果预测DdrO蛋白可能具有转录调控作用,参与DNA修复和复制,在耐辐射球菌的DNA损伤修复过程中发挥一定作用。     

烟草乙烯转录因子ERF基因NtTOE3的克隆、载体构建及表达分析

ERF（Ethylene-responsive factor）是一类具有AP2特征结构域的乙烯应答转录因子,在响应植物的逆境胁迫应答中起关键作用。为明确ERF家族成员TOE3在烟草中的生物学功能和调控机制,同源克隆普通烟草K326中NtTOE3基因cDNA全长,利用实时荧光定量PCR（qRT-PCR）表征基因表达模式,结合生物信息学分析,对基因功能和调控机制进行初步预测。结果表明,该基因全长1 356 bp,编码451个氨基酸残基,预测分子量为49.84 ku。生物信息学分析表明,该蛋白是一个亲水蛋白,可能定位在细胞核,且与美花烟草（Nicotiana sylvestris）NsTOE3有96%的同源性,故命名为NtTOE3。组织表达分析发现,该基因在烟草根、茎、叶、花中均有表达,其中茎中表达量最高。逆境胁迫试验表明,该基因能快速响应低钾、高盐、干旱、H₂O₂、ABA和4℃等逆境处理。表明NtTOE3转录因子在烟草非生物胁迫中起着重要调节作用。     

耐辐射球菌RNA聚合酶β亚基突变对基因表达全局的影响

RNA聚合酶的B亚基参与与利福平的结合,并在原核生物中高度保守．许多细菌中利福平耐药株的rpoB基因均有氨基酸改变．本实验室曾分离2两株耐辐射球菌（Deinococcus radiodurans）自发突变利福平耐药株（突变为：L420R和1258—669-bp缺失）．本研究发现,β亚基的变化对生长速度有独特的效果．利用DNA芯片技术和生化分析鉴定耐辐射球菌的利福平突变株如何调控基因表达改变生长速度的分子机制．参与代谢、细胞进程和信号传递以及信息贮存和处理的基因的转录在9bp缺失突变株中显著改变．9bp缺失突变株的上调基因的共有启动子序列为富含AT的序列．与野生株相比,L420R和9bp缺失突变株积聚了更多的活性自由基．这些结果初步探讨了D亚基突变调控基因的表达机制．     

人线粒体转录延伸因子蛋白结构与功能的生物信息学分析

[目的]基于生物信息学方法分析人线粒体转录延伸因子TEFM蛋白的结构和功能。[方法]检索Uniprot数据库中人线粒体转录终止因子TEFM蛋白的氨基酸序列,利用生物信息学方法对人TEFM的理化性质、物种间的蛋白质同源性、跨膜区、亲水性/疏水性、亚细胞定位、蛋白质二级结构、保守结构域、蛋白质三级结构、蛋白质相互作用进行预测与分析。[结果]人TEFM全长360个氨基酸,理论等电点9.39,属于TEFM蛋白超家族,不含跨膜区,属于亲水蛋白;人TEFM含有一个保守的螺旋-发夹-螺旋(HHH_3)结构域,二级结构以α-螺旋和无规则卷曲为主,三维建模空间结构与二级结构预测结果相符,进一步分析建模结果可靠。与人TEFM相互作用的蛋白质均为线粒体DNA转录因子或线粒体RNA聚合酶。[结论]人TEFM具有线粒体转录延伸因子蛋白超家族的典型结构,生物信息学分析结果对深入研究人TEFM在线粒体基因转录调控中的作用具有一定的理论指导意义。     

