mariner转座子pKKma的序列分析及转座性能

Characterization of a mariner transposon pK Kma

【目的】本研究针对携带mariner转座子的质粒pKKma,进行序列分析和功能注释。【方法】根据已知序列设计引物测定质粒序列。构建转座子突变文库,分析转座子转座效率。【结果】序列分析发现,质粒pKKma全长6879 bp,具有7个开放阅读框。其中,阅读框ORF6编码mariner转座酶(348 aa),属于mariner转座子Himar1转座酶的C9变种;pKKma有2个相同的27 bp的反向重复序列(inverted terminal repeats);阅读框ORF7为庆大霉素抗性基因aacC 1,位于转座子反向重复序列之间,与其它mariner转座子可转移序列比对发现,覆盖率仅为2.0%-47.7%,相应同源程度为3.2%-99.7%,可转移序列具有较大差异。转座效率分析显示,该转座子对于粘质沙雷氏菌的转座效率为(3.1×10-4)-(4.8×10-4),对于弗氏柠檬酸菌的转座效率为(1.3×10-3)-(1.7×10-3)。【结论】质粒pKKma携带一种新的mariner转座子,可在多种细菌中构建转座子文库,研究细菌基因的功能。

[Objective] This study was aimed at sequence analysis and function annotation of plasmid pKKma carrying a mariner transposon. [ Methods ] Primers were designed based on the partial known sequence and used for directly sequencing plasmid pKKma. Transposon mutagenesis libraries were constructed to analyze the mutagenesis efficiency of plasmid pKKma. [ Results] pKKma comprises 6879 bp with 7 open reading frames （ORFs）. Among them, ORF6 encodes a mariner transposase of 348 amino acids （aa） , a C9 variant of Himarl type transposase. Two inverted terminal repeats （ITRs） are identified and of 27 bp each. ORF7 encodes gentamycin resistance gene aacC1, locating between two ITRs. Transposable sequence alignment with other mariner transposons shows that the coverage is 2.0% -47.7% and the homology is 3.2% to 99.7%. The result indicates pKKma is significantly different from the other vectors with mariner transposon. The transposition efficiency is also analyzed. It＇ s （3.1 ×10^-4） - （4.8 ×10^-4） for S. marcescens and （ 1.3×10^-3） _ （ 1.7 ×10^-3） for C. freundii, respectively. [ Conclusion] pKKma carries a new mariner transposon and could be used to study the role of genes by constructing transposon libraries in bacteria.