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Functional analysis of chitinases of Aspergillus nidulans

构巢曲霉几丁质酶的功能分析

基本信息

批准号：
10660074
负责人：
HORIUCHI Hiroyuki
金额：
$ 2.24万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660074/
关键词：
Aspergillus chitinase tip growth autolysis

项目摘要

Chitin is a β-1, 4-linked polymer of N-acetyl-D-glucosamine (GlcNAc) and is one of the major cell wall components of many filamentous fungi. We cloned two chi genes (chiA and chiB) encoding chitinase-like proteins from Aspergillus nidulans. chiA gene encodes a protein of 961 amino acids which is homologous to fungal-type chitinases and has secretary signal-like sequence and long Ser/Thr/Pro rich sequence at its N- and C-termini, respectively. chiB encodes a protein of 416 amino acids homologous to bacterial-type chitinases. Disruptants of chiA could not grow on the medium containing 0.01% of SDS in the pyrGィイD1+ィエD1 background. Moreover, severe growth defects were observed under low osmotic condition in the pyrGィイD1-ィエD1 background even when uridine was added to the medium at the final concentration of 10 mM. Hyphal morphology of the double mutant of chiA and pyrG was aberrant and chitin distribution detected by Calcofluor white staining was abnormal under that condition. Lysis of hyphae at sub apical region was occasionally detected. On the other hand, chiB disruptants exhibited almost the same morphological phenotypes as the wild-type strain under various conditions. In the chiB-null mutant, however, the intracellular and extracellular chitinase activities were considerably reduced. Decrease in hyphal dry weight during autolysis occurred more slowly in the mutant than in the wild-type strain. Western blot analysis using anti-ChiB antiserum indicated that the synthesis of ChiB was largely induced when mycelia were transferred to media lacking carbon sources, a condition which induced hyphal autolysis. From these results, it is suggested that chiB plays a role in the process of autolysis in A. nidulans. Thus, it is suggested that the product of chiA is very important for formation of hyphae under some conditions, while contribution of that of chiB to it is small.

几丁质是n -乙酰- d -氨基葡萄糖（GlcNAc）的β- 1,4键聚合物，是许多丝状真菌细胞壁的主要成分之一。我们克隆了两个编码几丁质酶样蛋白的chi基因（chiA和chiB）。chiA基因编码一种961个氨基酸的蛋白质，与真菌型几丁质酶同源，其N端和c端分别具有秘书信号样序列和长Ser/Thr/Pro富序列。chiB编码一种由416个氨基酸组成的蛋白质，与细菌型几丁质酶同源。Disruptants贾无法生长的介质包含0.01%的SDS pyrGィイD1 +ィエD1背景。此外，在低渗透条件下，在pyrG γ γ γ D1- γ γ γ γ D1背景下，即使在终浓度为10 mM的培养基中加入尿苷，也观察到严重的生长缺陷。在这种条件下，chiA和pyrG双突变体的菌丝形态异常，calcofluewhite染色检测到的几丁质分布也异常。在亚根尖区偶见菌丝裂解。另一方面，chiB干扰素在不同条件下表现出与野生型菌株几乎相同的形态表型。然而，在chiB-null突变体中，细胞内和细胞外几丁质酶活性显著降低。突变体菌丝干重在自溶过程中的下降速度比野生型菌株慢。Western blot分析表明，将菌丝转移到缺乏碳源的培养基中，诱导菌丝自溶，大量诱导了ChiB的合成。这些结果表明，chiB在白莲的自溶过程中发挥了作用。因此，在某些条件下，chiA的产物对菌丝的形成有重要作用，而chiB的产物对菌丝的形成贡献较小。