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

1.在米曲霉（Aspergillus oryzae）中，编码Ku70和参与非同源末端连接（NHEJ）的DNA连接酶IV同源基因（LigD）分别被破坏。在ku70/ku80突变体中，我们有时无法获得预期的基因干扰物，可能依赖于感兴趣的基因。相反，如果将编码天冬氨酸蛋白酶及其调控基因prtR的pepA基因作为选择性标记替换，则在ligD干扰物中获得100%的基因靶向频率。此外，对A.oryzae基因组数据库中发现的5个MAP激酶基因进行基因置换，靶率也高达100%。因此，ligD缺失突变体是m.o ryzae中非常好的基因靶向工具。我们利用a-淀粉酶基因（amyB）启动子构建了一个转录因子条件过表达的突变文库，该启动子在麦芽糖存在的情况下引导感兴趣的基因过表达。初步筛选了40个转录因子，并对其余200个调控基因进行了过表达。基于上述构建的条件过表达突变体的蛋白酶活性平板分析，确定了一个可能参与细胞外蛋白水解基因表达的转录因子。利用覆盖了a.m oryzae 12,000个基因的寡核苷酸芯片进行表达分析表明，该基因（prtR）的过表达导致细胞外蛋白水解基因的表达增强，包括天冬氨酸、中性和碱性蛋白酶以及羧肽酶和氨基肽酶。转录调节基因malR位于malP-malT簇的下游，其n端具有典型的锌指基序，每个基因分别编码麦芽糖渗透酶和麦芽糖酶。对m.o ryzae中malP和malR基因的破坏分析表明，malR通过malP功能负责α-淀粉酶等淀粉水解基因的有效表达，麦芽糖通过malP功能被纳入细胞。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