Annotation Of Pheromone Signaling Pathway And Functional Study Of Gene Ste12 In Flammulina Filiformis

China is one of the earliest countries in the world to recognize and cultivate edible fungi.With abundant Flammulina filiformis germplasm resources,China is the birthplace of artificial cultivation of F.filiformis and the country with the largest yield.Mycelium growth and fruiting body development are important links in production.In the current process of industrial cultivation of edible fungi,improving the ability of mycelia to resist abiotic stress and degrade substrates can shorten the culture period and obtain better economic benefits.The MAPK cascade pathways exists widely in various eukaryotes and are involved in cell division,differentiation,apoptosis and other life processes.The pheromone signaling pathway is a part of the MAPK cascade signaling pathway.The gene functional of the pheromone signaling pathway have been identified in yeast and many pathogenic fungi,and it is found that this pathway is involved in the mating,growth and development,morphogenesis and other life activities of yeast and filamentous fungi.In particular,the transcription factor gene ste12downstream of pheromone signaling pathway is a key factor of growth and development,and is involved in the regulation of sexual reproduction and osmotic stress.In this study,the genes related to the pheromone signaling pathway of F.filiformis were annotated first,and the gene ste12 was selected for functional research through expression analysis.Then,the overexpression transformant was constructed for the gene ste12,and the transformant strain of F.filiformis was obtained by Agrobacterium-mediated genetic transformation.Finally,the abiotic stress ability and substrate degradation ability of transformant strains were analyzed.The main results obtained in this study:1.Based on the genomic and transcriptional data of F.filiformis,the pheromone signaling pathway and upstream and downstream genes were annotated.Combined with the differential expression of genes,a key gene ste12,was finally screened.Bioinformatics analysis of gene ste12 confirmed that it was a gene homologous to other large fungi.2.The overexpression plasmid ste12-oe of the gene ste12 was constructed and successfully transformed into F.filiformis.Finally,4 strains of F.filiformis gene ste12 overexpression transformants were obtained.Compared with the wild-type strain FL19,the expression levels of gene ste12 in the transformant strains Ste12^OE8,Ste12^OE10,Ste12^OE14 and Ste12^OE15 were up-regulated by about 195 times,25 times,80 times and 110 times,respectively,by q RT-PCR.3.The growth rate of the ste12 overexpressing transformant strain and the wild type strain was measured.It was found that there was no significant difference in the colony morphology and mycelial growth rate on the PDA medium;on the culture medium,the fruiting time of the transformant strain is slower than that of the wild type.4.Through abiotic stress tests on wild type and transformant strains,it was found that when the gene ste12 was overexpressed,the hyphae’s resistance to salt stress,high temperature,low temperature,oxidative stress and acid resistance ability was significantly enhanced,but the alkali resistance ability was lower than wild type.5.By measuring the enzyme activities of the main cultivation substrate degrading enzymes,it was found that the enzyme activities of the three main lignin degrading enzymes（Lac,Li P and Mn P）,hemicellulase and carboxymethyl cellulase showed a trend of first increasing and then decreasing.The enzyme activities of the three main lignin-degrading enzymes and hemicellulase overexpressing transformants were higher than those of the wild type at each stage.However,the enzyme activity of the carboxymethyl cellulose overexpressing transformant was lower than that of the wild type at each stage.This study annotated the F.filiformis pheromone signaling pathway using bioinformatics methods based on the F.filiformis genomic data.Then,the function of gene ste12 involved in resistance to abiotic stress and substrate degradation of F.filiformis was analyzed using molecular biology techniques.The results of this study can provide a certain data reference for the genetic breeding and production cultivation of F.filiformis and other edible fungi.