转录因子Fv-hmg1调控金针菇子实体形成的分子机制研究

China is the largest producer of Flammulina filiformis in the world. Because fruiting body is the production of Flammulina filiformis cultivation, the formation and development of fruiting body can determine yield and quality of Flammulina filiformis directly. The process of fruiting body formation and development is very complex, and the regulation mechanism is still unknown. Based on our previous study, overexpression of transcription factor Fv-hmg1 inhibited the expression of gene clp1 of A mating type pathway, reduced clamp connections, and prohibited fruiting body formation in Flammulina filiformis. But the Flammulina filiformis RNAi mutants formed more fruiting bodies in less time than wild type strain. Besides, the A mating pathway can up-regulate the expression of Fv-hmg1 and the B mating type pathway can inhibit it. Fv-hmg1 can interact with mating type pathway, then regulate the formation of fruiting body in Flammulina filiformis. In this study, we would identify the interaction between Fv-hmg1 and mating type pathway, and molecular mechanism of fruiting body formation regulated by transcription factor Fv-hmg1 using gene overexpression, RNAi, inducible fertility system, yeast one-hybrid system, immuno-co-precipitation, and so on. This research results will provide the theory evidence for molecular genetic breeding and highly efficient cultivation and production of Flammulina filiformis.

我国是世界上金针菇（Flammulina filiformis）产量最大的国家。金针菇以子实体为收获物，子实体形成和发育的状况直接决定着其产量与品质。金针菇的子实体形成与发育过程十分复杂，其调控机制仍不清楚。申请人前期研究显示，过表达金针菇转录因子Fv-hmg1能够抑制交配型A因子通路基因clp1表达，减少锁状联合的形成，进而阻碍金针菇子实体形成。而Fv-hmg1的RANi突变体比野生型菌株子实体形成时间显著缩短，数目增多。此外，Fv-hmg1基因的表达受到了交配型A因子上调和B因子沉默。因此，Fv-hmg1能够与交配型通路互作，调控金针菇子实体的形成。本项目拟采用基因过表达和RNAi、诱导型同核体可孕系统、酵母单杂交、免疫共沉淀等技术研究Fv-hmg1与交配型通路之间的互作，从而解析Fv-hmg1调控金针菇子实体形成的分子机制，为金针菇分子遗传育种和高效栽培生产奠定理论基础。

食用菌以子实体为收获物，子实体的形成和发育调控机制是重要的科学问题。本研究采用分子生物学技术，构建了Fv-hmg1基因的金针菇转化子菌株。过表达转化子与野生型相比，菌丝致密，生长速度慢；RNAi转化子与野生型相比，在菌落形态和生长速度上都没有显著差异。进一步对转化子出菇试验表明，RNAi转化子的子实体形成能力明显优于野生型菌株。而过表达菌株没有形成金针菇子实体。结合Fv-hmg1在双核菌丝和原基中的表达量被下调，说明Fv-hmg1在金针菇子实体形成过程中起到负调控的作用。Fv-hmg1的表达量受到了交配型A和B通路的调控。我们对交配型通路中的关键基因clp1和ste12的功能进行了分析。clp1基因是交配型A通路中的重要基因，在金针菇中过表达clp1抑制了菌丝的生长，但促进了子实体的发育。我们注释了交配型B位点下游的信息素受体通路，对通路下游的转录因子ste12构建了过表达突变体，发现该基因能够调控金针菇菌丝的非生物胁迫耐受性，还抑制了菌柄的发育。本项目所研究的HMG-box转录因子基因Fv-hmg1是一个新发现的调控基因。通过对HMG-box转录因子Fv-hmg1与交配型通路的相互关系进行研究，并明确了该转录因子与交配型通路共同调控金针菇子实体发育，为下一步研究食用菌子实体发育调控网络提供基础。本项目的相关研究已发表SCI论文4篇（另有1篇正在审稿中）、中文论文5篇，授权国家发明专利1项。