转录因子BcSmr1调控灰葡萄孢菌核休眠的分子机制研究

Dormancy is an important property for sclerotia, which has special biological significance for individual survival and species continuation. In nature, Botrytis cinerea usually produces black sclerotia. In previous studies, we reported six isolates of B. cinerea stably producing orange sclerotia. The orange sclerotia are lack of visible dormancy period and pretty weak in survival compared with the black sclerotia. Further studies confirmed that the function of transcription factor BcSmr1 is defected in these isolates. This results showed that the transcription factor BcSmr1 has an important biological function in the progress for regulation of sclerotial dormancy in B. cinerea. However, the molecular mechanism was remain totally unknown. In this project, we proposed to identify the key genes related to sclerotial dormancy regulated by BcSmr1 using transcriptome profiling with the sclerotia of OS isolate and their complemented mutant, and to characterize their expression patterns. The technology of gene knockout and complementation was used for functional verification. Meanwhile, the regulatory relationships between these key genes and BcSmr1 was elucidated using the method of transcriptional activation analysis and transcriptional binding site analysis. In theory, the results will reveal the molecular mechanism of sclerotial dormancy regulated by BcSmr1 in B. cinerea. It not only provide theory gist for control of gray mold diseases caused by B. cinerea, but also give a good reference for the study of the dormancy mechanism in other sclerotia producing fungi.

休眠是真菌菌核的重要生物学特性，对个体生存和物种延续有特殊的生物学意义。自然界中，灰葡萄孢一般产生黑色菌核。我们前期研究发现了6株产橙色菌核的灰葡萄孢菌株。与黑色菌核相比，橙色菌核缺乏休眠期，存活能力较差。随后证实橙色菌核性状的形成是转录因子BcSmr1功能缺陷引致的。可见，BcSmr1在调控灰葡萄孢菌核休眠过程中具有重要生物学功能，但其分子机制尚未解析。本项目提出对橙色菌株及其bcsmr1基因互补菌株成熟菌核开展转录组分析，鉴定受BcSmr1调控的“休眠”相关基因，确定其表达模式；进而采用基因敲除和互补试验验证其功能；采用转录激活和转录结合试验验证BcSmr1及这些关键基因之间的的调控关系。项目的完成将揭示BcSmr1调控灰葡萄孢菌核休眠的分子机制，不仅为防治灰霉病提供理论依据，也为其它产菌核真菌的菌核休眠机制研究提供借鉴。

休眠是真菌菌核的重要生物学特性，对真菌完成生活史具有特殊的生物学意义。前期研究结果发现Bcsmr1基因互补可以促进天然突变体XN-1的菌核休眠。在本研究中我们对XN-1及其Bcsmr1基因互补菌株成熟菌核开展了转录组学分析，从宏观上证实了氨基酸积累水平、胞内氧化还原状态、次生代谢产物含量等是促进菌核休眠的内在主要因素。通过差异表达筛选鉴定出受Bcsmr1调控的“休眠”相关基因36个。同时对其中10个差异表达极显著的基因进行了功能验证，其中有4个基因敲除转化子具有明显表型。生物学特性研究结果发现Bcbrn2、Bcscd1参与灰葡萄孢黑色素合成，且对菌核抗木霉寄生和非生物逆境具有重要作用；BcxlnR与灰葡萄孢菌核皮层细胞分化密切相关，该基因缺失后菌核极易被木霉寄生，对菌核存活具有重要作用；Bcsphl1与灰葡萄孢菌核形成有一定相关性。这些研究结果为揭示BcSmr1调控灰葡萄孢菌核休眠的分子机制奠定了基础，也为其它产菌核真菌的菌核休眠机制研究提供了借鉴。