【作者】 邱宁；

【导师】 喻子牛；

【作者基本信息】 华中农业大学 ， 微生物学， 2010， 博士

【摘要】 插入序列(Insertion Sequence, IS)是一类最为简单并具有高度可移动性的转座因子,是对基因组上一个或多个位点具有插入能力的小分子DNA片段。插入序列广泛存在于原核生物中,可通过剪切和插入影响功能基因的表达、突变和移动,同时也是导致原核微生物基因组重排的重要因素之一。本研究通过对苏云金芽胞杆菌(Bacillus thuringiensis)菌株YBT-1520全基因组序列的分析,注释了染色体上的134个转座酶基因,最终确定了68个完整的插入序列单元。在整体数量上明显多于其他已完成测序的蜡状芽胞杆菌群基因组。这68个拷贝分别属于6个IS家族中的14个不同的插入序列单元,其中有7个插入序列是由本研究新发现并命名的。为了从整体上了解插入序列在YBT-1520基因组中的分布和功能,本研究全面考察了YBT-1520染色体和质粒上所含有的每种插入序列的拷贝数、其在基因组上的定位、移码以及是否插入到功能基因中等。结果表明,菌株YBT-1520染色体中大量的插入序列主要是由IS231C, IS232A和ISBth166的多拷贝造成的。大部分插入到功能基因中的事件都是由IS231C介导的。而菌株YBT-1520基因组中唯一拷贝的铁载体蛋白(siderophore biosynthesis protein)编码基因被IS232A插入导致失活,从而使得YBT-1520菌株在贫铁环境中吸收铁离子的能力大大减弱。本研究以IS231C, IS232A, ISBth166这三个在YBT-1520染色体上拷贝数最高的插入序列为探针,对菌株YBT-1520和菌株HD-1这两株库斯塔克亚种(subsp.kurstaki)菌株进行了Southern杂交试验,结果显示,拷贝数最多且存在于多个功能基因内部的IS231C在两株菌中的定位基本一致。这表明IS231C的大量转座是发生在这两株菌分化之前的近期事件,IS231C并没有大范围介导这两株菌的同源重组。通过对多株不同亚种苏云金芽胞杆菌的Southern杂交试验还发现了ISBth66主要分布于库斯塔克亚种中,在本研究涉及的8个非库斯塔克亚种中则比较少见。本实验室正在测序的另外一个苏云金芽胞杆菌中华亚种CT-43的染色体上也存在不少于40个完整的插入序列单元。菌株CT-43染色体上不含有IS231C,但含有IS231A的多个拷贝；同时还含有不少于6个拷贝的IS232A；而已发现的菌株CT-43基因组中拷贝数最高的插入序列是属于IS110家族的ISBth13,有7个拷贝。这说明具有近期活性的插入序列在不同亚种的苏云金芽胞杆菌中存在着亚种差异性。此外,菌株CT-43基因组中还存在不少于4拷贝的与ISBth166有87%相似性的插入序列。蜡状芽胞杆菌群是完成全基因组测序最多的种群之一,现已有18株菌公布了全基因组序列。本研究通过重新注释,对这些插入序列所属家族进行了指定和修正。通过比较基因组的分析,对在蜡状芽胞杆菌群中广泛分布的插入序列家族进行了系统发育分析。结果表明与炭疽芽胞杆菌亲缘关系接近的菌株中的插入序列在分布和系统发育方面有着自身特有的进化分支,而大部分插入序列之间的进化关系和蜡状芽胞杆菌群各菌株之间的亲缘关系并不对应。这说明同一家族中的多个插入序列的分化发生在蜡状芽胞杆菌群分化之前,而插入序列的种类和进化可能与这些菌株特有的生境(niche)相关。插入序列在蜡状芽胞杆菌群菌株中分布的不均衡性可能是影响其进化的重要力量之一。运用上述对苏云金芽胞杆菌YBT-1520基因组中插入序列组的分析方法,本研究还针对一株致病菌鼻疽诺卡菌Nocardia farcinica) IFM10152中的细胞色素P450蛋白超家族进行了比较基因组分析。推测了鼻疽诺卡菌能合成抗生素的能力和其基因组中P450蛋白的多样性有很大关系。从对P450蛋白的聚类分析可以看出鼻疽诺卡菌是在进化上介于链霉菌和分支杆菌之间的菌株。更多还原

