Jasmonic Acid’s Effect On Primodium Formation In Flammulina Velutipes

Scratching is the essential process in Flammulina velutipes cultivation,which could promote primordium formation trimly.The molecule mechanism of scratching is dimness till now.Jasmonic acid(JA)is a signal molecule responding to mechanical wounding or herbivore attack in plant.Does it play the similar action in F.velutipes?This study identified homologous genes of the JA/SA signaling pathway of the Arabidopsis thaliana,and then we construct the JA/SA signaling pathway based on F.velutipes genome and transcriptome data.In order to study the function of JA signaling pathway in growth of mycelium and formation of fruiting body in F.velutipes,we added exogenous JA instead of scratching during F.velutipes cultivation experiment.Real-Time Quantitative PCR(RT-qPCR)was applied to detect the gene relative expression of JA signaling pathway member and high performance liquid chromatograph(HPLC)was applied to detect the content of endogenous JA.Besides,we performed several samples for RNA-Seq,and analysis the gene expression under the exogenous JA and scratching stress to find different expression genes.Moreover,we focused on the function prediction and metabolic pathways of these genes and the transcriptome sequencing results were verified by RT-qPCR.The main results are as follows:(1)Construct the JA/SA signaling pathway in F.velutipes and biological information analysis.Compared with amino acid sequences of F.velutipes using BLASTP and found 8 genes about JA/SA signaling pathway,including one allene oxide synthase(AOS)(encoding gene prediction No.gene2068),one COI1(coronatine insensitive 1)(encoding gene prediction No.gene2615)and two MYC2s(encoding gene prediction Nos.gene6724 and gene2796)in JA signaling pathway.Two pathogenesis_related 1(PR1)(encoding gene prediction No.gene 1937,gene5143)and two nonexpressor of pathogenesis_related genes 1(NPR1)(encoding gene prediction No.gene4378,gene8890).All the verified nucleotide sequences from this study have been submitted to GenBank under the accession ID numbers:KX827237,KX129930,KX129932,KX129933,KX257996,KX129927,KX129928,KX129929.And then we analysis the genes and its encoding protein by bioinformatics(gene structure,signal peptide prediction,transmenbrane structure prediction,cellular localization,conserved domain,phylogenetic analysis).The resultes showed that there exists JA/SA signaling pathway in F.velutipes.(2)We conducted experiments to investigate the response of F.velutipes to exogenous JA and antagonistic effection.The nitro blue tetrazolium(NBT)and RT-qPCR results demonstrated that JA/SA signaling pathways of F.velutipes resposed to exogenous JA/SA.In addition,the expression levels of these genes of JA/SA signaling pathway significantly increased under the treatment of exogenous 50 μmol·L-1 JA and 500 μmol·L-1 SA for 12 h respectively which showed that F.velutipes is more sensitive to exogenous JA of low concentration and exogenous SA of high concentration.JA signaling pathways in F.velutipes may also experience antagonistic effect of SA.When SA showed antagonistic effect to JA signaling pathway,gene AOS is the responsive gene and the strength of antagonistic effect depended on the concentration of SA.And JA has little antagonistic effect to SA signaling pathway.The resultes showed that JA signaling pathway can respose to exogenous JA,and SA show antagonistic effect to JA signaling pathway in F.velutipes.(3)To explore the effect of exogenous JA on vegetative growth and reproductive growth of F.velutipes.When strain FL19 grow in PDA with exogenous JA,mycelium growth was inhibited significantly.Then we added exogenous JA instead of scratching during F.velutipes cultivation experiment.In comparison with the control,scratching improved endogenous JA content in F.velutipes by HPLC checking and the gene expression of members in JA signaling pathway were up-regulated responsing to mechanical wounding(scratching)and exogenous JA.Although the gene expression pattern between scratching and exogenous JA were the same,scratching showed stronger stress to F.velutipes:the gene relative expression levels were all up-regulated,and reached their peak value after scratching 5 min while the time of JA was 120 min.Besides,the highest level of expression at 5 min was higher than that of 120 min.In F.velutipes cultivation experiment,we added exogenous JA instead of scratching.Compared with the control,we found that exogenous JA can shortened the time of fruiting body formation and ripening significantly as well as improve the number of fuiting body number of each bottle and consistent state of fruiting body which showed that exogenous JA may have the same function with sctatching on F.velutipes.All above showed that exogenous JA could inhibite mycelium growing and promote the formation of fruiting body in F.velutipes just as scratching.(4)This study used high-throughput sequencing technologies(Illumina)to analysis the difference among that three treatments,and the material came from the F.velutipes 2 days before fruiting body formation and the genes expression were up-regulated most at that time.There was 75%of genes whose FPKM of gene was up to 3.After difference expression gene analysis,we analyzed the genes expressing differently both after scratching and exogenous JA treatments by Wayne,Functional Categorization and KEGG Pathway analysis.Functional Categorization and KEGG Pathway analysis revealed the main biological function of differentially expressed genes and the biological process they involved in.There were more genes in metabolic process,biological process,catalytic activity as well as more genes in MAPK signaling pathway and Fatty Acid biosynthesis et al.which provided resources during fruiting body formation after scratching or exogenous JA.Finally,we verified some genes of MAPK signaling pathway and Fatty Acid biosynthesis by RT-qPCR.The results showed that consistent expression patterns with those findings in transcriptome analysis,which indicating the veracity of the Illumina sequences data.Besides,Fatty Acid biosynthesis can provide raw materials for endogenous JA synthetic by eighteen carbon alkane approach.MAPK and HOG signaling pathway answer to scratching and exogenous JA,which shows that many signaling pathways may play role in the formation of primodium in F.velutipes together.According to experimental results mentioned above,we concluded that JA signaling pathway played a definitive role in formation of fruiting body and growth of mycelium in F.velutipes.