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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-乙酰基-D-葡糖胺（GlcNAc）的β-1，4-连接的聚合物，并且是许多丝状真菌的主要细胞壁组分之一。我们从构巢曲霉中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961个氨基酸的蛋白质，与真菌类几丁质酶同源，N端和C端分别具有分泌信号样序列和富含Ser/Thr/Pro的长序列。chiB编码与细菌型几丁质酶同源的416个氨基酸的蛋白质。chiA的破坏菌不能在含有0.01%SDS的培养基上生长。此外，严重的生长缺陷，观察到在低渗透条件下的背景下，在pyrG pyrG D1-pyrG D1，即使当尿苷添加到培养基中的最终浓度为10 mM。菌丝形态的chiA和pyrG的双突变体是异常的，由Calcofluor白色染色检测几丁质分布是异常的条件下。菌丝在近顶端区域的溶解偶尔被检测到。另一方面，chiB破坏株在各种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无效突变体中，细胞内和细胞外几丁质酶活性大大降低。在自溶过程中菌丝干重的减少发生在突变体比野生型菌株更慢。使用抗ChiB抗血清的Western印迹分析表明，当菌丝体被转移到缺乏碳源的培养基中时，ChiB的合成在很大程度上被诱导，这是一种诱导菌丝自溶的条件。这些结果表明，chiB在A. nidulans。因此，在某些条件下，chiA的产物对菌丝的形成起着重要作用，而chiB的产物对菌丝的形成贡献很小。