【作者】 刘贵明；

【导师】 杨焕明；

【作者基本信息】 浙江大学 ， 生物信息学， 2008， 博士

【摘要】 本论文详细报道了对鳞翅目害虫有高毒力的苏云金芽孢杆菌(Bacillusthuringiensis)菌株YBT-1520的全基因组测序以及与炭疽芽孢杆菌(Bacillus anthracisAmes)、腊状芽孢杆菌(Bacillus cereus ATCC 14579)和苏云金芽孢杆菌(Bacillusthuringiensis 97-27)的比较基因组学分析。介绍了它们在染色体和质粒的保守性、致病因子、可移动因子、代谢和调控以及进化方面的异同点。YBT-1520基因组由1条染色体和9个质粒组成,其中染色体的长度为5,547,282bp,编码5440个ORF;9个质粒中,最大的为293,574 bp,编码298个ORF,最小的2062 bp,编码3个ORF,9个质粒的总长度为477,287bp,共编码523个ORF。直系同源簇比较表明YBT-1520与炭疽芽孢杆菌(Bacillus anthracis Ames)、腊状芽孢杆菌(Bacillus cereus ATCC 14579)和苏云金芽孢杆菌(Bacillus thuringiensis 97-27)染色体具有显著的保守性,是由共同的祖先进化而来。YBT-1520染色体编码腊状芽孢杆菌群共有的一些致病因子,如肠毒素、磷脂酶、InA蛋白、Enhancin蛋白、几丁质酶以及YBT-1520染色体特有的ZwA基因簇,这些致病因子对苏云金芽孢杆菌杀虫活性具有增效作用。与炭疽芽孢杆菌和腊状芽孢杆菌相比,YBT-1520编码更多的噬菌体DNA、插入序列和转座子,这些可移动元件使YBT-1520基因组具有很高的可塑性,能够与其它细菌的染色体、质粒进行遗传物质的交流。YBT-1520染色体含有32个双组分调控系统、59个受PlcR多效调节因子调控的基因以及310个具有调控作用结构域的基因。其中有5个双组分调控系统与细菌致病性直接相关,8个PlcR调控的基因也与YBT-1520的致病性相关。在分解代谢方面,YBT-1520碳水化合物的分解代谢能力较弱,只能利用有限的多糖如肝糖元、淀粉、几丁质和壳聚糖,缺少甘露糖代谢途径、树胶醛糖和鼠李糖代谢途径以及肌醇代谢的关键酶;相反地,YBT-1520编码丰富的蛋白酶、氨基酸膜转运蛋白以及绝大多数氨基酸合成代谢的关键酶,使其更容易地从环境中摄取氨基酸和核酸,这是腊状芽孢杆菌群共有的特点,因此腊状芽孢杆菌群祖先很可能不是土壤微生物,而是起源于昆虫病源菌。我们还分析了YBT-1520编码的特有基因,这些特有基因的主要功能是复制、重组和修复、转录以及氨基酸转运和代谢,其中包含一个尿素酶操纵子,这可能增加了YBT-1520对富含蛋白的酸环境的适应性。pBMB293是YBT-1520最重要的质粒,其编码的4个杀虫晶体蛋白基因(Cry1Aa,Cry1Ia,Cry2Aa和Cry2Ab)、1个营养期杀虫蛋白(Vip3A)和1个溶血性肠毒素操纵子都集中在pBMB293的一个致病岛上。除致病岛外的pBMB293序列与质粒pb10987和pPER272有高度的同源性,这个保守区域由共同的祖先进化而来。pBMB293还编码12个调控相关基因,包括2个能够调控芽孢形成的Rap-Phr调控系统。pBMB293编码的溶血性肠毒素操纵子和ORF8具有染色体编码的PlcR多效调节子的结合位点,可能受其调控。pBMB137编码的调控因子MerR能够对外界金属刺激做出快速反应,pBMB137编码的IS元件,如转座子Tn4430和Tn5044以及嗜热解烃菌同源插入序列使其更容易地进行遗传物质的传递交流。pBMB67编码的结合转移基因使其具有结合转移的特性。这些质粒更使该菌株具有高杀虫活性以及高度可塑性。更多还原

