Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1  

作　　者：LI Feng LI Ying JIANG Wei WANG ZhenFang LI JiLun 

机构地区：[1]State Key Laboratory for Agro-biotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China [2]School of Life Science, Anhui Key Laboratory of Plant Resources and Biology, Huaibei Coal Industry Teachers' College, Huaibei 235000, China

出　　处：《Chinese Science Bulletin》2009年第9期1522-1528,共7页

基　　金：Supported by National Natural Science Foundation of China (Grant No. 30570023);Scientific Research Project of Huaibei City, Anhui Province (Grant No. 070114)

摘　　要：A magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which had homology with the protein encoded by ORF1, were the cation transporter. Transmembrane domain analysis showed that the protein encoded by ORF1 contained four trans-membrane domains. It may be a transmembrane protein. The protein encoded by ORF1 contained two putative conserved domains: COG0053 and PRK09509. The MMT1 and FieF, containing conserved do-mains COG0053 and PRK09509 too, were Fe2+ transporter (cation diffusion facilitator superfamily). It was suggested that the protein encoded by ORF1 might take part in the magnetosomes biosynthesis as Fe2+ transporter.A magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which had homology with the protein encoded by ORF1, were the cation transporter. Transmembrane domain analysis showed that the protein encoded by ORF1 contained four transmembrane domains. It may be a transmembrane protein. The protein encoded by ORF1 contained two putative conserved domains： COG0053 and PRK09509. The MMT1 and FieF, containing conserved domains COG0053 and PRK09509 too, were Fe^2＋ transporter （cation diffusion facilitator superfamily）. It was suggested that the protein encoded by ORF1 might take part in the magnetosomes biosynthesis as Fe^2＋ transporter.

关 键 词：Tn5诱变 科学研究 缺失突变 序列分析 磁小体 海洋 迷你 克隆 

分 类 号：Q754[生物学—分子生物学] TQ466.3[化学工程—制药化工]