Hisada Hiromoto, Sano Motoaki, Ishida Hiroki, Hata Yoji, Abe Yasuhisa, Machida Masayuki
Research Institute, Gekkeikan Sake Co. Ltd. 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8361, Japan.
Appl Microbiol Biotechnol. 2006 Oct;72(5):1048-53. doi: 10.1007/s00253-006-0388-4. Epub 2006 Mar 18.
The manganese superoxide dismutase gene (sodM) is very highly expressed in Aspergillus oryzae. To elucidate the basis for this high-level expression, deletion analysis of the promoter was undertaken using beta-glucuronidase (GUS) as a reporter. Deletion of a 63-bp sequence from -200 to -138 in the 1,038-bp sodM promoter caused a drastic decrease in GUS activity. In addition, an electrophoretic gel mobility shift assay (EMSA) implicated a 30-bp element from -209 to -178 containing cis-element(s) in the high-level expression. The results of fine structure deletion analysis of this region were consistent with the EMSA results. To confirm these findings, we constructed enhanced sodM promoters by incorporating tandem repeats of this region, which resulted in an approximate twofold increase in expression relative to the native sodM promoter.
锰超氧化物歧化酶基因（sodM）在米曲霉中表达水平非常高。为了阐明这种高水平表达的基础，我们以β-葡萄糖醛酸酶（GUS）作为报告基因对启动子进行了缺失分析。在1038 bp的sodM启动子中，从-200至-138缺失一段63 bp的序列导致GUS活性急剧下降。此外，电泳凝胶迁移率变动分析（EMSA）表明，从-209至-178的一段30 bp元件含有与高水平表达相关的顺式元件。该区域精细结构缺失分析的结果与EMSA结果一致。为了证实这些发现，我们通过并入该区域的串联重复序列构建了增强型sodM启动子，相对于天然sodM启动子，其表达量增加了约两倍。