【Abstract】 In this thesis,we reported the whole genome sequence of Bacillus thuringiensis strain YBT-1520,which is highly toxic to lepidopteran pests and performed comparative analysis with the genomes of Bacillus anthracis Ames,Bacillus cereus ATCC 14579 and Bacillus thuringiensis 97-27.Comparison of these genomes and their plasmids reveals the similarities and differences in terms of conservation,virulence,mobile elements,metabolic competence and regulatory mechanisms.The genome of YBT-1520 is composed of a chromosome of 5,547,282bp and nine plasmids of total length of 477,287 bp,harbouring 5440 and 523 open reading frames (ORF),respectively.Of the 9 plasmids,the largest is 293,574 bp and harbors 298 ORFs, the smallest one is 2062 bp and harbors 30RFs.The analysis of orthologous genes reveals high conservation of genome content among YBT-1520,Bacillus anthracis Ames,Bacillus cereus ATCC 14579 and Bacillus thuringiensis 97-27,suggesting that they could have been inherited from a common ancestor.The chromosomally encoded virulence factors in YBT-1520 are common to the B.cereus group of bacteria,including enterotoxin, phospholipase,immune inhibitor A,Enhancin protein and chitinase,except that the ZwA gene cluster is specific to YBT-1520.Those virulence factors may enhance insecticidal toxicity to pests.Compared with Bacillus anthracis and Bacillus cereus,YBT-1520 encods more phages,IS elements and transposons,which may account for the genome plasticity and genetic exchange between the plasmid and chromosome.The YBT-1520 chromosome encods 32 putative two-component regulatory systems(5 genes involved in pathogenicity), 59 genes regulated by the pleiotropic regulator PlcR(8 genes involved in pathogenicity) and 310 genes with regulator motifs.In metabolic competence,YBT-1520 has a reduced capacity for sugar utilization and the polysaccharides which can be degraded is limited to glycogen and starch,chitin and chitosan.It lacks mannose,arabinose and rhamnose pathways and also lacks the key enzymes of inositol degradation pathway.In contrast, YBT-1520 encods abundant proteolytic enzymes,peptide and amino acid transporters and enzymes for amino acid metabolism,which is common to the B.cereus group,suggesting that their ancestor should be an opportunistic insect pathogen rather than a benign soil bacterium.We also performed analysis of the specific genes for YBT-1520,most of which are associated with replication and repair,transcription and amino acid metabolism.There is a urease operon specific for YBT-1520,which may be related to its adaptation to the protein-rich environment.The most important plasmid pBMB293 harbors a pathogenicity island consisting of five insecticidal crystal proteins(Cry1Aa,Cry1Ia,Cry2Aa andCry2Ab),one vegetative insecticidal protein(Vip3A)and one haemolytic enterotoxin operon.Comparative analysis identified a high conserved core region among pBMB293,pb10987 and pPER272, suggesting that these plasmids might have evolved from a common ancestral plasmid and formed a unique plasmid family,pBMB293 carries 12 genes related to regulation, including 2 Rap-Phr regulators for the sporulation process.There are 2 putative PlcR-binding motifs,which harbor downstream of the enterotoxin operon and the conserved hypothetical protein ORF8.Plasmid pBMB137 encodes a MerR regulator responding to environmental stimuli.The IS,such as Tn4430,Tn5044 and IS homolog to Geobacillus thermodenitrificans NG80-2p facilitate the genetic exchange for pBMB137. Plasmid pBMB137 encodes several genes involved in conjugation.The presence of nine plasmids could account for the genome plasticity and high pathogenicity for YBT-1520.更多还原