Functional analysis of chitinases of Aspergillus nidulans

Chitin是N-乙酰-D-葡萄糖胺（GlcNAc）中的β-1，4-连接聚合物，是多种碱性真菌的主要细胞壁成分之一。我们克隆了两个基因（chiA和chiB）编码来自曲霉的类黄酮蛋白。chiA基因编码了961氨基酸的蛋白质，其中Fungal型chitinases和具有Secretary Signal样序列，并且在其N-和C-末端具有长Ser/Thr/Pro丰富序列，尊重。chiB编码一种416氨基酸同源的蛋白质，使其成为细菌类型的衣原体。Disruptants of chiA could not grow on the medium containing 0. 01%of SDS in the pyrGイイD1+イエD1 background。Moreover，严重的增长影响在PyrG D1-YE D1的低运动条件下观察到的背景，即使在10毫米的最终浓度下，Uridine也被添加到了最后浓度下。根据Calcofluor的白色突变体的超形态学被发现为不正常状态下。在亚apical区域的Lysis被偶尔探测到。在另一方面， chiB干扰剂被展示出几乎相同的形态学类型在各种条件下的野生类型约束。在chiB-无突变，如何，内源性和提取性的chitinase活性似乎被减少了。在野生类型的应变中，在高强度干重期间，自动化只会减少比突变更缓慢。Western blot分析使用反ChiB反指标，即ChiB的综合结果被广泛地诱导，当梅西利亚转移到媒体缺失的碳源时，一种诱导的hyphal autolysis的条件。从这些结果中，我认为chiB在自动化过程中扮演了一个角色。Nidulans。因此，我认为ChiA的产品对某些条件下的hyphae形成非常重要，但由于ChiB对它的贡献很小。

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.