巢湖铜绿微囊藻Chao 1910的基因组学和磷代谢通路分析

【背景】铜绿微囊藻(Microcystis aeruginosa)广泛分布于温带湖泊,因产生微囊藻毒素且易成为蓝藻水华优势藻株而备受关注。【目的】基于全基因组序列分析和基因转录水平验证,阐明从巢湖新分离的铜绿微囊藻Chao 1910的主要代谢通路和磷营养高效利用机制。【方法】通过第三代测序技术拼接获得Chao1910的全基因组序列,完成主要代谢通路的基因注释,并对与蓝藻水华优势藻株形成相关的磷代谢通路进行深入分析。【结果】比较基因组学表明,Chao1910藻株与日本铜绿微囊藻NIES-843的亲缘关系最近,其糖酵解、磷酸戊糖途径和核苷酸合成等代谢通路的基因组成非常保守,同时具有完整的磷转运、磷吸收、多聚磷酸盐合成/分解等磷营养高效利用的通路。不同于其他铜绿微囊藻,Chao 1910藻株不具有微囊藻毒素合成基因簇,推测其主要依靠对磷营养的高效利用获取生存竞争优势。【结论】Chao1910藻株是巢湖首株完成全基因组测序的铜绿微囊藻,这将有助于揭示其获得生存竞争优势的分子机制,为遏制巢湖蓝藻水华暴发提供依据。     

10株铜绿假单胞菌的全基因组序列分析

目的 了解不同分离来源铜绿假单胞菌的全基因组基本特征,以此分析基因组多态性及其遗传进化关系。方法 选择10株医源性和食源性来源的铜绿假单胞菌代表性菌株,应用Solexa高通量测序技术对其进行全基因组测序,以此进行多位点序列分型(multilocus sequence typing, MLST),比较各菌株基因组中携带的耐药基因、毒力基因及插入序列(insertion sequence, IS)元件,并通过比较基因组学分析方法拟合泛基因组和核心基因组积累曲线,筛选核心基因SNP构建系统发育分子进化树。结果 10株菌的基因组从6.3～7.0 Mbp大小不一,包含5 868～6 598个基因,平均G+C含量为67.1%;发现10个菌株各具不同的ST型。在这10个菌株的基因组中,共检测到75种耐药基因,包括抗β-内酰胺酶类、抗氨基糖苷类、抗氟喹诺酮类等;共发现188种毒力基因,不同来源菌株间无明显差异;各菌株之间IS元件种类和数量差异较大。分析发现,铜绿假单胞菌具有开放型泛基因组和稳定型核心基因组;10株菌可分为3个进化分支,且不同分离时间和来源无明显相关性。结论 本研究获得10株不同分离来源的铜绿假单胞菌的全基因组序列,初步证实食品及患者分离来源菌株基因组数据无明显相关性,为后续铜绿假单胞菌的分子流行病学和致病性机制研究提供数据参考。     

携带armA基因的铜绿假单胞菌耐药性及基因周边环境

目的了解多重耐药(MDR)铜绿假单胞菌armA基因与可移动遗传元件的携带情况及其相关性;分析armA基因的周边环境,探讨armA基因转移的可能机制。方法收集MDR铜绿假单胞菌98株,琼脂稀释法测定MIC,PCR方法检测16S rRNA甲基化酶基因armA、I型整合子、可移动元件IS26及重要耐药基因侧翼基因环境,测序并拼接PCR产物明确耐药基因座位排列,并对armA基因进行周边序列分析。结果 98株MDR铜绿假单胞菌检出5株armA基因PCR扩增阳性,携带armA基因的菌株对庆大霉素和阿米卡星全耐药;检出20株携带I型整合子,17株携带可移动元件IS26;armA基因扩增阳性的菌株均携带I型整合子和IS26;序列测序显示armA定位于Tn1548相关区域,位于插入序列ISCR1的下游,该序列含多种移动元件。结论大连市氨基糖苷类高水平耐药基因armA广泛分布在MDR铜绿假单胞菌中,均对庆大霉素和阿米卡星高度耐药;该基因定位在转座子Tn1548的质粒上,提示16S rRNA甲基化酶基因armA的广泛播散可能是可移动元件ISCR1-armA-IS26结构参与其中。     