一个新的家蚕转录相关锌带蛋白基因的克隆及序列分析

锌带蛋白(zinc ribbon protein )是锌指类蛋白的一种,它的Cys4 Zn(2+)结合位点由3个β₂片层折叠而成,而不是α螺旋结构。锌带结构与锌指结构同为转录因子结合核酸的结构域,锌带蛋白作为转录相关因子在调节基因表达活性等方面具有重要作用。在对家蚕 Bombyx mori蛹cDNA文库测序中,发现一个新的编码家蚕锌带蛋白基因的EST序列（GenBank 登录号DY230964）,以此序列为信息探针检索家蚕EST数据库,通过同源筛选,获得一个新的家蚕锌带蛋白基因cDNA全序列并经RT-PCR检测和克隆、测序验证,结果表明与电子克隆序列完全一致。我们将其命名为 BmZNRD1 (Zinc Ribbon Domain Containing 1)（GenBank登录号DQ432055）。该基因全长为675 bp,由363 bp的开放阅读框序列(ORF)、10 bp的5′端非翻译区序列(5′UTR)和302 bp 的3′端非编码区序列(3′ UTR)组成,其编码的120个氨基酸序列与其他真核生物间具有较高的同源性（达60%左右）,预测分子量为13.54 kD, 等电点为6.8。BmZNRD1编码的氨基酸序列是一种锌带蛋白,推测有2个功能结构域,分别是位于N-端的Cx₂Cx₁₄Cx₂C和C-端的Cx₂Cx₂₄Cx₂C,其中C-端保守氨基酸序列Cx₂Cx₆Yx₃QxRSADEx₂TxFx₂Cx₂C在生物进化中保守性很高,从酵母、果蝇、线虫到两栖类、哺乳类都有发现该结构域的存在,与酵母RNA聚合酶A亚单位9和转录相关蛋白有很高的相似性,推测其具有相同的功能。将BmZNRD1基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有3个外显子,2个内含子,外显子/内含子边界符合经典的GT-AG规则。 关键词： 家蚕; 锌带蛋白基因; 电子克隆; 基因克隆; 序列分析     

白及丝氨酸羟甲基转移酶(SHMT)基因的序列分析

丝氨酸羟甲基转移酶（SHMT）是植物细胞光合作用和一碳代谢的关键酶之一,研究SHMT基因的序列信息对揭示其蛋白结构与功能具有重要指导意义。从白及转录组数据库中分离得到SHMT基因（NCBI登录号：MG544187）,运用生物信息学软件对该基因进行序列分析。结果显示：该基因长度为1 953 bp,编码的蛋白质长度为471aa、分子量为51.861 06 kD、理论等电点为7.17,SHMT二级结构主要由无规则卷曲结构和α螺旋结构组成,核苷酸和氨基酸序列与铁皮石斛SHMT相似性最高,达93%,结构域分析发现该蛋白具有高度保守结构域,三级结构预测为四聚体结构,跨膜区及信号肽分析发现该蛋白无跨膜区及信号肽,亚细胞定位分析发现该蛋白主要位于细胞质、叶绿体亚细胞器位置。本分析结果为白及SHMT的应用研究提供了基础数据,也为植物SHMT基因的分子研究提供了理论依据及基础资料。     

真菌中锌簇蛋白的结构和功能

锌簇家族蛋白即Zn₂Cys₆类锌指蛋白,是真菌中特有的一类蛋白,它们属于转录因子类,广泛参与真菌中初级和次级代谢、胁迫应答和细胞分裂等生命活动的调控。锌簇蛋白主要包括N端的DNA结合结构域、中间的调节结构域和C端的酸性区域,其中DNA结合结构域包含锌指基序并负责结合靶基因的启动子。目前已经解析了多个锌簇家族转录因子DNA结合结构域的三维结构,并发现该家族中一些蛋白能够参与调控多个基因的表达,但缺乏对其结构、动力学和功能关系的全面分析。本文综合分析了不同锌簇蛋白与DNA结合的结构特征,总结其结构域与功能的关系,指出锌簇蛋白研究的重要方向,旨在为锌簇家族蛋白的深入研究提供思路。     

红花花青素合成酶基因的克隆及表达分析

根据红花转录物测序结果中得到的中间序列,采用R11-PCR和RACE方法从红花花瓣中克隆到1个4嬲基因的全长cDNA,该基因全长序列1226bp,具有完整的开放阅读框（ORF）,共1050bp,编码349个氨基酸。生物信息学软件分析显示,该基因编码的蛋白理论分子量约为82．27kDa,等电点为5．09,序列里含有典型的加尾信号序列AATAA和Poly（A）。保守结构域预测表明,该基因编码的蛋白具有典型的ANS蛋白功能结构域,其保守结构域中含有铁离子及2．0-酮戊二酸结合位点。结合其他物种的臌因构建系统树表日月,红花ANS蛋基因与其他物种氨基酸具有一定的同源性,其中与芍药的亲缘关系最近。应用实时荧光定量PCR分析表明,ANS基因在红花的初花期和盛花期的表达量最高。     

