Qiao Tianmin

桑氏链霉菌几丁质酶 ChiKJ40 基因的克隆表达及其抑菌作用

背景目前关于桑氏链霉菌(Streptomyces sampsonii)生防基因的研究不多，仅从其基因组中克隆了2个几丁质酶基因片段，其单个几丁质酶的完整基因序列相关研究未见报道。 目的克隆S. sampsonii KJ40的几丁质酶基因ChiKJ40并进行原核表达，纯化重组蛋白并研究其抑菌作用。 方法采用PCR扩增法从S. sampsonii KJ40中克隆几丁质酶基因ChiKJ40，连接到表达载体pET-32a，导入Escherichia coli BL21(DE3)进行诱导表达。使用His标记蛋白质微量纯化试剂盒对重组几丁质酶进行纯化，Bradford蛋白浓度测定试剂盒测定粗酶液和纯化酶液的浓度，几丁质酶试剂盒测定粗酶液和纯化酶液的几丁质酶活性。观察重组几丁质酶对桉树焦枯病菌(Cylindrocladium scoparium)、栗疫病菌(Cryphonectria parasitica)、链格孢菌(Alternaria alternate)、紫丝核菌(Rhizoctonia violacea)几种致病真菌的抑菌作用。 结果ChiKJ40基因(登录号为MF434484)在E. coli中经IPTG诱导表达，获得42 kD的重组几丁质酶，不同浓度IPTG在37 ℃诱导3 h，蛋白产量无明显变化。0.2 mmol/L IPTG 16 ℃诱导过夜，重组几丁质酶主要以可溶性形式存在于上清，小部分以包涵体存在于沉淀中。粗酶液几丁质酶活性为0.080 U/mL，酶比活力为0.041 U/mg，纯化酶液几丁质酶活性为0.046 U/mL，酶比活力为0.115 U/mg，纯化倍数为2.8，酶活回收率为57.5%。重组几丁质酶处理后，C. scoparium、C. parasitica和A. alternata菌丝细胞出现分节、膨胀，R. violacea菌丝溶解且部分被破坏成碎片。 结论ChiKJ40基因的研究补充了S. sampsonii的生防背景，为几丁质酶基因找到了新的来源，并为其应用奠定了理论基础。

Diversity of epiphytic fungi on the diseased and healthy leaves of Bambusa

Based on the sampling on permanent sample plots in different seasons, quantities of epiphytic fungi, seasonal changes of fungal species, fungal community composition, and the relationship of epiphytic fungi and microenvironment of Bambusa pervariabilis × Dendrocalamopisis grandis were studied by plate dilution, morphological and molecular biological methods. The above four aspects were also comparatively analyzed between healthy leaves and diseased leaves. The results show that quantities of epiphytic fungi in diseased leaves was more than those of healthy leaves; fungal species was at most in summer and autumn and the isolation rate of Fusarium avenaceum and Microdochium sp. 3 was higher than the others. Diversity index of fungi in different kinds of leaves indicated they had different richness and evenness degrees. Physiological index showed that the correlation was significant between epiphytic fungi quantities on diseased leaves and content of soluble protein, and had a significant positive correlation between fungal quantities in healthy leaves and content of soluble sugar.