原生动物基因组转座元件的研究进展

转座元件是一类广泛分布于真核生物的可移动的遗传因子, 可以引起基因重组和变异, 在物种进化及遗传改良中起着重要作用。针对近年来原生动物全基因组序列中大量发现的转座元件, 文章着重比较了转座元件在锥虫、利什曼虫、微孢子虫、变形虫和滴虫基因组序列中的存在种类、分布特征及其功能意义。原生动物转座元件以LINE 和SINE为主, 其次是DNA转座元件和LTR反转座元件, 部分转座元件在高A+T含量区富集, 预示着转座元件与基因组序列A+T含量有着紧密联系。根据不同种微孢子虫基因组之间转座元件的差异, 推测在微孢子虫基因组进化过程中, 至少经历了一次转座元件的丢失事件。最后对转座元件在原生动物寄生虫的进一步研究和应用作了展望。     

Y1转座酶关联转座子在大肠杆菌和沙门氏菌基因组中的分布和结构特征

Y1转座酶关联转座子(Y1ATs)的活性催化位点为一个酪氨酸,能够切割和连接单链DNA,在原核生物分布广泛。为探究Y1转座酶关联转座子在大肠杆菌(Escherichia coli, E. coli)与沙门氏菌(Salmonella enterica, S. ente)基因组中系统进化特性,通过Hmmsearch程序对Y1转座酶关联转座子进行了挖掘分析。结果表明,Y1转座酶关联转座子广泛分布于96.84%大肠杆菌基因组和80.4%沙门氏菌基因组。根据序列比对和蛋白结构域预测将Y1转座酶关联转座子分为10类,均隶属于IS200/IS605超家族,其中11 645个属于IS200家族,4 811个属于IS605家族。IS200家族广泛分布于S. ente基因组中(72.24%),而IS605家族广泛分布于E. coli基因组中(89.38%)。IS200拷贝数以及完整拷贝数显著高于IS605。IS200家族仅含有一个Y1转座酶编码区,而IS605家族含两个开放阅读框,分别编码Y1转座酶和TnpB蛋白。IS200家族的Y1氨基酸序列高度保守(95.3%),而IS605家族的Y1和TnpB具有较高遗传多样性,为研究转座子在原核生物的遗传进化模式提供重要参考。IS200家族具有高度保守的Y1转座酶,且完整拷贝数比例较高,提示该类转座子可能具有转座活性,对其活性的挖掘有利于研制转座子介导的新型高效基因编辑工具。     

植物转座元件注释及生物学功能研究进展

转座元件是指在基因组中能够移动、复制并重新整合到基因组新位点的DNA片段。在植物中,多种类型的转座元件,特别是占比较高的LTR类逆转录转座元件,可以通过产生新基因和转录本、提供调节元件、改变基因结构等多种途径广泛调控基因表达,最终多维度有效推动基因组进化。同时,基因组测序组装技术的快速发展也为转座元件的检测、注释提供了良好契机。本文从结构分类、全基因组检测、功能研究、基因组进化4个方面对当前植物转座元件的研究进展进行综述,同时对今后的研究方向进行了展望。     

昆虫RNA-Seq数据的分析流程

随着高通量RNA测序(RNA-Seq)技术的发展和测序成本迅速下降,RNA-Seq技术已经成为生物学研究的重要工具,为生物学家全面地了解和研究转录组提供了机遇。高通量测序具有读长短、存在一定比例的测序错误、数据量大等特点,因此RNA-Seq数据分析与基因组分析和传统的EST数据分析有所不同。本文通过介绍不同的测序平台、原始数据产生和低质量数据过滤的计算流程,对短序列比对、转录组拼接、功能注释、以及差异表达分析进行了研究和分析,最后对RNA-Seq在昆虫学研究中的应用进行了综述,并对RNA-Seq技术进行了总结和展望。     

两种方法对大熊猫基因组重复序列的注释比较

重复序列是动物基因组的重要组分,对于基因组结构多样性、调节基因表达和介导多种遗传疾病具有重要影响。本研究采用了2种策略:基于序列比对的Repeat Masker(RM)和从头预测的Repeat Scout(RS),对大熊猫Ailuropoda melanoleura基因组中的重复序列进行鉴定与注释,详细阐明了其转座子元件(TE)的组成、类型、数量、亚家族、长度分布、分化率等。比较2种注释方法的结果,RM注释到的TE数量在绝大部分亚家族中均多于RS,而在某些亚家族中则少于RS;RS注释到的TE亚家族类型及平均长度均小于RM。此外,RS构建的大熊猫TE一致性序列中,有20%不属于现有的重复序列类型,可能包含大熊猫特有的TE类型。研究结果对于阐明大熊猫重复序列的特征及其生物学功能奠定了重要基础。     