毛尖紫萼藓抗旱相关基因Gp-LEA的克隆与表达分析

以毛尖紫萼藓干旱cDNA文库中获得的一段与LEA基因同源性较高的EST序列为基础,采用RACE技术分离该基因cDNA全长序列,命名为Gp-LEA。Gp-LEA基因的cDNA全长814bp,开放阅读框456bp,编码含151个氨基酸蛋白质。生物信息学分析结果显示,Gp-LEA蛋白为稳定蛋白,分子质量为16.612kD,理论等电点（pI）为5.06,含有LEA₂功能结构域,不属于跨膜蛋白且不存在信号肽。系统发生分析表明,Gp-LEA基因编码蛋白与花旗松LEA蛋白亲缘关系最近。荧光定量PCR分析显示,Gp-LEA基因在复水和快速干旱模式下均能表达。推测Gp-LEA基因在毛尖紫萼藓的复水和干旱过程中起着重要作用。     

从猕猴肝脏组织扩增黄嘌呤脱氢酶／氧化酶基因片段

RT-PCR扩增猕猴黄嘌呤脱氢酶／氧化酶（XDH／XO）基因片段,为进一步开展相关研究提供实验资料。方法提取猕猴新鲜肝脏组织总RNA,用RT-PCR二步法进行XDH／XO基因片段扩增,对获得的目的片段进行序列测定,与GenBank上发表的人类（Homosapiens）、小鼠（Musmusculus）、家鼠（Rattusnorvegicus）、野猪（Susscrofa）等物种XDH／XO基因进行该序列同源性比对分析,DNAMAN软件预测该段核苷酸的氨基酸序列,Inter-ProScan及SWISS-MODEL工具进行XDH／XO的编码蛋白结构域及功能预测及三维结构构建。结果RT-PCR产物电泳检测得到了与设计大小相一致的目的条带,序列测定共测到683个核苷酸,DNAMAN软件预测该段核苷酸的氨基酸序列包括了1个编码53个氨基酸的开放阅读框（ORF）,通过该软件包中Multiplealignment对目的基因片段的核苷酸序列与NCBI报道的人类、小鼠、家鼠、野猪XDH／XO基因mRNA互补的cDNA核苷酸序列同源性进行同源性比较分析,结果显示所扩增得到的目的片段与人类同源性最高,为95．6％,与小鼠、家鼠、野猪的同源性分别为85．2％、84．3％、86．1％,说明获得的基因片段是猕猴的XDH／XO基因片段,且该基因在物种间具有较高的相似性。生物信息学预测该段XDH／XO编码蛋白含有醛氧化／脱氢酶的钼喋呤结合点结构域及黄嘌呤脱氢酶结构域。结论在体外成功扩增出猕猴XDH／XO基因片段,为进一步开展高尿酸血症致病机理研究,抗高尿酸血症新药研发奠定工作基础。     

Comparison of the genomes of deinococcal species using oligonucleotide microarrays

The bacterium Deinococcus radiodurans is one of the most resistant organisms to ionizing radiation and other DNAdamaging agents. Although, at present, 30 Deinococcus species have been identified, the whole-genome sequences of most species remain unknown, with the exception of D. radiodurans (DRD), D. geothermalis, and D. deserti. In this study, comparative genomic hybridization (CGH) microarray analysis of three Deinococcus species, D. radiopugnans (DRP), D. proteolyticus (DPL), and D. radiophilus (DRPH), was performed using oligonucleotide arrays based on DRD. Approximately 28%, 14%, and 15% of 3,128 open reading frames (ORFs) of DRD were absent in the genomes of DRP, DPL, and DRPH, respectively. In addition, 162 DRD ORFs were absent in all three species. The absence of 17 randomly selected ORFs was confirmed by a Southern blot. Functional classification showed that the absent genes spanned a variety of functional categories: some genes involved in amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, and DNA metabolism were not present in any of the three deinococcal species tested. Finally, comparative genomic data showed that 120 genes were Deinococcus-specific, not the 230 reported previously. Specifically, ddrD, ddrO, and ddrH genes, previously identified as Deinococcus-specific, were not present in DRP, DPL, or DRPH, suggesting that only a portion of ddr genes are shared by all members of the genus Deinococcus.     

