Identification Of Key Gene Controling Pileus Color And Preliminary Study Of Its Role In Lightening The Color Of Oyster Mushrooms Under High Temperature

Oyster mushrooms（Pleurotus spp.）are widely cultivated and consumed worldwide for their easy cultivation,high biological efficiency as well as the high edible value and the medicinal purposes value.Pileus color is an important commercial characteristic for oyster mushrooms.Oyster mushrooms of different color that fit consumer?s preferences will enhance the economic benefits of their cultivation.Little is known about the mechanisms of pileus color formation in oyster mushroom,which limits molecular breeding for this essential trait.In this paper,the chemical composition and molecular mechanism of pileus color formation were analyzed by using oyster mushrooms with typical pileus color,i.e.,Pleurotus cornucopiae,Pleurotus citrinopileatus,Pleurotus djamor with blackish grey,yellow and pink pileus color respectively.The mainly results are as follows:1.Isolation and identifcation of pigments from oyster mushrooms.In this study,pigments were extracted and identified in oyster mushrooms of three different species with different pileus color,i.e.,P.cornucopiae,P.citrinopileatus and P.djamor with blackish grey,yellow and pink pileus,respectively.The pigments were extracted by acids/bases method,appearing the three color types correspondingly to their pileus color.The characteristics of solubility and chemical reaction of the pigments of oyster mushrooms were typically conformed to that of melanin.The pigments of three oyster mushrooms were all identified as melanin using a panel of spectroscopic and physical/imaging techniques,mainly containing phenolic hydroxyl,carboxyl,amidogen,carbonyl,methylene and methyl groups.Nevertheless,HPLC（high-performance liquid chromatography）and elemental analysis indicated that the melanin in oyster mushrooms was actually a mixture of eumelanin and phaeomelanin.The differences of quantities and relative proportions of eumelanin and phaeomelanin resulted in differences in pileus color.The overwhelming content of eumelanin in P.cornucopiae determined the pileus color of blackish grey.The higher content of pheomelanin of P.citrinopileatus and P.djamor adjusted the color of pileus into yellow and pink.Electron microscopy studies showed that the melanin units were likely located in the cell wall.2.Fine mapping and functional analysis of the gene PcTYR involved in control of pileus color of P.cornucopiae.We selected a dark homokaryon（BPC067）from the previous segregating population and a pure white homokaryon 358P1 as homokaryotic parental lines which generated a population that segregates for both dark and white alleles.A 0.8-Mb major QTL region controlling pileus color in the oyster mushroom P.cornucopiae was mapped on chromosome 7 through bulked-segregant analysis sequencing（BSA-seq）and extreme-phenotype genome-wide association studies（XP-GWAS）.Candidate genes were further selected by comparative transcriptome analysis,and a tyrosinase gene（PcTYR）was identified as the most confident candidate gene.q PCR analysis suggested that the expression level of PcTYR was negatively correlated with the WI of pileus color（r=-0.727,p<0.01）.Overexpression of PcTYR resulted in a significantly darker pileus color,while the pileus color of RNA interference（RNAi）strains for this gene was significantly lighter than that of the wide type（WT）strains,suggesting that PcTYR plays an essential role in pileus color formation.Four SNPs were detected through sequence comparisons of PcTYR between the dark and white strains,which may affect gene expression.3.The molecular mechanism of high temperature lightening pileus color of oyster mushroom was clarified preliminarily.The pileus color of P.cornucopiae was significantly lighter under 26°C than that under 18°C at both of young and mature fruiting body stage.This color difference was larger at young stage.The expression level of PcTYR gene and tyrosinase（TYR）activity decreased significantly under high temperature,which decreased by 28.73%and 47.62%,respectively,indicating that high temperature lightened the pileus color by inhibiting the expression level of PcTYR,thus reducing TYR activity.In order to further explore the pathways regulating PcTYR gene under high temperature,pileipellis of young fruiting body at 18°C and 26°C were collected for transcriptome analysis.A total of199 differentially expressed genes（DEGs）were identified.GO and KEGG functional enrichment analysis showed that DEGs were significantly enriched in the pathways of cell metabolism,membrane part,signal transduction and amino acid,glucose and lipid metabolism.High temperature significantly affected the respiratory electron transport chain pathway and redox homeostasis,suggesting that ROS accumulation was induced by high temperature.Meanwhile,the pathway of protein processing in endoplasmic reticulum（ER）was significantly up-regulated under high temperature,suggesting that high temperature induced ER stress.The above two pathways were verified:Firstly,H₂O₂ content was significantly increased by 9.6 times at high temperature,proving that high temperature promoted ROS accumulation.The expression level of PcTYR gene and TYR activity were reduced significantly by exogenous addition of H₂O₂,while addition of ROS scavenge agent（DMTU）alleviated the downward trend of both,suggesting that ROS accumulation reduced the pileus color by inhibiting the expression level of PcTYR gene and TYR activity.Secondly,marker genes of ER stress Bi P,PDI and CNX were significantly upregulated by8.2,3.93 and 1.53 times,respectively,proving that high temperature induced ER stress.Experiments with exogenous addition of H₂O₂ and DMTU further indicated that high temperature induced ER stress via ROS accumulation.Exogenous addition of tunicamycin（TM）simulated ER stress,which resulted in lighter pileus color.However,the overexpression of the Bi P gene could significantly alleviate the lightening of pileus color under high temperature,suggesting that ER stress negatively regulated the formation of pileus color.q PCR and enzyme activity assay showed that ER stress did not affect the expression level of PcTYR,but inhibited TYR activity under high temperature.In conclusion,high temperature down-regulated the expression level of PcTYR by promoting ROS accumulation,thus reducing TYR activity;Meanwhile,ER stress induced by ROS accumulation under high temperature reduced TYR activity,leading to lighter pileus color.In this study,pigments in oyster mushrooms were extracted and identified;the key gene controlling pileus color in oyster mushrooms was fine mapped,and the gene function was verified;the molecular mechanism of high temperature lightening the pileus color in oyster mushroom was clarified primarily.These results will enhance our understanding on the formation mechanism of pileus color in oyster mushrooms and will facilitate molecular breeding for this essential trait.