Protein Arginine Methyltransferase PRMT5 Participates In Regulation Of Ganderic Acids Biosynthesis In Ganoderma Lucidum Under Heat Stress

Ganoderma lucidum has high economic value,and its secondary metabolite triterpenoids（Ganoderic acids,GAs）are its main medicinal ingredients.With the completion of the Ganoderma genome sequencing and the continuous improvement of the genetic system,it has provided a basis for the study of the molecular mechanism regulating the biosynthesis of GAs.During the growth and development of microorganisms,it is affected by the environmental stress of heat stress.It is particularly important to study how microorganisms perceive heat stress and then change secondary metabolism.Protein arginine methyltransferase PRMT5 is a methylase that can modify protein arginine sites,mainly regulates the structure of chromosomes and gene transcription through symmetrically dimethylated histone H4/H2A N-terminal arginine residues and affects the activity of target proteins by methylating non-histone substrates.This paper investigated the role of the protein arginine methyltransferase PRMT5 in regulating the biosynthesis of GAs under heat stress and response to heat stress.First,this study cloned the GlPrmt5 gene in Ganoderma lucidum,constructed GlPrmt5-silenced and overexpressing strains,and studied the basic physiological functions of GlPrmt5 in Ganoderma.The results of phenotypic analysis showed that:1)the mycelium growth of the GlPrmt5-silenced strain was slower,and the mycelial growth of the overexpressing strain was unchanged;When subjected to heat stress,cell wall stress and oxidative stress,the growth of the GlPrmt5-silenced strain slowed down,while the growth rate of the overexpressed strain was higher than that of the wild type;2)GAs content of GlPrmt5-silenced strains increases,while GAs of overexpressing strains do not change;3)H4R3me2S and SmD3me2S protein levels of GlPrmt5-silenced strains decrease,while H4R3me2S protein levels of overexpressing strains increase,there was no change in SmD3me2S protein levels.These results indicate that GlPrmt5 is involved in regulating the growth,the biosynthesis of GAs,and stress response of Ganoderma lucidum.Next,in order to explore the mechanism of GlPrmt5 involved in regulating the growth,secondary metabolism and stress response of Ganoderma lucidum,H4R3me2S antibody was used to perform chromatin immunoprecipitation（ChIP）experiments,and ChIP-seq and RNA-seq combined analysis were performed.Studies on the involvement of GlPrmt5 in the regulation of GAs under heat stress found that:1)Under heat stress,the level of H4R3me2S protein in wild type gradually increased with the increase of heat shock time,however,the level of H4R3me2S protein in GlPrmt5-silenced strains were no longer elevated;2)the content of GAs in the GlPrmt5-silenced strain increase;moreover,and the content of GAs in GlPrmt5-silenced strains were higher than wild-type under heat stress;3)analysis of ChIPseq and RNA-seq results shows that under heat strss,the level of H4R3me2S modification near the promoter of HMGR and CYP51 in the GlPrmt5-silenced strain became lower,which are two key genes in the triterpene metabolic pathways,but the transcription levels were increased.These results collectively indicate that GlPrmt5 is involved in regulating the biosynthesis of GAs under heat stress by modifying H4R3me2S of HMGR and CYP51.In addition,research on the mechanism of GlPrmt5 in response to heat stress found that:1)Under heat stress,the SmD3me2S protein level in wild type gradually decreased with the increase of heat shock time,but the SmD3me2S protein level no longer decreased after heat shock in GlPrmt5 overexpressing strains.2)Scanning electron microscope observed that the GlPrmt5-silenced strain shrank compared with the wild type strain,and was more obvious under heat stress;3)The GlPrmt5-silenced strain had a thinner cell wall than the wild type strain;4)Analysis of RNA-Structural variants in RNA-seq found that the introns of the receptor protein kinase NIK1 in the cell membrane surface of the GlPrmt5-silenced strain and the heat-shocked wild-type strain were abnormally spliced,and transcriptome data showed the expression levels of most genes in the MAPK cascade signal pathway decreased.The above results indicate that GlPrmt5 responds to heat stress and regulates cell wall synthesis through alternative splicing of NIK1.