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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.

甲壳素是N-乙酰-D-氨基葡萄糖的β-1，4-连接聚合物，是许多丝状真菌的主要细胞壁成分之一。我们从尼杜拉曲霉中克隆了编码几丁质酶样蛋白的两个chi基因(chia和chib)。ChIA基因编码961个氨基酸，与真菌几丁质酶同源，在其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编码一个416个氨基酸的蛋白质，与细菌型几丁质酶同源。在含有0.01%十二烷基硫酸钠的PYRGィイD_1+ィエD_1背景下，CHIA的破碎物不能生长。此外，在低渗条件下，即使在终浓度为10 mM的培养液中加入尿苷，也能在ィイD_1-ィエD_1背景下观察到严重的生长缺陷。在此条件下，chia和pyrG双突变体的菌丝形态发生了异常变化，并且经硫代荧光白染色检测到甲壳素的分布也出现异常。偶见根尖下区菌丝溶解。另一方面，在不同条件下，ChiB破碎物表现出与野生型菌株几乎相同的形态表型。然而，在chib缺失突变体中，细胞内和细胞外几丁质酶的活性显著降低。自溶期间菌丝干重的下降在突变体中比在野生型中发生得更慢。用抗ChiB抗血清进行Western印迹分析表明，当菌丝转移到无碳源的培养基上时，大量诱导了ChiB的合成，这是一种诱导菌丝自溶的条件。这些结果表明，ChiB在多核拟青霉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是非常重要的，而CHIB的产物对菌丝的形成贡献很小。