Analysis Of Genes Associated With Cold-induced Fruiting In Flammulina Velutipes And Genome Editing Using CRISPR-Cas9

Flammulina velutips is one of most actively cultured mushroom in China.However,the commercial culture of F.velutipes requires cold treatment for a long period to induce fruiting.The energy of cost of this process is high.One strategy for conserving energy is to achieve high-temperature tolerant strains.Cold-induced fruiting of F.velutips were investigated by some approaches in this study to provide theoretical basis for development of high-temperature tolerant strains.Main results as following:(1)Mycelium,grown on sawdust medium in the dark,was harvested.Primordia was collected after cold induction.De novo RNA-seq was carried out.There were 26,888,494 and 26,275,146 clean reads obtained from mycelium and primordia of F.velutipes.20157All-Unigenes were de novo assembled and 15058 of them were annotated.7935 AllUnigenes were differentially expressed between mycelium and primordia,4025 AllUnigenes of them were up-regulated and 3910 were down-regulated.(2)GO and KEGG pathway analysis of the differentially expressed All-Unigenes indicated that functional group associated with two-component system,MAPK pathway,unsaturated fatty acid metabolism,calcium related pathway paly important role in coldinduced fruiting of F.velutipes.Five All-Unigenes of histidine kinase were differentially expressed between mycelium and primordia,three of them up-regulated and two of them down-regulated.16 differentially expressed All-Unigenes involved in MAPK pathway.AllUnigenes encoding Hsp70 and Hsp90 from KEGG pathway(protein processing in endoplasmic reticulum)were down-regulated and Hsp40 up-regulated in cytoplasm.However,All-Unigene encoding Hsp40 was down-regulated in endoplasmic reticulum.35 differentially expressed All-Unigenes were categorized into the KEGG pathway“biosynthesis of unsaturated fatty acids”.Five differentially expressed All-Unigenes were annotated to Delta 9-fatty acid desaturase.Differentially expressed All-Unigenes(unigene3897 and unigene 2842)involved in calcium related pathway were found.(3)A total of 643 EST-SSRs identified in 20157 All-Unigenes.And 1560 primer pairs were designed.Moreover,5548 and 5955 SNPs were detected in mycelium and primordia library.(4)Micro RNA sequencing was applied for discovery of microRNA associated withcold-induced fruiting in F.velutipes.A total of 9.7M and 8.4M clean reads were obtained from mycelium and primordia library.12 significant differentially expressed microRNAs were obtained,seven of them up-regulated and five of them down-regulated.Prediction and functional annotation of microRNA targets were performed.Genes from PI3K-Akt pathway(Hsp90,S6 and PP2A),protein processing in endoplasmic reticulum(Hsp70,UGGT,Bip,GRP94)and calcium signal pathway(ANT)were important microRNA targets.(5)Genes encoding DCL and AGO proteins were identified from genomic database and cloned.The open reading frame of dcl is 4077 bp,3183bp and 4458 bp.DCL proteins contained RNase III and Dicer conserved domain.The open reading frame of ago is 2445 bp,3039bp,2802 bp and 2772 bp.AGO proteins contained Piwi and PAZ domain.Genes encoding DRK1 and Mga2 protein were identified and cloned.DRK1 contained HAMP domain.Mga2 contained ANK domain.(6)U6 snRNA of F.velutipes were identified from genomic database.The sequence similarity of Fv U6 snRNA and human snRNA was extremely high.However,the sequence similarity of Fv U6 promoter and U6 promoter of Aspergillus oryzae was very low.H1 promoter,Fv U6 promoter and gpd promoter were used for expression of g RNA in this study.Codon optimization of Cas9 was performed for expression in F.velutipes.(7)drk1 gene was chosen as target gene in this work.A series of vectors including pgfvs-hCas9-hph-gpd-D1/-D2/-D3 and pgfvs-hCas9-hph-gpd-D1 were constructed.These plasmids were transformed to protoplast of YX74 strain by PEG method.Six transformants expressing hCas9 were harvested after Hygromycin B selection,but no mutants were obtained.(8)A series of vectors including p0390-hph-Fv Cas9-gpd-D1/-D5,p0390-hph-Fv Cas9-Fv U6-1-D4/-D5,p0390-hph-Fv Cas9-Fv U6-2-D5 and p0390-hph-Fv Cas9-Fv U6-3-D5 were constructed.These plasmids were transformed to protoplast of YX74 strain by Agrobacterium-mediated transformation method.27 transformants expressing Fv Cas9 were harvested after Hygromycin B selection,but no drk1 mutants were obtained.(9)PyrG gene was chosen as target gene.Results of RT-q PCR,western blot and CDS PCR confirmed that Fv Cas9 had expressed in Fv Cas9-1 strain.g RNAs were transformed to protoplast of Fv Cas9-1 strain by PEG and electroporation method.A total of 153 transformants were harvested,but no PyrG mutants were obtained.(10)Preparation of Sp Cas9-g RNA ribonucleoprotein complexes in vitro.Ribonucleoprotein complexes were delivered to protoplast of YX74 by electroporation method.A total of 53 transformants were identified,but no PyrG mutants were obtained inthis study.