摘要
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 agglomerans andrimid biosynthetic gene cluster （AY192157）. The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 （the thioesterase domain of type I polyketide synthase） coding region on BSF. 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 BS, and the antagonistic substances produced by B8 are andrimid and/or its analogs.
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.
基金
Supported by the National Natural Science Foundation of China,No. 39870034the National High Technology Research and Development Program of China, the 863 Program, No. 104-04-01-01the Major Project of Science and Technology Development of Zhejiang Province, No. 021102529
