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Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8 被引量:1
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作者 Xu-Ping Yu Jun-Li Zhu +5 位作者 Xue-Ping Yao Shi-Cheng He Hai-Ning Huang Wei-Liang Chen Yong-Hao Hu De-Bao Li 《World Journal of Gastroenterology》 SCIE CAS CSCD 2005年第39期6152-6158,共7页
AIM: To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism.METHODS: Transposon-mediated mutagenesis and tagging method and cassette PCR-ba... AIM: To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism.METHODS: Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain(s) of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame (ORF) finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www. ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used.RESULTS: A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglornerans andrimid biosynthetic gene cluster (AY192157). The Tn5was inserted at 2 420 bp of the gene corresponding to the COG3319 (the thioesterase domain of type Ⅰ polyketide synthase) coding region on B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F.CONCLUSION: The anrFgene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs. 展开更多
关键词 anrF基因 异体同形 染色体 遗传因子
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阴沟肠杆菌B8拮抗活性基因‘admA’及上游调控序列的克隆与功能鉴定 被引量:1
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作者 朱军莉 李德葆 余旭平 《遗传》 CAS CSCD 北大核心 2012年第4期495-502,共8页
为了阐明水稻白叶枯病拮抗菌阴沟肠杆菌B8的作用机理,文章采用转座子标签法和染色体步移技术克隆到突变株B8B中Tn5插入位点周边拮抗活性相关片段,并通过基因敲除验证了获得的拮抗相关片段admA’上游调控序列的功能。以转座子中Kan抗性... 为了阐明水稻白叶枯病拮抗菌阴沟肠杆菌B8的作用机理,文章采用转座子标签法和染色体步移技术克隆到突变株B8B中Tn5插入位点周边拮抗活性相关片段,并通过基因敲除验证了获得的拮抗相关片段admA’上游调控序列的功能。以转座子中Kan抗性基因为标签,克隆了B8B菌株中Tn5插入位点左侧2 608 bp序列,经两次染色体步移得到Tn5插入位点右侧的2 354 bp序列。序列拼接后获得B8菌株拮抗相关序列4 611 bp的Bcontig。生物信息学分析显示该序列含有7个ORF,分别对应于3-磷酸甘油醛脱氢酶(GADPH)基因的部分编码区、2个LysR家族转录调控因子、弧菌假设蛋白VSWAT3-20465及成团泛菌(Pantoea agglomerans)andrimid生物合成基因簇的admA、admB和部分admC基因序列。B8B菌株Tn5插入分别位于同源于弧菌假设蛋白的anrPORF及‘admA’基因上游200 bp和894 bp处。通过同源重组技术,借助敲除质粒pMB-BG,获得拮抗活性消失的突变株B-1和B-3。结果表明B8B突变株中Tn5的插入可能影响了anrP蛋白的转录和表达,进而调控拮抗物质编码基因簇的生物合成。B8菌株中拮抗物质相关基因是类似于andrimid生物合成基因簇的基因家族,其上游调控区对该抗生素的生物合成具有重要的作用。 展开更多
关键词 阴沟肠杆菌B8 拮抗机理 andrimid 染色体步移
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阴沟肠杆菌B8拮抗活性相关基因的克隆与分析
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作者 余旭平 朱军莉 +4 位作者 姚学萍 何世成 黄海宁 陈卫良 李德葆 《科学通报》 EI CAS CSCD 北大核心 2004年第12期1139-1144,共6页
为研究具有广谱拮抗活性的阴沟肠杆菌B8的拮抗机理,应用自杀性质粒pZJ25,将Tn5转座到B8的染色体上,筛选到2株拮抗活性丧失的菌株.选择其中B8F突变株,应用Tn5上的Kanr基因作为标签,对Tn5插入位点右侧的拮抗活性相关基因片段进行了克隆,... 为研究具有广谱拮抗活性的阴沟肠杆菌B8的拮抗机理,应用自杀性质粒pZJ25,将Tn5转座到B8的染色体上,筛选到2株拮抗活性丧失的菌株.选择其中B8F突变株,应用Tn5上的Kanr基因作为标签,对Tn5插入位点右侧的拮抗活性相关基因片段进行了克隆,筛选得到质粒pTLF,经亚克隆后测序,获得F片段735bp的序列.提取原始菌株B8基因组DNA,酶切后与PstⅠ接头连接,应用接头引物和根据F片段设计的特异引物,在F片段的左右两侧进行染色体步行,获得了F片段(Tn5插入位点)左侧的1106bp序列和F片段右侧的664bp序列.生物信息学分析显示,获得的B8菌株拮抗相关基因序列包含3个ORF,与Pantoeaagglomerans的andrimid生物合成基因簇(基因GenBank登录号AY192157)有较高的同源性,分别对应于admM,admN和admO共3个基因.被Tn5插入破坏的ORF(命名为anrF基因)对应于admM(Polyketide合酶基因),插入位点位于终止密码前214bp处.由此证明,anrF基因与B8菌株的拮抗活性相关,推测B8菌株产生的拮抗物质为andrimid. 展开更多
关键词 阴沟肠杆菌B8 拮抗机理 anrF基因 andrimid生物合成基因簇 基因克隆 生物防治
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Cloning and analysis of the antagonistic related genes of Enterobacter cloacae B8 被引量:2
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作者 YUXuping ZHUJunli +4 位作者 YAOXunping HEShicheng HUANGHaining CHENWeiliang LIDebao 《Chinese Science Bulletin》 SCIE EI CAS 2004年第13期1370-1375,共6页
To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonist... To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid. 展开更多
关键词 无性繁殖 拮抗机理 DNA 肠细菌学B8 生物控制 生物合成酶 药物残留
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