Takaya N, Yamazaki D, Horiuchi H, Ohta A, Takagi M
Department of Biotechnology, University of Tokyo, Japan.
Biosci Biotechnol Biochem. 1998 Jan;62(1):60-5. doi: 10.1271/bbb.62.60.
We cloned a chitinase-encoding gene from Aspergillus nidulans by polymerase chain reaction using degenerated oligonucleotide primers designed from the conserved amino acid sequences among chitinases from yeasts and Rhizopus spp. The cloned gene, named chiA, encoded a polypeptide consisting of 660 amino acids. Disruption of chiA had no effect on hyphal or conidiophore morphology, but germination frequency and hyphal growth rate decreased substantially. Expression of chiA was investigated using Escherichia coli beta-galactosidase as a reporter enzyme. The beta-galactosidase activity was present during hyphal growth and increased twice as the conidiophores developed. In situ staining of beta-galactosidase activity found high expression in metulae, phialides, and conidia during conidiophore development, indicating that the expression of chiA is developmentally regulated. This is the first report to isolate a chitinase gene from A. nidulans and investigate its functions using the gene disruption technique and gene fusion methods in filamentous fungi.
我们使用从酵母和根霉属几丁质酶的保守氨基酸序列设计的简并寡核苷酸引物，通过聚合酶链反应从构巢曲霉中克隆了一个编码几丁质酶的基因。克隆的基因名为chiA，编码一个由660个氨基酸组成的多肽。chiA的破坏对菌丝或分生孢子梗形态没有影响，但萌发频率和菌丝生长速率显著降低。使用大肠杆菌β-半乳糖苷酶作为报告酶研究了chiA的表达。β-半乳糖苷酶活性在菌丝生长期间存在，并在分生孢子梗发育时增加了两倍。β-半乳糖苷酶活性的原位染色发现在分生孢子梗发育期间，在梗基、小梗和分生孢子中有高表达，表明chiA的表达受发育调控。这是首次从构巢曲霉中分离几丁质酶基因并在丝状真菌中使用基因破坏技术和基因融合方法研究其功能的报告。
