Expression Analysis Of Zn₂Cys₆ Transcription Factor Family And Functional Analysis Of PoZCP26 In Pleurotus Ostreatus

Pleurotus ostreatus（Jacq.）P.Kumm.is one of the most important cultivated mushrooms in China.The regulatory mechanisms of fruiting body development and the heat stress response of P.ostreatus have been a research hotspot.Zn₂Cys₆ transcription factor family is one of the largest transcription factor families in fungi which plays crucial roles in response to multiple abiotic stress and fruiting body development.However,the functional analysis of this family in P.ostreatus is very limited.In this thesis,Zn₂Cys₆ transcription factor family members in P.ostreatus（PoZCPs）were identified using bioinformatics methods.The expression profiles of these family members during fruiting body development and under heat stress were further analyzed.Moreover,the function and downstream regulation mechanism of PoZCP26 which take an active part in fruiting body development and heat stress response were investigated.The main results are as follows:1.We identified 66 ZCP family members named PoZCP01-PoZCP66 in P.ostreatus through a genome-wide search method.The family members were classified into 15 types（A-O）according to their zinc cluster domain.Physical and chemical property analyses showed a huge diversity among the family members and most of them were located in the nucleus.Phylogenetic analysis of PoZCPs classified these proteins into six groups,and conserved motif combinations and similar gene structures were found in each group.2.The expression profiles of these PoZCP genes during different developmental stages and under heat stress were further investigated using RNA-sequencing,and the expression patterns were diverse among PoZCPs.Thirteen PoZCPs were selected for validation of their expression levels through real-time quantitative PCR（RT-qPCR）analysis using stable reference genes tub and actin1.The results showed that PoZCP15,PoZCP26 and PoZCP31 were upregulated dramatically during heat stress as well as PoZCP10,PoZCP26,PoZCP31,and PoZCP40 were upregulated dramatically during fruiting body development.PoZCP26 and Po ZCP31 were involved in both heat stress response and fruiting body development.3.The overexpression and RNA interference transformants of PoZCP26 were constructed with the agrobacterium-mediated transformation method which target fragments inserted randomly and integrated into the genome of P.ostreatus.We analyzed the functions of PoZCP26 on mycelia growth,thermostability and fruiting body development.The results showed that PoZCP26 regulated the mycelial growth rate and recovery ability after heat stress positively.The role of PoZCP26 in mycelial resistance in heat stress might be due to its tolerance to H₂O₂.Furthermore,PoZCP26 positively regulated the mycelial growth rate in the cultivation substrate.After PoZCP26 was overexpressed,the primordial formation periods and the fruiting body morphology showed no significantly different with wild type.After Po ZCP26 was interfered,the formation of primordium was markedly delayed and its differentiation was inhibited,which led to fruiting body malformation or development retardation.4.PoZCP26 might regulate the expression of G-protein,hydrophobin,DnaJ and other genes involved in protein ubiquitination and carbohydrate metabolism in response to mycelia growth,heat stress and the fruiting body development.Some motifs probably combined by PoZCP26 were also selected after analyzation.This study laid the foundation for exploring the functions of ZCP family members in P.ostreatus.It provided new insight into the molecular mechanism of the heat stress response and fruiting body development in P.ostreatus.Besides,it also set a target for molecular breeding of P.ostreatus and other edible fungi.