Breeding Of Thermotolerant Strains Of Stropharia Rugosoannulata And Study On Its Thermotolerant Mechanism

Stropharia rugosoannulata has good taste and color,and has high edible and medicinal value.In recent years,it has been favored by the market and consumers.The artificial cultivation area has expanded year by year and the industry has developed rapidly.As the cultivation area continues to expand,the constraints that restrict the development of the industry are increasingly prominent.When the temperature of the cultivation material exceeds30℃for a long time,mycelium is prone to"burns"and pollution and other undesirable phenomena.When the air temperature exceeds 25℃,the fruit body quality deteriorates and the cap is easy to open.Therefore,the breeding of thermotolerant varieties of Stropharia rugosoannulata is greatly significant to broaden the cultivation season of Stropharia rugosoannulata and improve its yield and quality.Exploring the thermotolerant mechanism of Stropharia rugosoannulata thermotolerant strains has important scientific significance and application value for future breeding work.The main findings are as follows:1.Research results of monospore hybrid breedingUsing single spore hybrid breeding technology,they have undergone primary screening,double screening,antagonistic tests,subculture,SRAP based on molecular marker genetic analysis and mushroom test,high-quality and high-yield high-temperature-resistant strain J8L4 was finally selected.The mushroom grew well under the conditions of the highest material temperature of 27℃～34℃and the highest temperature of 40℃～47℃for 7 days.The mushrooms were produced at 40 days,yield of 14.56kg/m~2,and the proportion and biotransformation rate of Grade 1 mushrooms reached as high as 66.21%and 91.03%.An invention patent has been applied(Shanongqiupai No.3,201910546457.5)to provide high-quality,high-yield,high-temperature-resistant strains for industrial development.2.Research results on thermotolerant mechanism of Stropharia rugosoannulata(1)Determination of physiological indexes of different heat-resistant Stropharia rugosoannulata mycelium in high temperatureWe used thermotolerant strains and heat-sensitive strains as materials,the content of trehalose,thiobarbituric acid reactant(TBARS),superoxide dismutase(SOD)and catalase in the mycelium under different temperature stress(CAT),peroxidase(POD)and glutathione reductase(GR)activities were measured to study the response of different heat-sensitive Stropharia rugosoannulata to high temperature stress.The results show that the heat resistance mechanism of trehalose and TBARS,CAT,SOD,GR,and POD in the mycelium of different thermotolerant strains was revealed under the high temperature stress.The TBARS content of the two strains increased first and then decreased,and the TBARS content of the heat-sensitive strains increased significantly at 0～60h of high temperature stress.The overall activity of CAT,GR,and POD changed first increased and then decreased,while SOD activity did not change greatly before 0 to 36h of stress,and continued to increase after 36h of high temperature stress.The four antioxidant enzyme activities of thermotolerant strains were higher than those of heat-sensitive strains.Among them,the CAT activity increased the most.The trehalose content in the mycelium of the thermotolerant strains increased first and then decreased.The trehalose content reached the highest at 36h under high temperature stress.The trehalose accumulation of the heat-sensitive strain showed a trend of rising first,then falling,and then rising and then falling.It reached the highest value at 6h under high temperature stress.The increase in trehalose content of the thermotolerant strain was significantly larger than that of the heat-sensitive strain,and the change trend was relatively stable.The test results showed that under high temperature stress,the mycelium can alleviate oxidative damage caused by high temperature and improve its thermostability by adjusting the activity of autogenous antioxidant enzymes and the content of trehalose.(2)Transcriptome analysis of different thermotolerant Stropharia rugosoannulata under high temperature stressTranscriptome sequencing of thermotolerant strains and heat-sensitive strains after high-temperature treatment at 38℃for 0h and 24h was performed to investigate the response mechanism of Stropharia rugosoannulata under high temperature stress through gene expression analysis and provide theoretical reference for related breeding research.The GO functional cluster analysis showed that differentially expressed genes were mainly concentrated in cell walls,cell membranes,components of the endoplasmic reticulum,biological processes,molecular functions,and pheromone receptor activity.KEGG functional enrichment showed that the differential genes were mainly concentrated in the pathways of glycerophospholipid metabolism,starch and sucrose metabolism,and protein processing in the endoplasmic reticulum.Among them,HSP20,HSP40,HSP70,HSP90 and other heat shock proteins,HSP70 nucleotide exchange factor Hsp BP1,chaperone protein Gro EL,pyruvate decarboxylase(PDC),endoplasmic reticulum type I mannosidase(ERMan I),catalase(CAT),superoxide dismutase(SOD)and other genes were up-regulated,while sphingomyelin phosphodiesterase 1(SMPD1)and trehalose-6-phosphate esterase(TPP)were down-regulated.The mobilization range of the thermotolerant strains was significantly higher than that of heat-sensitive strains.It was speculated that the above genes were related to the thermal tolerance of Stropharia rugosoannulata.At the same time,RT-q PCR was used to verify the expression of related genes,and the expression levels of related genes were consistent with the results of transcriptome analysis.