【作者】 姚学萍；

【导师】 胡永浩； 余旭平；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2002， 硕士

【摘要】 以广谱拮抗菌阴沟肠杆菌B8菌株和拮抗活性缺失菌株B8B、B8F及从B8B和B8F二菌株克隆获得的重组质粒pB、pF为基础，对阴沟肠杆菌B8菌株拮抗相关的B和F基因片段进行序列分析。结果显示，F片段长度为728bp，与现有生物数据库的BLAST比较分析，发现该序列仅有局部短于1OObp的区域与polyketide合成酶基因或与脂肪酸合成酶基因有低的同源性，推测为一新基因；B片段长约4kb，序列拼接结果推测靠近Tn5插入位点部位有重复序列，对B片段Tn5远端的部分序列进行BLAST比较，发现它与小肠结肠炎耶尔森氏菌的强毒力岛有一定的同源性。 鉴于B片段的BLAST分析结果，对B8、B8B和B8F菌株进行了动物试验，测定各菌株毒力。结果显示，B8和B8F具有弱的一过性毒力，而B8B菌株无毒力。 试验研究设计并合成了由40和44个碱基的寡聚脱氧核苷酸组成的染色体爬行接头，在接头序列和测定的F片段近Tn5的序列上，设计了2对染色体爬行用的PCR引物，从B8菌株中提取基因组DNA，BamHI酶切，与染色体爬行接头连接，依次用2对引物进行PCR，扩增出239bp产物，经克隆、测序，发现其中18bp为扩增的相应于F片段在B8F菌株Tn5插入位点对面的序列，其余则为F片段728bp序列的一部分，为进一步进行染色体爬行，克隆和测定整个B和F基因，揭示阳菌株的拮抗分子机制提供了技术资料贮备。更多还原

【Abstract】 Using Enterobacter cloacae B8, the mutated strains B8B and B8F, and the recombinant clones pB and pF, we try to sequence the antagonistic-related genes of Enterobacter cloacae B8 by subcloning and Genome Primering System.The acquired sequences were analyzed with BLAST Program to find any homology to sequences deposited in Genebank. The results showed that the F fragment ,728bp in length, could be a new gene with a little homology to the genes coding for polyketide synthetase or fatty-acid synthetase and the B fragment, about 4kb in length, is inferred to have repeat sequences around Tn5 insertion site,in which there is homology to the WA 314 right arm of the high-pathogeniciry island of Yersinia enterocolitica.To reveal any pathogenicity of Enterobacter cloacae B8 and its mutated strains B8B and B8F to animals, the experiment with mice was carried out. The result showed that B8 and its mutated strain B8F had weak transient virulence but the mutated strain B8F had no virulence.The chromosome walking adapter consisting of 40nt and 44nt oligonucleotides was designed and synthesized. And the two pah’s of PCR primers that bind to the adapter and the sequence of F fragment close by Tn5 respectively were also designed. The genomic DNA of B8 was isolated, digested with BamH I, and ligated to the adapter.Using the two pairs of the primers ,two rounds of PCR were performed hi turn and a fragment of 239bp was amplified successfully.lt was proved by cloning and sequencing that 18bp of the fragment is the sequence opposite to F fragment on the left of Tn5 insertion site in B8F,the other is part of the 728 bp of F fragment. This result makes it possible to continue to carry out chromosome walking ,to clone and sequence the wholegenes of B fragment and F fragment ,and to reveal the antagonistic molecular mechanism of B8.更多还原