广谱拮抗菌阴沟肠杆菌B8菌株拮抗相关基因片段的克隆及序列分析
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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。
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 whole
genes of B fragment and F fragment ,and to reveal the antagonistic molecular mechanism of B8.
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。
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 whole
genes of B fragment and F fragment ,and to reveal the antagonistic molecular mechanism of B8.
引文
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4．莫照兰．弧菌拮抗菌的筛选．青岛海洋大学学报．2001，31(2)：225～231
5．乳酸杆菌细菌素防治幼畜腹泻上的应用试验．现代化农业．1990，14～15
6. Cook R.J,et al. Molecular mechannism for biolgical control of plant pathogens,Phytopathology, 1995,31:53～80
7. Davies J.E. What are antibiotics?Achaic functions for modern activities,Mol.Microbiolgy ,1990,4:1227～1232.
8．许长蔼：荧光假单孢菌的生防改良．天津农业科学．2001，(7)：4～7
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14．陈卫良．抗水稻白叶枯病菌拮抗物质的研究．浙江大学博士论文．杭州，2001年
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16．黄海宁．用转座子标鉴法克隆阴沟肠杆菌B8对白叶枯病菌拮抗相关基因片段．浙江农业大学硕士论文，杭州1997年
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38． Auerswald E.A. Structural analysis of Tn5 Cold Spring Harbsymb.Quant. Biol,1981,45:107-113
39． Baan R.A,et al. Specifiv in situ cleavage of 16S ribosomal RNA of Escherichia coli interferes with the functional of initiation factor IF-I.Proc Natl Acad Sci US A,1976 73:702-706
40． Berg D.E.et The prokayotiv transpsable element Tn5． Biotechnology, 1983,4:4170-4350
41． Bimboim H.C. A rapid alkline extraction method for the isolation of plasmid DNA.Meth.Enzymol ,1983,100:243-255
42． Davagnino J.et. The DNA replication inhibitor microcin B17 is a rofty-three-amino-acid protein containing sixty percent glycine.Bacteriol,1998,167:439-446
43． De lorenzo.V,et al. Mini-Tn5 Transposon derivatives for insertion mutagenesis, promoter probing and chromosomal insertion of cloned DNA in gram-negative bacteria.J. Bacterio,1990,172:6568-6572
44． Frey J.et. Physical and genetic analysis of the col D plasmid. J.Bacteriol.1986,166(1) :15-19
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46． Handlhan D,et. Studies on transformation of Escherichia coli with plasmid J.Mol.Biol, 1983,166: 557
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2．吴健生，王金生．植物病原菌的细菌素．微生物通报．1996，23：95～97
3．SA30蜡样芽孢杆菌在体外对几种致病菌的生物拮抗试验．中国兽医杂志．1994，(20)2：12～14
4．莫照兰．弧菌拮抗菌的筛选．青岛海洋大学学报．2001，31(2)：225～231
5．乳酸杆菌细菌素防治幼畜腹泻上的应用试验．现代化农业．1990，14～15
6. Cook R.J,et al. Molecular mechannism for biolgical control of plant pathogens,Phytopathology, 1995,31:53～80
7. Davies J.E. What are antibiotics?Achaic functions for modern activities,Mol.Microbiolgy ,1990,4:1227～1232.
8．许长蔼：荧光假单孢菌的生防改良．天津农业科学．2001，(7)：4～7
9．Vondohoren H.et al.Multifunction peptide synthetase,Chem.rev,1997,97:2676～2705
10．方定一．仔猪大肠杆菌病的防治研究概况．家畜传染病．1980，(1)：66～73
11．何明清．仔猪黄痢及其生物制剂预防研究．四川农业科技．1980，(5)：26～29
12. Cheetham BF, Katz ME. A role for bacteriophages in the evolution and transfer of bacterial virulence determinants.Mol Microbiol, 1995, 18:201～208
13. Ladha J.K. Isolation and identification of nitrogen fixing Enterobacter cloacae and Klebsiella planticola associated with rice plants. Can. J.Microbiol, 1983,29:1301～1308.