【Abstract】 Insertion sequences (ISs) are small transposable DNA fragments that can insert into one or multiple loci of genome.It is well recognized that IS elements sometimes inactivate genes upon insertion or activate and/or enhance the expression of nearby genes. Meanwhile, ISs have played an important role in genome reshuffling and evolution by facilitating horizontal gene transfer and mediating homologous recombination between multiple copies present in a given genome.Sequencing of YBT-1520 genome revealed 134 transposase coding genes (at least 68 intact genes) on the chromosome which is one of the most notable features of YBT-1520. In this work,fourteen kinds of intact ISs including seven new-named elements were identified in YBT-1520.A detailed characterization of these ISs distributed into 6 families is presented. Moreover, a comparative analysis of their counterparts in 18 finished Bacillus cereus group genomes as well as in different B. thuringiensis strains has been done in order to understand the distribution and evolution of these IS elements.The IS elements of YBT-1520 were subjected to a careful analysis of IS copies, location and insertion into function genes.Furthermore, these ISs were compared to the elements of the other B. cereus group genomes, in an attempt to demonstrate the distribution and evolution of IS elements in these closely related genomes. YBT-1520 chromosome contains a larger number of transposase coding genes than that of other B. cereus group genomes mainly due to the multi-copies of IS231C, IS232A and ISBth166. Although most of the IS elements randomly distributed throughout the genome were inserted in non-coding regions, at least 9 CDSs were disrupted by the insertion of IS elements (mostly by IS231C). The only copy of siderophore biosynthesis protein coding gene, which is a growth-determining factor under iron-limited conditions in the host insect gut, was inserted by IS232A.To further assess the involvement of IS elements in promoting genome reorganization between two B. thuringiensis kurstaki strains (YBT-1520 and B. thuringiensis HD-1),hybridization experiments were performed using fragments of three most abundant IS elements (IS231C, IS232A and ISBth166) of different families as probes. The result showed that the most multitudinous IS231C had minor different hybridization profiles between these two isolates, which indicated that it did not cause large-scale rearrangements.The distribution of ISBth166 among B. thuringiensis serovars was further examined by Southern blot analysis which suggesting that ISBth166 is widely distributed among kurstaki strains.Genome survey of B. thuringiensis CT-43 revealed at least 40 intact IS elements. Multi-copies of IS231A, in stead of IS231C, were found in CT-43 genome. Meanwhile,4 copies of ISs show 87% similarity to ISBth166.All these results showed species specificity of ISs in B. thuringiensis and was in concert with the conclusion we’ve drawn before.To data, eighteen B. cereus group genomes have been completely sequenced and published in Genbank. A careful analysis and re-annotation were performed to the IS complement in B. cereus group. The extended survey of all IS elements among the B. cereus group genomes showed some correlation between MLST phylogenetic relatedness and distribution of IS families.IS3 family transposases of subgroup I members were clustered together. The different existence of IS4 and IS110 family elements were also found between subgroup I and the other two groups. This indicated that the IS elements of subgroup I B. anthracis-related genomes may have their distinct phylogenetic lineage, which may affected by their different ecological niche. The disequilibrium in distribution and copy numbers of ISs among B. cereus group genomes is probably one of the major forces of genome evolution.Genome analyses of cytochrome P450 complement of Nocardia farcinica genome were further performed using the same method. Comparative analyses of these cytochrome P450 proteins with their courterparts in related Actinomycetes showed different function relationship among them. The unique features of Nocardia farcinica genome were probably due to the abundant and various cytochrome P450 members.更多还原