Characterization And Function Analysis Of Catalase Genes In Pleurotus Ostreatus

Pleurotus ostreatus is one of the most cultivated mushrooms in China,and has the third largest production levels.The development from mycelia to fruiting body of P.ostreatus is a complex process which is regulated by various factors both inside and outside.P.ostreatus is mostly cultivated under horticultural facilities,and hence high temperature,especially in summer,can affect its yield and quality.Catalase?CAT?is the most important enzyme for H₂O₂ decomposition,and abundant studies have shown that it is involved in stress response and developmental process of fungi.Study of CAT in edible fungi is limited,thus we combined omics and experimental method to conduct the study of CAT of P.ostreatus.The main contents are as follows:1.Genetic variation,gene structure and evolutionary status of catalase genes of P.ostreatus:CCMSSC03989 was selected to conduct de novo genome sequencing,and assembly of CCMSSC00389was improved.Two catalase genes were annotated in genome of P.ostreatus,and they were named CAT-1 and CAT-2,respectively.CAT-1 was located on chromosome 4 and CAT-2 was located on chromosome10.CAT-1 and CAT-2 showed much higher SNP density than the average level of the whole genome,and promoter region of CAT-1 had rich In/Dels.CAT-1?group II?encoded putative peptides of 745 amino acids,CAT-2?group III?encoded putative peptides of 528 amino acids.Furthermore,the gene structures were variant between CAT-1 and CAT-2.2.Expression patterns of CAT-1 and CAT-2 during development and under stress:During the developmental stages of mycelia?primordia?fruiting body?spores,CAT-1/CAT-1 was barely detected in mycelia,accumulated gradually during the development,and had maximal level in spores;CAT-2/CAT-2 had maximal accumulation in mycelia,decreased gradually during the development.CAT-2 activity can be detected in all the developmental stages,but CAT-1 activity was only detected in spores.CAT responded most obviously to heat stress than other abiotic stresses,such as oxidative stress,osmotic stress and acid-base stress.But CAT-1 and CAT-2 responded differently to heat stress,CAT-1 was up-regulated28-255 fold after 40°C heat treatment while CAT-2 showed up-regulation or down-regulation within 4fold depending on the different heat temperature or strains.3.Verification of gene function through overexpression:overexpression transformants of CAT-1 and CAT-2 were obtained through ATMT.The CAT-1 expression level of transformants OE_CAT1₅,OE_CAT1₉ and OE_CAT1₁8 were 26.7,15.8,and 19.7 fold than that of WT;the CAT-2 expression level of transformants OE_CAT2₉,OE_CAT2₁7 and OE_CAT2_K16 were 2.0,1.6 and 1.5 fold than that of WT.Overexpression of CAT-1 increased the expression levels of SOD,GSH-px and TrxR genes significantly,and inhibited the activity of CAT-2 to some extent.Overexpression of CAT genes enhanced the H₂O₂ resistance of mycelia to some extent,but the heat and 3-AT resistant ability were not increased.Primordium differentiation was hindered when CAT-1 was overexpressed.Transformants of CAT-2showed no obvious differences on development and agronomic traits compared with WT.However,the differentiation of primordium was hindered when CAT-2 activity was decreased by using 3-AT.Conclusion:the rich genetic variation,significantly different gene structure and evolutionary status of the catalase genes of P.ostreatus were confirmed.CAT-1 and CAT-2 showed different expression patterns during development and they both participated in the formation of primordium.CAT-2 is main catalase gene and CAT-1 is spore-specific catalase gene of P.ostreatus.Overexpression of CAT-1 exhibited an important influence on the formation of primordium.CAT responded most obviously to heat stress,but CAT-1 and CAT-2 showed different performance,and overexpression of CAT did not improve the heat resistant ability of mycelia.