金针菇低温诱导子实体发育的信号传导和调控机制研究

Flammulina velutipes is the largest industrialized cultivation mushroom in china. At present, there are two major problems in the Flammulina velutipes production: One is the low and unstable yield, poor quality of fruiting body. Another is huge energy consumption in the Flammulina velutipes industrialized cultivation because the fruiting body differentiation needs the induction by very low temperature. The reason why there exist these problems is that the mechanism of differentiation and development of fruiting body of Flammulina velutipes is still not understood. In this project, Flammulina velutipes strain Fv1 and its mutation strain F1C4 with higher temperature tolerance will be used as materials for the comparison and analyses of their transcriptomes under both normal and low temperature conditions by applying RNA-Seq technology. The functions of related genes, transcription factors, signal donors and receptors involved in fruiting body differentiation and development of Flammulina velutipes will be characterized by applying TALEN targeted gene knockout technology and RNAi method. The key function genes, signal transduction pathways and regulation factors in the differentiation and development of Flammulina velutipes fruiting body induced by low temperature will be obtained and elucidated.so that the mechanism of differentiation and development of fruiting body of Flammulina velutipes will be revealed and understood. This research is of great scientific theoretical significance and practical application value for directed modification of Flammulina velutipes genome to develop energy-saving varieties with high temperature tolerance, for effective regulation of growth and development of Flammulina velutipes, for increase of fruiting body yield and quality, and for reduction of energy consumption in Flammulina velutipes industrialized cultivation.

金针菇是我国最大宗的工厂化栽培菇类。目前金针菇产业存在的两大问题是：①子实体产量低且不稳定、品质差；②金针菇子实体分化需要低温刺激诱导，工厂化栽培能源消耗巨大。原因是人们对金针菇子实体分化发育的机理还不了解。本项目拟以白色金针菇Fv1菌株及其诱变育成的高温节能型菌株F1C4为材料，采用RNA-Seq技术对这两个菌株在常温及低温诱导条件下的转录组学进行比较分析研究，采用TALEN靶向基因敲除技术和RNAi技术鉴定基因功能和转录因子、信号受体的功能，探明低温诱导金针菇子实体分化发育的关键功能基因、信号传导途径以及调控因子，揭示金针菇低温诱导子实体分化发育的机制。这对定向改造金针菇基因组培育高温节能型新品种、有效调控金针菇生长发育过程、提高金针菇子实体产量和品质、减少金针菇工厂化栽培的能源消耗将具有重大的科学理论意义和实际应用价值。

本课题对冷诱导金针菇子实体形成机理开展了研究，获得的主要结果：1.对金针菇黑暗条件下获得的菌丝和黑暗及低温条件下获得的原基进行de novo转录组测序，组装获得20157个unigenes，其中15058个unigenes得到功能注释，并且菌丝和原基间共有7935个差异表达基因，4025个上调，3910个下调；2.对差异表达基因进行KEGG pathway分析和GO功能注释，双组份系统转导途径、MAPK通路、 GATA转录因子、MADS转录因子、热激蛋白、非饱和脂肪酸途径、钙传导通路等可能在冷诱导金针菇子实体形成中发挥重要作用；3.构建CRISPR-Cas9载体对7个拟冷诱导金针菇子实体形成基因进行突变，PEG法转化金针菇单核菌株YX74及筛选后，获得33个含hcas9的转化子菌株；4.构建CRISPR-Cas9载体对3个拟冷诱导金针菇子实体形成基因进行突变，ATMT转化金针菇单核菌株YX74及筛选后，获得73个含FvCas9的转化子菌株，最后在mRNA和蛋白质水平对FvCas9进行检测。