Molecular biological characterization of inducible expression of filamentous fungal genes and its application

以米曲霉为代表的丝状真菌在日本发酵工业中扮演着重要的角色。本研究以Taka-淀粉酶A基因为模型基因，从分子水平上对丝状真菌中参与诱导表达的转录激活因子和转录抑制因子进行了研究，克隆并测定了两个基因组基因的核苷酸序列和米曲霉JCM02239中一个编码Taka-淀粉酶A的基因。利用根瘤拟青霉作为中间宿主，对其表达特性进行了研究，结果表明，启动子区域的CCAAT序列和CREA蛋白识别的顺式元件对多年生拟青霉的Taka-淀粉酶A基因的表达起调控作用。虽然我们一直试图通过各种方法克隆ANCP基因，如利用人NF-YA基因DNA结合区的西南杂交法等，但目前还没有克隆到ANCP基因。同时，我们尝试用DNA亲和层析柱从细胞核中分离纯化ANCP因子。在CREA阻遏蛋白方面，我们对启动子区域的CREA结合序列进行了修饰，并将其用于转化空腹拟青霉。在不久的将来，我们将获得哪些CREA结合序列对碳分解代谢抑制具有重要作用的信息。此外，利用麦芽糖亲和层析柱分离了麦芽糖结合蛋白，并检测到两种结合蛋白，即35 kDa和80 kDa蛋白，目前正在进行化学鉴定。

Filamentous fungi represented by Aspergillus oryzae have been playd important roles in Japanese fermentation industries. In this resarch project, we aimed to utilize genes efficiently in filamentous fungi by characterizing at the molecular levels transcriptional activator and repressor ptoteins involved in the inducible expression of filamentous fungal genes using Taka-amylase A gene as a model gene.We have cloned and determined the nucleotide sequences of two genomic genes and a cDNA encoding Taka-amylaseA from Aspergillus oryzae JCM02239. By using A.nidulans as an intermediate host, the expression properties of Taka-amylase A gene have been examined, which showed that CCAAT sequence and cis element recognized by CREA protein in the promoter region appeared to regulate Taka-amylase A gene expression.In this project, AnCP,CCAAT binding factor, has been shown to recognize CCAAT sequence of amdS and gatA genes of A.nidulans in addition to Taka-amylase A gene, which indicates AnCP in one of wide domain regulatory proteins in A.nidulans. Although we have been trying to clone the gene coding for AnCP by various methods such as South-Western or hybridization using DNA binding region of human NF-YA gene, AnCP gene has not been yet cloned. At the same time, we are trying to purify AnCP factor from nuclei by using DNA affinity column. As for CREA repressor protein, CREA binding sequences in the promoter region have been modified and uesd to transform A.nidulans. In near future, we will get information which CREA binding sequences are important to exert to carbon catabolite repression. Furthermore, maltose binding proteins have been isolated by using maltose affinity column and two binding proteins, 35 and 80kDa proteins, were detected which are now under chemical characterization.