甘蓝型油菜MADS-box基因家族的鉴定与系统进化分析

MADS-box基因家族参与调控开花时间、花器官分化、根系生长、分生组织分化、子房和配子发育、果实膨大及衰老等植物生长发育的重要过程。基于甘蓝型油菜(Brassica napus)基因组测序数据,利用生物信息学方法对甘蓝型油菜MADS-box基因家族进行鉴定和注释及基因结构与系统进化分析。结果显示,在甘蓝型油菜中鉴定出307个MADS-box基因家族成员,根据进化关系可将其分为两大类型,I型(M-type)包含α、β、γ三个亚家族,II型(MIKC-type)包括MIKCC和MIKC*两个亚家族,MIKCC可进一步分为13个小类;甘蓝型油菜A基因组染色体上分布的MADS-box基因多于C基因组。在基因结构上,MIKC-type亚家族基因序列普遍比M-type长且含有较多的外显子;M-type亚家族蛋白序列中的motif数量为2–5个,MIKC-type亚家族蛋白序列中平均含有7个motif。拟南芥(Arabidopsis thaliana)与甘蓝型油菜MADS-box基因共线性分析结果显示,全基因组复制事件对MADS-box基因家族尤其是MIKC亚家族的扩张起重要作用;MIKC亚家族基因在进化过程中受到的选择压力约为M-type的2倍,这表明MIKC-type亚家族在进化过程中被选择性保留。     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特 的关键作用,因而也称为微囊藻毒素去毒酶. 从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列. 序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920 bp,其中5′-UTR长74 bp,3′-UTR长174 bp,编码区长672 bp,编码223个氨基酸. 应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878 bp序列. 与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

Genome-wide comparison of cyanobacterial transposable elements, potential genetic diversity indicators

Transposable elements are widely distributed in archaea, bacteria and eukarya domains. Considerable discrepancies of transposable elements in eukaryotes have been reported, however, the studies focusing on the diversity of transposable element systems in prokaryotes were scarce. Understanding the transposable element system in cyanobacteria by the genome-wide analysis will greatly improve the knowledge of cyanobacterial diversity. In this study, the transposable elements of seventeen cyanobacterial genomes were analyzed. The abundance of insertion sequence (IS) elements differs significantly among the cyanobacterial genomes examined. In particular, water bloom forming Microcystis aeruginosa NIES843 was shown to have the highest abundance of IS elements reaching 10.85% of the genome. IS family is a widely acceptable IS classification unit, and IS subfamily, based on probe sequences, was firstly proposed as the basic classification unit for IS element system, therefore both IS family and IS subfamily were suggested as the two hierarchical units for evaluating the IS element system diversity. In total, 1980 predicted IS elements, within 21 IS families and 132 subfamilies, were identified in the examined cyanobacterial genomes. Families IS4, IS5, IS630 and IS200-605 are widely distributed, and therefore supposed to be the ancestral IS families. Analysis on the intactness of IS elements showed that the percentage of the intact IS differs largely among these cyanobacterial strains. Higher percentage of the intact IS detected in the two hot spring cyanobacterial strains implied that the intactness of IS elements may be related to the genomic stabilization of cyanobacteria inhabiting in the extreme environments. The frequencies between IS elements and miniature inverted-repeat transposable elements (MITEs) were shown to have a linear positive correlation. The transposable element system in cyanobacterial genomes is of hypervariability. With characterization of easy definition and stability, IS subfamily is considered as a reliable lower classification unit in IS element system. The abundance of intact IS, the composition of IS families and subfamilies, the sequence diversity of IS element nucleotide and transposase amino acid are informative and suitable as the indicators for studies on cyanobacterial diversity. Practically, the transposable system may provide us a new perspective to realize the diversity and evolution of populations of water bloom forming cyanobacterial species.     

The bacteriophage Mu transposase protein can form high-affinity protein-DNA complexes with the ends of transposable elements of the Tn 3 family

The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome. A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements. These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism. Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101. No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size. These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related.     

The splicing of transposable elements and its role in intron evolution

Recent studies have demonstrated that transposable elements in maize and Drosophila are spliced from pre-mRNA. These transposable element introns represent the first examples of recent addition of introns into nuclear genes. The eight reported examples of transposable element splicing include members of the maize Ac/Ds and Spm/dSpm and the Drosophila P and 412 element families. The details of the splicing of these transposable elements and their relevance to models of intron origin are discussed.     

