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

1.已在黑曲霉中进行了各自编码Ku 70和参与非同源末端连接（NHEJ）的DNA连接酶IV同源物（LigD）的基因的破坏。在ku 70/ku 80突变体中，我们有时未能获得预期的基因破坏物，这可能取决于目的基因。相反，使用构巢曲霉sC基因作为选择标记，编码天冬氨酸蛋白酶的pepA基因及其调控基因prtR的基因置换导致ligD破坏物中100%的基因靶向频率。此外，在A. peculiata基因组数据库中发现的5个MAP激酶基因的基因置换也显示出高达100%的靶向率。因此，ligD缺失突变体是非常好的工具，在水稻基因打靶。2.我们已经构建了一个条件性过表达的转录因子的突变体库，使用α-淀粉酶基因（amyB）启动子，指导目的基因在麦芽糖存在下的过表达。起初作为一名飞行员 ...更多信息 3.通过平板蛋白酶活性测定，筛选出一个与胞外蛋白水解基因表达相关的转录因子。使用覆盖12，000个A. pasta基因的寡核苷酸微阵列进行的表达分析表明，该基因（prtR）的过表达导致胞外蛋白水解基因的表达增强，所述胞外蛋白水解基因包括天冬氨酸蛋白酶、中性蛋白酶和碱性蛋白酶以及羧肽酶和氨肽酶。在malP-malT簇的下游分别编码麦芽糖通透酶和麦芽糖酶。对A.a.中的malP和malR基因的破坏分析表明，malR负责通过malP功能将麦芽糖掺入细胞中来有效表达淀粉分解基因如α-淀粉酶。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