ANALYSIS OF TRANSCRITIONAL REGULATORY NETWORKS IN ASPERGILLUS ORYZAE USING REGULATORY MUTANT LIBRARY AND DNA MICROARRAY

1.米曲霉中涉及非同源末端连接(NHEJ)的Ku70和DNA连接酶IV同源基因(LigD)的破坏。在ku70/ku80突变体中，我们有时无法获得预期的基因中断，这可能取决于目的基因。相反，编码天冬氨酸蛋白酶的PepA基因及其调控基因prtR的基因置换，使LigD干扰物的基因打靶频率达到100%。此外，对米曲霉基因组数据库中发现的5个MAPK基因进行了基因替换，其靶向率也高达100%。2.我们利用α-淀粉酶基因启动子构建了转录因子条件性过表达突变文库，该基因启动子在麦芽糖存在的情况下引导目的基因的过表达。最初作为飞行员S…筛选出了40个以上的转录因子，其余200个调控基因已经过表达。3.在以上构建的条件性过表达突变体中，通过平板法检测其蛋白酶活性，确定了一个可能参与胞外蛋白分解基因表达的转录因子。利用涵盖12,000个米曲霉基因的寡核苷酸芯片进行的表达分析表明，该基因(PrtR)的过表达导致胞外蛋白分解基因的表达增强，包括天冬氨酸、中性和碱性蛋白水解酶以及羧肽酶和氨基肽酶。4.转录调控基因MalR位于MALP-MALT簇的下游，每个基因分别编码麦芽糖渗透酶和麦芽糖酶。对米曲霉MALP和MARR基因的中断分析表明，MARR通过麦芽糖被结合到细胞内的MALP功能，负责α-淀粉酶等淀粉酶基因的高效表达。较少

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 … More 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