Expression of cmg1, an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans, Increases during Sclerotial Parasitism

During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml(−1), respectively. A single copy of the cmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.

在核盘菌(Sclerotinia sclerotiorum)的菌核侵染过程中，菌寄生真菌(mycoparasite)盾壳霉(Coniothyrium minitans)可穿透以β-1,3-葡聚糖(β-1,3-glucan)为主要成分的寄主细胞壁。本研究采用基于聚合酶链式反应(PCR)的策略，从该真菌的互补脱氧核糖核酸(cDNA)文库中克隆得到一个编码β-1,3-葡聚糖酶(β-1,3-glucanase)的基因，命名为cmg1。该基因的核苷酸序列及推导的氨基酸序列与其他真菌外切β-1,3-葡聚糖酶(exo-β-1,3-glucanase)基因序列具有较高的同源性。推导得到的蛋白质（去除预测的24个氨基酸的N端分泌信号肽后）的理论分子质量为83346道尔顿(Da)，理论等电点(pI)为4.73。表达cmg1基因的酿酒酵母(Saccharomyces cerevisiae) INVSc1菌株可将分子量约为100 kDa的β-1,3-葡聚糖酶分泌至培养基中，该结果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)验证。对纯化的重组酶进行N端序列分析后发现，分泌型酶的N端起始于第32位丙氨酸(Ala-32)，较预测的信号肽酶切割位点下游偏移了7个氨基酸。纯化后的重组葡聚糖酶在体外可抑制核盘菌(Sclerotinia sclerotiorum)的菌丝生长：当浓度为300 μg·mL⁻¹时抑制率为35%，浓度为600 μg·mL⁻¹时抑制率可达85%。盾壳霉(Coniothyrium minitans)的基因组中仅含有单拷贝的cmg1基因。Northern印迹分析(Northern blot analysis)结果显示，碳源饥饿(carbon starvation)环境及添加核盘菌粉碎菌核均可上调cmg1基因的转录水平；而在添加2%葡萄糖的培养基中，cmg1基因仅出现轻微的表达抑制。在与核盘菌发生重寄生互作的过程中，cmg1基因的表达水平会升高。