漆酶在荷叶离褶伞子实体分化中的作用机制研究

Lyophyllum decastes is a kind of rare edible mushroom, which is difficult to commercial cultivation due to its weak mycelium decomposition substrates and difficult fruiting body differentiation. Fruiting body differentiation is an important part of its life cycle, and laccase plays an important role in this process.Previous researches have shown that fruiting body differentiation of mushroom was correlated with its laccase activity, but the mechanism of laccase is still unclear. In our previous study,we screened 2 laccase genes by comparing the transcriptome differences of fruiting body differentiation period. In this study, Lyophyllum decastes strain "ZY48-1"and“HY1022-01" were used as material. We construct mutant strains of lowexpression of laccase gene and study physiological and molecular mechanism about laccase regulating fruiting body differentiation in Lyophyllum decastes through molecular biology technologies of RNAi, RT-PCR and transcriptome sequencing.In order to obtain low expression mutant strains of laccase gene of Lyophyllum decastes, to reveal the change rule of laccase activity in the development of its fruiting body and clarify the role and the mechanism of laccase in its fruiting body differentiation. This study will provide the foundation for revealing mechanism of fruiting body differentiation in Lyophyllum decastes.

荷叶离褶伞是珍稀食用菌，是祁连山优势种群，目前我们已驯化成功，由于其菌丝体分解培养料能力弱，子实体分化困难，很难商业化栽培，漆酶在食用菌子实体分化中具有重要作用。已有研究表明漆酶与子实体分化存在一定的相关性，但漆酶对子实体分化的调控机制尚不清楚，我们前期研究发现漆酶与子实体分化相关，通过子实体分化差异转录组测序筛选到2个漆酶基因，本项目以荷叶离褶伞菌株ZY48-1和诱变漆酶高产株HY1022-01为研究材料，采用RNAi、RT-PCR、差异转录组测序和差异蛋白组测序技术，进行荷叶离褶伞漆酶基因同源克隆、漆酶基因低表达突变株构建，漆酶与子实体分化生理机制研究和漆酶调控子实体分化分子机制研究，以期获得荷叶离褶伞漆酶基因低表达突变株，揭示荷叶离褶伞子实体发育中漆酶活力变化规律，探索漆酶基因在子实体分化中的功能，阐明子实体分化中漆酶作用的机制，为揭示荷叶离褶伞子实体分化机制奠定基础。

荷叶离褶伞是珍稀食用菌，子实体分化困难，漆酶在食用菌子实体分化中具有重要作用。为探讨荷叶离褶伞漆酶在形态发育过程中的功能，本研究开展了荷叶离褶伞漆酶基因Ldlac3同源克隆及漆酶基因低表达突变株构建研究，应用转录组技术分析了荷叶离褶伞漆酶基因家族不同发育时期差异表达基因，进行了漆酶基因家族的基因功能研究及漆酶基因家族的生物信息学分析、荷叶离褶伞漆酶基因同源克隆及原核表达分析、荷叶离褶伞漆酶基因RNAi载体构建、农杆菌介导的荷叶离褶伞遗传转化体系建立，构建了漆酶基因低表达突变株；开展了荷叶离褶伞漆酶与子实体分化生理、分子机制研究，研究了荷叶离褶伞在半袋期、满袋期、原基期、幼菇期、成菇期5个不同生长期漆酶的酶活性变化规律，利用Illumina HiSeq平台对荷叶离褶伞营养菌丝期（VM）、原基期（PI）、幼子实体期（YF）、中等子实体期（MSF）和成熟子实体期（MF）5个不同发育时期的菌丝或子实体进行了比较转录组分析，发现在子实体分化发育期胞外漆酶活性变化具有明显阶段性， PI与VM、YF与PI、MSF与YF、MF与MSF之间分别有9612、1838、426和3683个差异表达基因，推测荷叶离褶伞胞外酶相关基因、转录因子和信号通路可能与子实体的形成有关；对荷叶离褶伞野生株和漆酶干扰突变株进行了比较转录组学研究，发现有734个差异表达基因，其中干扰突变株上调基因 314个，下调基因 420个，Ldlac 3基因参与了细胞过程（cellular processes）的过氧化物酶体途径的转运和分解代谢、基因组形成（Genetic Information Processing）的氨酰-tRNA合成途径的转运。因此，通过调控基因代谢过程（Metabolism）途径的氨基酸新陈代谢、脂类代谢、辅助因子和辅酶新陈代谢、能量代谢和糖代谢实现对荷叶离褶伞菌株生长发育的调控。