Evolutionary forces generating sequence homogeneity and heterogeneity within retrotransposon families

Genome projects allow us to sample copies of a retrotransposon sequence family residing in a host genome. The variation in DNA sequence between these individual copies will reflect the evolutionary process that has spread the sequences through the genome. Here I review quantitatively the expected diversity of elements belonging to a transposable genetic element family. I use a simple neutral model for replicative mobile DNAs such as retrotransposons to predict the extent of sequence variability between members of a single family of transposable elements, both within and between species. The effects of horizontal transfer are also explored. I also consider the impact on these distributions of an increase in transposition rate arising from a mutational change in copy of the sequence. In addition, I consider the question of the interaction between retrotransposons and their hosts, and the causes of the abundance of transposable elements in the genomes that they occupy.     

Survey of transposable elements from rice genomic sequences

Oryza sativa L. (domesticated rice) is a monocotyledonous plant, and its 430 Mb genome has been targeted for complete sequencing. We performed a high-resolution computer-based survey for transposable elements on 910 Kb of rice genomic DNA sequences. Both class I and II transposable elements were present, contributing 19.9% of the sequences surveyed. Class II elements greatly outnumbered class I elements (166 versus 22), although class I elements made up a greater percentage (12.2% versus 6.6%) of nucleotides surveyed. Several Mutator-like elements (MULEs) were identified, including rice elements that harbor truncated host cellular genes. MITEs (miniature inverted-repeat transposable elements) account for 71.6% of the mined transposable elements and are clearly the predominant type of transposable element in the sequences examined. Moreover, a putative Stowaway transposase has been identified based on shared sequence similarity with the mined MITEs and previously identified plant mariner-like elements (MLEs). Members of a group of novel rice elements resembling the structurally unusual members of the Basho family in Arabidopsis suggest a wide distribution of these transposons among plants. Our survey provides a preview of transposable element diversity and abundance in rice, and allows for comparison with genomes of other plant species.     

Transposition of a bacterial insertion sequence in chloroplasts

Bacterial transposable elements (IS elements, transposons) represent an important determinant of genome structure and dynamics, and are a major force driving genome evolution. Here, we have tested whether bacterial insertion sequences (IS elements) can transpose in a prokaryotic compartment of the plant cell, the plastid (chloroplast). Using plastid transformation, we have integrated different versions of the Escherichia coli IS element IS 150 into the plastid genome of tobacco ( Nicotiana tabacum ) plants. We show that IS 150 is faithfully mobilized inside the chloroplast, and that enormous quantities of transposition intermediates accumulate. As synthesis of the IS 150 transposase is dependent upon programmed ribosomal frame shifting, our data indicate that this process also occurs in chloroplasts. Interestingly, all insertion events detected affect a single site in the plastid genome, suggesting that the integration of IS 150 is highly sequence dependent. In contrast, the initiation of the transposition process was found to be independent of the sequence context. Finally, our data also demonstrate that plastids lack the capacity to repair double-strand breaks in their genomes by non-homologous end joining, a finding that has important implications for genome stability, and which may explain the peculiar immunity of the plastid to invading promiscuous DNA sequences of nuclear and mitochondrial origin.     

Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence

A repeating element of DNA has been isolated and sequenced from the genome of Bordetella pertussis. Restriction map analysis of this element shows single internal ClaI, SphI, BstEII and SalI sites. Over 40 DNA fragments are seen in ClaI digests of B. pertussis genomic DNA to which the repetitive DNA sequence hybridizes. Sequence analysis of the repeat reveals that it has properties consistent with bacterial insertion sequence (IS) elements. These properties include its length of 1053 bp, multiple copy number and presence of 28 bp of near-perfect inverted repeats at its termini. Unlike most IS elements, the presence of this element in the B. pertussis genome is not associated with a short duplication in the target DNA sequence. This repeating element is not found in the genomes of B. parapertussis or B. bronchiseptica. Analysis of a DNA fragment adjacent to one copy of the repetitive DNA sequence has identified a different repeating element which is found in nine copies in B. parapertussis and four copies in B. pertussis, suggesting that there may be other repeating DNA elements in the different Bordetella species. Computer analysis of the B. pertussis repetitive DNA element has revealed no significant nucleotide homology between it and any other bacterial transposable elements, suggesting that this repetitive sequence is specific for B. pertussis.     

Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2.

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.