内生菌球毛壳ND35的分子标记及其在杨树苗期的种群动态监测

项目名称： 内生菌球毛壳ND35的分子标记及其在杨树苗期的种群动态监测

项目编号： No.30872024

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 高克祥

作者单位： 山东农业大学

项目金额： 30万元

中文摘要： 本研究在建立RAPD优化体系的基础上，利用随机引物对毛壳菌属14个菌株和6个土传植物病原菌株进行分析，筛选到球毛壳ND35特异性RAPD条带，并根据测序结果设计特异PCR引物，得到引物对Cg ND35-F/Cg ND35-R，获得了有实用价值的特异SCAR检测标记并对杨树、小麦和生姜等大田作物中接种定殖的球毛壳ND35菌株进行了分子检测。又根据SCAR标记获得的球毛壳ND35菌株特异片段设计了另一对用于实时荧光定量PCR检测的引物ND35-1/ND35-2，以球毛壳ND35菌株基因组DNA为模板，利用此引物建立并优化了SYBR Green Ⅰ#23454;时荧光定量PCR反应的最佳体系，并据此对杨树和黄瓜两种植物中ND35菌株的定殖情况进行了检测。结果表明该引物对可以用于实时荧光定量PCR检测球毛壳ND35种群数量动态变化。另外，还进行了球毛壳ND35侵染定殖后对植物生长的影响和生防机理研究。这为深入了解内生菌的生防机制，制定更切实际的防治策略，提高内生菌毛壳的生防效率奠定了基础。

中文关键词： 球毛壳ND35菌株; 分子检测; 实时荧光定量PCR; SCAR标记

英文摘要： 14 strains of Chaetomium and 6 strains of plant pathogens have been analyzed with random primer based on optimal RAPD system and screened specific fragment of ND35. A pair of primers (Cg ND35-F/Cg ND35-R) has been designed based on gene sequencing result. The meaningful SCAR marker has been first got from this study. Positive results have been got through recovery test on specificity and sensitivity. Molecular detection of C. globosum strain ND35 colonized in poplar, wheat and ginger in the field has been investigated by SCAR marker. Another pair of primers (ND35-1/ND35-2) used in real-time PCR has been designed based on the specific fragment from SCAR marker. Being taken the genomic DNA of C. globosum ND35 as template, a best technique of quantitative Real-time PCR by SYBR GreenⅠas developed and optimized. Then use this technique we detected the colonization of strain ND35 in cucumber and poplar. The results shows that this pair of specific primers could be used for real-time PCR determination of C. globosum ND35. On the other hand,biological control mechanism and effect on growth of the host plant infected and colonized by C. globosum ND35 have been investigated. So this lay a foundation for understanding biocontrol mechanism of endophytic fungi, formulating more suitable control tactics and increasing biocontrol efficacy of endophytic Chaetomium.

英文关键词： Chaetomium globosum strain ND35; molecular detection; real-time PCR; SCAR marker