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发挥着重要作用。本课题以taka -淀粉酶A基因为模式基因，在分子水平上分析丝状真菌基因诱导表达的转录激活因子和抑制因子，以期有效利用丝状真菌基因。我们克隆并确定了米曲霉JCM02239的两个基因组基因的核苷酸序列和编码Taka-amylaseA的cDNA。以空心拟木糖为中间寄主，研究了taka -淀粉酶A基因的表达特性，发现CCAAT序列和CREA蛋白在启动子区识别的顺式元件可能调控taka -淀粉酶A基因的表达。本项目发现，除了taka -淀粉酶A基因外，CCAAT结合因子AnCP还能识别假单胞菌amdS和gatA基因的CCAAT序列，表明AnCP是假单胞菌的宽结构域调控蛋白之一。虽然我们一直试图通过西南杂交或利用人NF-YA基因结合区杂交等多种方法克隆出编码AnCP的基因，但AnCP基因尚未被克隆出来。同时，我们尝试用DNA亲和柱从细胞核中纯化AnCP因子。对于CREA抑制蛋白，已经对启动子区域的CREA结合序列进行了修饰，并用于转化假芽孢杆菌。在不久的将来，我们将得到有关CREA结合序列在碳分解代谢抑制中发挥重要作用的信息。此外，利用麦芽糖亲和柱分离出麦芽糖结合蛋白，检测到35和80kDa两个结合蛋白，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.