Identification Of Regulatory Function Of Flammulina Velutipes PKAC-2 Gene In Responding To Stress By Neurospora Crassa

In this study,the gene fv-pkac-2(gene 8023)encoding protein kinase A catalytic subunit,which is the key composition of cAMP/PKA signal transduction pathway in Flammulina velutipes was identified based on genomic and transcriptomic data of flammulina velutipes.In order to reveal the function of this subunit,we explore the relationship between cAMP/PKA signal transduction pathway and Flammulina velutipes growth responding to stress furthermore.We carried out following experiments:Computational analysis on Flammulina velutipes gene fv-pkac-2;Analyzing the differential expression of this gene on mononuclear body,heterocaryon,and different growing period of mycelium;Analyzing the differential expression of this gene when treated with abiotic stress like temperature and sodium hydrogen peroxide;Constructing the fixed-point complement expression vector of Neurospora crassa based on the consequence analyzed by bioinformatics.Transferring the Flammulina velutipes gene fv-pkac-2 into Neurospora crassa pkac-2 gene deleted mutant strain(Apkac-2)through electrochemistry,and then confirmed the phenotype of positive transformants,analyzed the stress response and transcriptome.The main results are as follows:The ORF length of gene fv-pkac-2 is 1692bp,encoding 494 amino acids and containing 4 exons and 3 introns,and the length of these introns are 55bp,48bp and 122bp.We named the protein as FV-PKAC-2 and submitted it to EBI protein database to forecast its homologous domain;The phylogenetic tree showed that the protein holds together with other homologous protein of basidiomycetes and has high homology with Trametes versicolor,which illustrates the protein FV-PKAC-2 in Flammulina velutipesa has certain degree of conservatism.Real-Time Quantitative PCR(qRT-PCR)was used to analysis the expression of gene fv-pkac-2 in Flammulina velutipes,and the results showed that the expression of heterokaryon strains was higher than that of homokaryon strains;In different growth stages of hyphae,the expression of gene fv-pkac-2 significantly improved in cultivation material.Treated with the temperature and hydrogen peroxide stress,the gene fv-pkac-2 of Flammulina velutipes was up-regulated,while a certain down-regulated in sodium chloride stress,these results suggest that gene fv-pkac-2 has certain effect on mycelial growth,fruitbody development and resist abiotic stress.The monokaryotization of pkac-2 gene deletion Neurospora crassa strains(Apkac-2).Through the screening medium,pkac-2 single mutant homokaryon Neurospora crassa strain was screened out,and they were used as the experimental material for genetic transformation.The construction of site-specific compensation vector of Neurospora crassa.Using Neurospora crassa vector pCB1532 as the starting plasmid,the recombinant vector was constructed by enzyme digestion-ligation and one-step synthesis,and was named as fvpkac-2C.The site-specific compensation vector(fvpkac-2C)was transformed into Neurospora crassa pkac-2 homokaryon strains by electrotransformation,and positive transformants detected by PCR and gene expression were obtained,named Cpkac-2.The verification of Neurospora crassa transformant phenotype.The result indicated that the wide type(WT)and complement pkac-2 mutation(Cpkac-2)of Neurospora crassa had better mycelial growth than pkac-2 gene deletion Neurospora crassa strains(Δpkac-2).And the mycelial of Cpkac-2 was more dense,the mycelial morphology tended to move close to Flammulina velutipes.Analysis of Neurospora crassa transformant responding to abiotic stress.We treated the Neurospora crassa with hydrogen peroxide,sodium chloride and temperature stress to to explore the Flammulina velutipes pkac-2 gene expression in Neurospora crassa and the role of pkac-2 gene in growth and stress response,and found that the growth of wild type(WT)and complement strains(Cpkac-2)were better than that of sodium chloride and hydrogen peroxide,also,the survival rate of wild type strain(WT)and complement(Cpkac-2)was higher than that of gene deletion mutant in temperature stress.The results showed that Cpkac-2 was able to restore the stress tolerance.Transcriptome analysis of Neurospora crassa.We performed transcriptome analysis on the Neurospora crassa wild type(WT)、Neurospora crassa pkac-2 gene deletion strains(Δpkac-2),and fv-pkac-2 of Flammulina velutipes complement strain(Cpkac-2).After Verifying the accuracy of transcriptome sequencing,the homologous gene of Flammulina velutipes was found.After KEGG Gene annotation,we found the differentially expressed genes and the homologous gene of Flammulina velutipes may be involved in the metabolic pathways,biosynthesis of secondary metabolites,and biosynthesis of amino acids.Based on the above results,we speculate that Flammulina velutipes protein kinase A catalytic subunit encoding gene fv-pkac-2 plays important roles in mycelium growth and fruiting body development and also plays an important role in response to abiotic stress;At the same time,the fv-pkac-2 gene may play a role in the regulation of genes involved in gene transcription,amino acid synthesis and so on.