2005 Fiscal Year Final Research Report Summary
ANALYSIS OF TRANSCRITIONAL REGULATORY NETWORKS IN ASPERGILLUS ORYZAE USING REGULATORY MUTANT LIBRARY AND DNA MICROARRAY
| Project/Area Number |
15380055
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
GOMI Katsuya Tohoku University, Graduate school of Agricultural Science, Professor, 大学院農学研究科, 教授 (60302197)
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| Co-Investigator(Kenkyū-buntansha) |
ABE Keietsu TOHOKU UNIVERSITY, Graduate school of Agricultural Science, Associate Professor, 大学院農学研究科, 助教授 (50312624)
MACHIDA Masayuki NATIONAL INSTITUTE OF ADVANCED SCIENCE AND TECHNOLOGY, TEAM LEADER, 生物機能工学研究部門, 主任研究官 (30358006)
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| Project Period (FY) |
2003 – 2005
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| Keywords | Aspergillus oryzae / transcription factor / conditional overexpression / gene disruption / non-homologous end joining / homologous recombination / DNA microarray |
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| Research Abstract |
1.Disruption of the genes that each encodes Ku70 and DNA ligase IV homolog (LigD) involved in the non-homologous end joining (NHEJ) has been carried out in Aspergillus oryzae. In ku70/ku80 mutant, we sometimes failed to obtain an expected gene disruptant, probably dependent on the gene of interest. On the contrary, gene replacement of the pepA gene encoding an aspartic protease and its regulatory gene, prtR, using A.nidulans sC gene as a selectable marker resulted in 100% of gene targeting frequency in the ligD disruptant. In addition, gene replacement of five MAP kinase genes found in A.oryzae genome database also showed the targeting rates as high as 100%. Consequently, the ligD deletion mutants are quite excellent tools for gene targeting in A.oryzae.
2.We have constructed a mutant library of conditional overexpression of transcription factors using a-amylase gene (amyB) promoter that directed an overexpression of the gene of interest in the presence of maltose. At first as a pilot s
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cale construction 40 transcription factors were selected, and then the rest 200 regulatory genes have been overexpressed.
3.Based on the plate assay for protease activity among the conditional overexpression mutants constructed above, a putative transcription factor involved in expression of extracellular proteolytic genes was identified. Expression analysis using an oligonucleotide microarray covering 12,000 genes of A.oryzae showed that overexpression of this gene (prtR) resulted in enhanced expression of extracellular proteolytic genes, including aspartic, neutral, and alkaline proteases as well as carboxypeptidases and aminopeptidase.
4.A transcriptional regulator gene, malR, with a typical zinc finger motif at N-terminus is located at downstream of the malP-malT cluster in which each gene encodes maltose permease and maltase, respectively. Disruption analyses of the malP and malR genes in A.oryzae showed that the malR is responsible for efficient expression of the amylolytic genes such as α-amylase through the malP function by which maltose is incorporated into the cell. Less
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Research Products
(4 results)