LexA analog (dra0074) is a regulatory protein that is irrelevant to recA induction

The protein DRA0074 is suggested to be another LexA in Deinococcus radiodurans, having similar motifs and RecA-mediated cleavage activity to D. radiodurans LexA (dra0344). However, its function has not been studied. We disrupted the gene dra0074 and measured its effect on RecA induction using fusion translation, immunoblot, and proteomic analysis. Results showed that the product of gene dra0074 is not involved in RecA induction, but is a regulator of other metabolisms in D. radiodurans.     

Horizontal Transfer of Archaeal Genes into the Deinococcaceae: Detection by Molecular and Computer-Based Approaches

Members of the Deinococcaceae (e.g., Thermus, Meiothermus, Deinococcus) contain A/V-ATPases typically found in Archaea or Eukaryotes which were probably acquired by horizontal gene transfer. Two methods were used to quantify the extent to which archaeal or eukaryotic genes have been acquired by this lineage. Screening of a Meiothermus ruber library with probes made against Thermoplasma acidophilum DNA yielded a number of clones which hybridized more strongly than background. One of these contained the prolyl tRNA synthetase (RS) gene. Phylogenetic analysis shows the M. ruber and D. radiodurans prolyl RS to be more closely related to archaeal and eukaryal forms of this gene than to the typical bacterial type. Using a bioinformatics approach, putative open reading frames (ORFs) from the prerelease version of the D. radiodurans genome were screened for genes more closely related to archaeal or eukaryotic genes. Putative ORFs were searched against representative genomes from each of the three domains using automated BLAST. ORFs showing the highest matches against archaeal and eukaryotic genes were collected and ranked. Among the top-ranked hits were the A/V-ATPase catalytic and noncatalytic subunits and the prolyl RS genes. Using phylogenetic methods, ORFs were analyzed and trees assessed for evidence of horizontal gene transfer. Of the 45 genes examined, 20 showed topologies in which D. radiodurans homologues clearly group with eukaryotic or archaeal homologues, and 17 additional trees were found to show probable evidence of horizontal gene transfer. Compared to the total number of ORFs in the genome, those that can be identified as having been acquired from Archaea or Eukaryotes are relatively few (approximately 1%), suggesting that interdomain transfer is rare.     

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).     

A comparative proteomic approach to better define Deinococcus nucleoid specificities

Compared to radiation-sensitive bacteria, the nucleoids of radiation-resistant Deinococcus species show a higher degree of compaction. Such a condensed nucleoid may contribute to the extreme radiation resistance of Deinococcus by limiting dispersion of radiation-induced DNA fragments. Architectural proteins may play a role in this high degree of nucleoid compaction, but comparative genomics revealed only a limited number of Deinococcus homologs of known nucleoid-associated proteins (NAPs) from other species such as Escherichia coli. A comparative proteomic approach was used to identify potentially novel proteins from isolated nucleoids of Deinococcus radiodurans and Deinococcus deserti. Proteins in nucleoid enriched fractions were identified and semi-quantified by shotgun proteomics. Based on normalized spectral counts, the histone-like DNA-binding protein HU appeared to be the most abundant among candidate NAPs from both micro-organisms. By immunofluorescence microscopy, D. radiodurans HU and both DNA gyrase subunits were shown to be distributed throughout the nucleoid structure and absent from the cytoplasm. Taken together, our results suggest that D. radiodurans and D. deserti bacteria contain a very low diversity of NAPs, with HU and DNA gyrase being the main proteins involved in the organization of the Deinococcus nucleoids.     

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.     

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

耐辐射奇球菌超氧化物歧化酶基因的克隆与序列分析

By using a 453 bp length gene fragment of superoxide dismutase(SOD)as a probe,which was firstly amplified from Deinococcus radiodurans genomic DNA by PCR with degenerate oligonucleotide primers corresponding to the conservative regions of known SODs,a putative SOD gene was identified from the database of D.radiodurans whole genome.Its 636 bp length open reading frame and 5′ and 3′ flanking sequence was determined.The conventional E.coli ribosomal and RNA polymerase binding sites were found upstream from SOD encoding region and an inverted repeat sequence downstream of the termination codon.The deduced 211 amino acid sequence of the structural gene showed a high similarity to other manganese and iron containing SODs in normally conserve regions.