竹黄菌侵染桂竹互作机制研究

Shiraia bambusicola, a species obligate parasitic pathogen with tissue specificity on bamboo, is a medicinal fungus with very high economic value. We have got breakthough success on artificial cultivation, then effective material could be provided for scientific research of the fungus. However, relationship between S. bambusicola and its host bamboo is known a little. Relative research includes mophology,anotomy and gene's interaction on the fungus and it's host bamboo should be studied futherly.In this project, green fluorescent protein (GFP) will be used to make the isolate of GFP-S. bambusicola, then to inoculate the isolate on to tissue cultured bamboo Phyllostachys bambusoides. Morphological characteristics during development stages of the fungus on the host bamboo will be observed by using fluorescence microscopy and confocal laser scanning microscope. Differentially expressed genes of S. bambusicola and P. bambusoides.during the fungus development will be focused and studied by analysis method of genome, transcriptome and bioinformatics. Those genes are of very importance for the fungus to distinguish the host bamboo and the specific tissue, and for the host bamboo to follow the procedure, which will be further studied. Functional verification of the key genes will be carried out by use gene overexpression technology and gene knockout technology to reveal interact mechanism between S. bambusicola and P. bambusoides. Research result will be of scientific significance on interaction between the fungus and it's host bamboo, and practical value for the guidance of the resource protection and artificial breeding for S. bambusicola.

竹黄是一种具组织特异性的竹类植物专性寄生菌，也是一种具有很高经济价值的药用真菌。尽管我们人工接种获得突破性成功，可为科学研究提供有效的实验材料，但对竹黄菌与寄主竹子之间的相互关系缺乏透彻了解，故有必要从形态学、组织解剖学，基因互作等方面进行深入研究。本项目拟采用绿色荧光蛋白（GFP）标记竹黄菌，将GFP-竹黄菌株接种至组培桂竹与非寄主楠竹对比观察，阐明竹黄菌侵染寄主各时期的过程；通过竹黄菌基因组初级组装进行基因功能预测,同时对竹黄菌浸染成功与否的菌丝体和竹子材料进行转录组分析，找到竹黄菌与寄主相互识别特异性选择中起重要作用的差异表达基因；利用基因超表达和敲除技术对竹黄菌-桂竹互作相关性较大的基因进行功能验证，揭示竹黄菌与寄主桂竹互作的初步机制。研究结果不仅具有竹黄菌与寄主互作的科学意义、也具有指导竹黄的资源保护、人工繁育推广的实际价值。

项目在基金31460199的资助下，围绕竹黄菌侵染及发育过程研究、竹黄菌与桂竹互作相关基因研究以及目的基因功能和互作关系研究等关键科学问题，开展了竹黄菌寄主桂竹的组培、绿色荧光蛋白（GFP）基因转化竹黄菌、竹黄菌基因组测序、竹黄菌转录组分析、竹黄菌不同发育时期代谢组学分析和竹黄菌与桂竹互作相关基因的功能等方面的研究。由于GFP基因转化受到竹黄菌抑菌影响未能实现，使用了普通显微镜、荧光显微镜和扫描电镜进行系统的显微观察，基本阐明了竹黄菌侵染桂竹并在桂竹上生长发育的过程。项目首次全面系统的分析了竹黄菌的基因组信息，首次从基因组水平确证了竹黄菌的分类地位，分析了竹黄菌致病和次级代谢相关的基因信息，为进一步挖掘竹黄菌致病和次级代谢相关基因奠定了基础。通过基因组测序、转录组测序和生物信息学分析，成功发现并筛选出5个未报道过的在竹黄菌与寄主竹子相互作用过程中起重要作用的相关的基因。首次采用广泛靶向代谢组学方法，对不同时期的竹黄进行的代谢组学分析，此次代谢组分析检测出多种未在竹黄中报道过的化合物，为后续竹黄中众多有效成分的分离纯化鉴定、活性成分的药性研究以及竹黄新药理作用的发现提供重要参考。成功构建了ku70、veA和PKS基因敲除载体。筛选到一套适合于竹黄菌的原生质体制备方法。已发表论文4篇，另有1篇已接收，1篇已投稿。培养硕士研究生4人（3人已毕业）。本项目的成果对于研究竹黄菌与桂竹互作机制和竹黄菌次级代谢生物合成机制等具有十分重要的意义。