【作者】 刘小锦；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 博士

【摘要】 苏云金芽胞杆菌(Bacillus thuringiensis)菌株YBT-1520的全基因组测序结果表明它含有1条染色体和11个质粒。毒素抗毒素系统(TA系统)最初是在大肠杆菌的低拷贝大质粒上发现的,此后的研究多集中于G~-细菌的大质粒上,对G~+的质粒尤其是小质粒的研究非常少。本研究从NCBI数据库中收集了562条毒素或者抗毒素蛋白质的序列,通过穷举比对在YBT-1520基因组上发现了多种TA系统。1.质粒pBMB8240上潜在TA系统kyAB的研究本研究在YBT-1520菌株中发现了新质粒pBMB8240上有两个邻近的开放读码框,分别命名为KyA和KyB蛋白,通过序列分析,发现KyA和KyB分别与已知抗毒素、毒素蛋白有序列一致性。KyA和kyB基因的克隆实验表明kyA基因很容易克隆;而多次努力克隆到的kyB基因区域都发生了基因突变。KyAB系统装载到不稳定的载体pBMB0631上去之后发现重组质粒的稳定性大大升高。以上结果表明kyAB系统是一个潜在的TA系统,并且能够增强不稳定质粒的稳定性。这是第一个克隆到大肠杆菌中的来自苏云金芽胞杆菌的质粒TA系统。2.质粒pBMB7635上潜在TA系统kyCD的研究本研究发现YBT-1520中的质粒pBMB7635与本实验室以前克隆的质粒pBMB9741有很高的相似性,对pBMB7635重新测序,并将质粒pBMB9741重新命名为pBMB7635。质粒pBMB7635上有两个邻近的开放读码框,与已经鉴定的TA系统有较高相似性,将这两个分别编码94和133个氨基酸大小的蛋白质分别命名为KyC和KyD蛋白。KyC、kyD和kyCD系统及临近区域的克隆实验表明kyC基因很容易克隆;而多次努力克隆kyD基因和kyCD系统及临近区域只得到少量转化子,而且这些转化子中kyD区域都发生了基因突变,表明kyCD很可能是TA系统,且不能在大肠杆菌中克隆。3.质粒pBMB2062的克隆和分析克隆并分析了菌株YBT-1520中的多拷贝小质粒pBMB2062,序列分析表明,pBMB2062含有2个ORFs(大于75个氨基酸),缺失实验证明ORF1(289aa)对于质粒在芽胞杆菌中的复制和稳定是必需的,ORF2则不是。研究随机选取了7个不同血清型菌株的小质粒进行了克隆和测序,发现这些小质粒的序列高度保守。斑点杂交实验表明该质粒在目前鉴定的84个Bt血清型菌株中的分布为41%。4.YBT-1520染色体上mazEF系统的克隆和分析在YBT-1520染色体上找到一对mazEF系统,本实验对mazEF系统进行了敲除突变,改变MazEF的表达水平来探索MazEF的表达与PCD之间的的相互作用,从而进一步研究mazEF的功能。研究发现,敲除了mazEF系统的菌株在45℃培养4天之后,菌体量逐渐减少,取样镜检发现菌体大部分死亡。从另外一个角度证明了染色体上的mazEF系统在PCD中起作用。本实验通过基因组间上TA系统的比较分析,为进一步研究质粒在不同菌株(包括其它芽胞杆菌群菌株)的分布、进化等提供一定的遗传信息和理论依据,对多拷贝小质粒的稳定机制以及苏云金芽胞杆菌中隐蔽型质粒的功能提出了新的认识。更多还原

【Abstract】 Bacillus thuringiensis strain YBT-1520,which is highly toxic to lepidopteran pests, was isolated by our lab.In this thesis,we reported the sequence determination and initial analysis of the genome of this strain.The results revealed that this genome consisted of one chromosome and nine plasmids.TA systems have been extensively studied on low-copy-number plasmids in Gram-negative bacteria as addiction systems because of their ability to maintain the plasmids during cell division and a few TA systems have been described on small plasmids in Gram-positive bacteria.We identified 562 TA loci belonging to the seven known TA gene families and present the results from an exhaustive search for TA loci in the completely sequenced Bacillus genomes.The putative TA loci from YBT-1520 genomes were studied and the putative TA loci from other 6 completely sequenced Bacillus genomes were found by bioinformatics analyse.1.The study on the putative kyAB TA system from plasmid pBMB8240Based on the shotgun sequence analysis of the strain YBT-1520 plasmid pBMB8240 was found to be a novel cryptic plasmid.In pBMB8240,two contiguous orfs(this gene pair was tentatively named kyAB for strain kurstaki YBT-1520) were noticed.The putative protein KyA and KyB displays sequence similarities with other antitoxin and toxin protein,respectively.Attempts to clone the kyA in E.coli DH5αwere easy and successful.In kyB cloning case,all recombinants harbored mutations in the kyB gene,suggesting the toxic activity of KyB protein in E.coli.Based on sequence homology with other toxin-antitoxin system genes and the lethal activity of KyB in Escherichia coli,the segregational stability kyAB cassette was identified to be the first functional putative TA segregational stability system from B. thuringiensis.2.The study on the putative kyCD TA system from plasmid pBMB7635A database sequence search revealed that the B.thuringiensis H1.1 plasmid pGI1 (8254 bp) had DNA sequences and organizations similar to plasmid pBMB9741 except for a fragment of 1.6 kb located upstream from the rep-gene.We designed a pair of contiguous primers to verify the DNA sequence upstream from the rep-gene.Here we named the ’new’ plasmid as pBMB7635(7635 bp).Two orfs(orf94 and orf133) present in pBMB7635 but absent in pBMB9741. ORF94 shares 100%amino acid identity with the TasA putative antitoxin protein of pGI1, while its neighbor ORF133 shares 98%amino acid identity with the TasB putative toxin protein of pGI1.The orf94/orf133 system is denoted kyCD system.Attempts to clone the kyC in E.coli DH5αwere easy and successful,while continuing efforts to clone kyD gene and kyCD system in E.coli DH5αhave not yet been successful,suggesting the toxic activity of KyD protein,suggesting the KyD protein failed to inhibit the lethal activity of the cognate toxin and kyCD system appeared to be functional but unregulated in E.coli.3.Plasmid pBMB2062 from YBT-1520 strain was cloned and characterizedA multicopy plasmid pBMB2062 from Bacillus thuringiensis subsp,kurstaki YBT-1520 strain was cloned and characterized by dot-blot analysis with the ORF1 fragment as a probe,and it was found to be present in 41%B.thuringiensis strains.The sequences of 7 pBMB2062-like plasmids from randomly selected B.thuringiensis strains (positive signal in the dot-blot analysis) were highly conserved.Two orfs,orf1 and orf2, were present in this plasmid.Orf1 was found to be necessary for plasmid replication, whereas Orf2 did not play a role in replication or stability.Based on its sequence homology,ORF2 was putative solitary antitoxin belonged to MetJ/Arc/CopG family.4.The mazEF system from YBT-1520 chromosome was cloned and characterizedBased on the sequences homology with other mazEF toxin-antitoxin systems,a novel mazEF system was identified on the YBT-1520 chromosome.To investigate the role of mazEF system in Bacillus thuringiensis,the mazEF system on B.thuringiensis strain BMB171 was knocked out.The conclusion was that the mazEF system was involved in the PCD of the B.thuringiensis strain BMB171.更多还原