| Pleurotus ostreatus,commonly known as oyster mushroom,is one of the most important edible mushrooms with third largest yield in China,and its production and quality directly affects the economic benefits of farmers.Therefore,the growth and development of the fruiting body of P.ostreatus has become a research hotspot.The previous research has revealed that the transcription factor Po ZCP26 promotes the development of fruiting body of P.ostreatus.In this study,the wild-type(WT)strain and two over-expression strains(OE3 and OE5)as well as three RNA interference strains(Ri2,Ri12,and Ri15)of P.ostreatus Po ZPC26 were used as materials and the transcriptomic data of six strains during 3 development stages including mycelia(M),primordia(P),and fruiting body(F)were analyzed using high-throughput sequencing.The key genes that may modulate the development of mushroom and be regulated by Po ZCP26 meanwhile were screened out.The relative expression levels of these key genes were verified by RT-q PCR.The overexpression strains of these two key genes were constructed using Agrobacterium tumefaciens-mediated transformation to verify the effects of these genes on the growth and development of P.ostreatus,commonly known as the oyster mushroom.The results of the experiments are as follows:1.Analysis of transcriptomic data and comparison of intersecting sets(positively regulatory comparison group WT-MP-up VS OE3-MP-up VS OE5-MP-up VS Ri15-MP-down,negatively regulatory comparison group WT-MP-down VS OE3-MP-down VS OE5-MP-down VS Ri15-MP-up)were used to screen for genes that are positively or negatively regulated by Po ZCP26 during the M to P developmental stage,with FC(fold change)≥2 and p-Value<0.05.A total of 54 genes positively regulating development from M to P as well as positively regulated by Po ZCP26 and 51 genes negatively regulating development from M to P as well as positively regulated by Po ZCP26 were screened.Seven key genes(g11718,g11781,g4216,g3416,g1438,g4751,g5090)were ultimately selected based on validation through fluorescence quantitative PCR,which showed that the relative expression levels of these genes were consistent with the transcriptomic data trends.Among them,two genes(g11718 and g1438)were selected for further study.2.The c DNA from wild-type mycelia(WT-M)was used as a template and the c DNA fragments of two key genes(g11718 and g1438)were PCR cloned and then inserted to the over-expressin vector Po-gpd OE using infusion cloning technology.Two over-expression vectors of target genes were succcessfully constructed.After confirming the accuracy of the sequence,the overexpression vectors were transformed into P.ostreatus wild-type strain(WT)using Agrobacterium tumefaciens-mediated transformation(ATMT).Three positive transformants for each gene were identified by PCR.RT-q PCR was used to detect the relative expression levels of the key genes,and positive transformants with increased expression levels were selected as over-expression strains and were named as OE11718-1,OE11718-3,OE1438-1,and OE1438-10.3.The effect of overexpression of the two key genes on P.ostreatus mycelial growth on the plates and heat tolerance was evaluated.During cultivation on PDA plates at 28°C,the mycelial growth rate of the overexpression strains was significantly faster than that of the wild-type strain.During the 37°C heat shock period,the mycelial growth rate of the overexpression strains was significantly slower than that of the wild-type strain.However,during the recovery period after heat shock,the mycelial growth rate of the overexpression strains was significantly higher than that of the wild-type strain.These results indicated that the two genes,g11718 and g1438,promote mycelial growth at the optimal temperature but inhibit mycelial growth under heat shock.4.The impact of overexpression of the two genes on the growth and development of fruiting body development was investigated.During the mycelial and primordia growth stage,the over-expression strains showed significantly faster mycelial growth than the wild-type strain,and slightly higher numbers of primordia.During the early stage of fruiting body development,the overexpression strains exhibited distorted cap shapes with irregular surface protrusions,indicating that the two genes,11718 and 1438,have a negatively regulatory roles in the development of cap shape and surface morphology. |