14．陈卫良．抗水稻白叶枯病菌拮抗物质的研究．浙江大学博士论文．杭州，2001年
15. Barefoot S.F.et. Detection and activity of lactacto bacteriocin produced by Lactobacillus alidophilus.App.Ewviron. Microbiol, 1983,45:1808～1815
16．黄海宁．用转座子标鉴法克隆阴沟肠杆菌B8对白叶枯病菌拮抗相关基因片段．浙江农业大学硕士论文，杭州1997年
17. Blum G. Ott M,Lischewski A,Ritter A,Imrich H,Tschape H, Hacher J. Excision of large DNA regions termed pathogenicity islands from tRNA-specific locin the chromosome of an Escherichia coli wild-type pathogen. Infect. Immun. 1994,62: 606～614
18. Hentschel U,Steinert M,Hacher J.Common mechanisms of symbiosis and pathogenesis. Trends Microbiol. 2000,228:226～231
19. Ochman H, Lawrence J.G,Groisman E.A. Lateral gene transfer and the nature of bacterial innovation.Nature ,2000,405:299～304
20. Hacher J, Kaper J.B. Pathogenicity islands and the evolution of microbes, Ann. Rev.microbial, 2000,54:641～679
21. Hacker J,kaper J.B (Eds.). Pathogenicity islands and other mobile virulence determinants,ASM Press. Washing DC, 1999,1～352
22. Hacker J, Blum-Ochler G.muhldorfer I,Tschape H. Pathogenicity islands of virulent bacteria Stucture ruction and impact on microlbial evolution. Mol.Microbiol, 1997,23: 1089～1097
23. Projan S, Novick R, Archer. G, Crossley K.(Eds). Staphylococci in Human disease,Churchill Livingstone. New York, 1997,55～81
24. Betley M.J, Borst D.w, Regassa L.B. Staphylococcal enterotoxins, toxic shock syndrome toxin and streptococcal pyrogenic exotoxins:a comparative study of their molecular biology. Chem.Immunol, 1992,55:1～35
25．康白，何明清．蜡样芽孢杆菌DM423对羔羊、仔猪、鸡腹泻控制的流行病学分析．中国微生态学杂志．1992，4(4)：40～48
26．张冬梅．小肠结肠炎耶尔森氏菌强毒力岛的结构与功能．国外医学-流行病学传染病分册，2000，2：109～112
27．梁荣．动物微生态理论与应用．动物医学进展，1997，18(1)：38～42997，(6)9～10
28．解志刚．枯草杆菌与活菌制剂，微生物学杂志．1996，16(2)48～50
29．陈代荣．何明清．调痢生治疗家兔肠炎的研究．中国畜禽传染病．1996，2：34～35
30．何明清．我国动物微生态制剂的起源、发展战略几应用前景．中国微生态学杂志．2001，13(3)：174～176
31．易发平．微生态制剂的理论基础及其应用现状．中国家禽．2001，23(10)：12～13
32．叶嗣颖．医学微生物中的生态学问题．中国微生态学杂志．1998，10(4)：253～256
33．雷祚荣．细菌毒素分子生物学．北京：中国科学技术出版社1993
34．徐建国．毒力岛和细菌毒力的进化．中华微生物学和免疫学杂志．1999，2：169～171
35． J.萨姆布鲁克等著,金冬雁等译.分子克隆实验指南(第二版).北京:科学出版社,1995
36． Ahmed A. The revised nucleotide sequence of Tn5． gene.l995,154:129-130
37． Akuydu A.H, Masaki and T.Ohta. Molecular structure and immunity specificty of colicin E6． and evolutionary intermediate between E-group colicins and cloacin DF13． J Bacteriol 1989,171:6430-6436
38． Auerswald E.A. Structural analysis of Tn5 Cold Spring Harbsymb.Quant. Biol,1981,45:107-113
39． Baan R.A,et al. Specifiv in situ cleavage of 16S ribosomal RNA of Escherichia coli interferes with the functional of initiation factor IF-I.Proc Natl Acad Sci US A,1976 73:702-706
40． Berg D.E.et The prokayotiv transpsable element Tn5． Biotechnology, 1983,4:4170-4350
41． Bimboim H.C. A rapid alkline extraction method for the isolation of plasmid DNA.Meth.Enzymol ,1983,100:243-255
42． Davagnino J.et. The DNA replication inhibitor microcin B17 is a rofty-three-amino-acid protein containing sixty percent glycine.Bacteriol,1998,167:439-446
43． De lorenzo.V,et al. Mini-Tn5 Transposon derivatives for insertion mutagenesis, promoter probing and chromosomal insertion of cloned DNA in gram-negative bacteria.J. Bacterio,1990,172:6568-6572
44． Frey J.et. Physical and genetic analysis of the col D plasmid. J.Bacteriol.1986,166(1) :15-19
45． Gould J.M, Gramer W. A. Studies on the depolarization of Escherichia coli cell menbrans by colicin E.J. Biol. Chen. 1977,252:5491-5497
46． Handlhan D,et. Studies on transformation of Escherichia coli with plasmid J.Mol.Biol, 1983,166: 557
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