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B contig。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anrP ORF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。
To reveal the antagonistic mechanism of B8 strain to Xanthomonas oryzae pv. oryzae, transposon tagging method and chromosome walking were deployed to clone antagonistic related fragments around Tn5 insertion site in the mutant strain B8B. The function of up-stream regulatory sequence of gene ‘admA’ involved in the antagonistic activity was further identified by gene knocking out technique. An antagonistic related left fragment of Tn5 insertion site, 2 608 bp in length, was obtained by tagging with Kan resistance gene of Tn5. A 2 354 bp right fragment of Tn5 insertion site was amplified with 2 rounds of chromosome walking. The length of the B contig around the Tn5 insertion site was 4 611 bp, containing 7 open reading frames (ORFs). Bioinformatic analysis revealed that these ORFs corresponded to the partial coding regions of glyceraldehyde-3-phosphate dehydrogenase, two LysR family transcriptional regulators, hypothetical protein VSWAT3-20465 of Vibrionales and admA, admB, and partial sequence of admC gene of Pantoea agglomerans biosynthetic gene cluster, respectively. Tn5 was inserted in the up-stream of 200 bp or 894 bp of the sequence corresponding to anrP ORF or admA gene on B8B, respec-tively. The B-1 and B-2 mutants that lost antagonistic activity were selected by homeologuous recombination technology in association with knocking out plasmid pMB-BG. These results suggested that the transcription and expression of anrP gene might be disrupted as a result of the knocking out of up-stream regulatory sequence by Tn5 in B8B strain, further causing biosythesis regulation of the antagonistic related gene cluster. Thus, the antagonistic related genes in B8 strain is a gene family similar as andrimid biosynthetic gene cluster, and the upstream regulatory region appears to be criti-cal for the antibiotics biosynthesis.
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