Analysis Of The Transcriptome At Various Phases Of "Masangjun"(Lentinula Edodes) Furit Body Development And Identification Of Genes Associated With Pileus Opening

The pileus opening process,in which the spores fall off as the pileus opens,is essential to the growth of macrofungals.The substrates that are not opened by unpileusped mushrooms are chosen early in the commercialization and selling process in order to prolong post-harvest storage and boost economic efficiency.However,the quality of the substrates,including nutrition and appearance,decreases sharply after pileus opening.We discovered the trait of facile opening of the fruit body pileus of the Le.M-sd2 strain during artificial culture in this work by domesticating the wild Lentinula edodes(Le.M-sd2)and comparing it with other shiitake mushroom species,and we specifically targeted the early bud stage(EBS),young fruit body stage(YFB)when the mycelium is about to tear,and mature fruit body stage(MFB)of Le.M-sd2 fruit bodys.The transcriptome was sequenced over the course of the three distinct periods,and enrichment analysis was used to identify the major differentially expressed genes at the time of membrane tearing.Agrobacterium transformation was then used to confirm the impact of candidate genes on the phenotype of pileus opening.The following are the key conclusions:1.The strain "Masangjun" Le.M-sd2 was identified as belonging to the genus Shiitake using morphological and ITS sequence identification.The biological traits of Le.M-sd2 were examined after the strain had been purified,and it was determined that no light,24°C,p H=7-8,and fructose,beef paste,and magnesium sulfate were the optimum sources of carbon,nitrogen,and inorganic salt for mycelial production.2.Comparison testing of different strain variations was carried out.According to the findings,Le.M-sd2’s mycelium was more active under the same growth circumstances,and the tuberous bulge first developed in the sticks.Le.M-sd2 pileus was shown to have a greater amino acid content than other mushroom species,accounting for 40.50±0.72%(4,876.67 mg/100g)and 7.23±0.33%(870.00 mg/100g)of the total free amino acids,respectively.This may help to explain why the intake of Le.M-sd2 pileus This may clear up the fundamental misunderstanding of how eating "Masangjun" makes you feel more pleasant and delightful.Also,compared to the other two marketed mushroom species,Le.808 and Le.0912,Le.M-sd2 had a considerably lower crude fat content and a higher concentration of crude polysaccharides(p < 0.05).3.A KEGG enrichment analysis of transcriptome data was used to identify the pileus opening process(membrane tearing)from the level of gene transcriptional differences,which were primarily enriched for pentose glucuronide conversion,the longevity regulation pathway,starch and sucrose metabolism,and glycerophospholipid metabolism.From the enriched pathways,we screened the genes that were differentially expressed for three characteristics.The enrichment route was used to screen the highly variable open pileus candidate genes hsp70,α-amy4,and psd,which respectively encode heat shock protein 70,alpha-amylase,and phosphatidylserine decarboxylase.4.The antibiotic susceptibility experiment with the Le.M-sd2 strain revealed Hyg larger than 4 μg/m L as the deadly concentration for following positive strain screening and also found that Cef would not be lethal to Agrobacterium marcescens mycelium,but there would be some growth inhibition.A Cef concentration of 400μg/m L was chosen for the subsequent test to wash the co-culture blocks in order to completely inhibit the growth of Agrobacterium equi EHA105 and prevent the presence of Agrobacterium contamination from influencing the test results,according to the sensitivity experiment of Agrobacterium equi EHA105 to Cef.5.Three previously screened open candidate genes,hsp70,α-amy4,and psd,were homologously cloned from strain Le.M-sd2 using high fidelity(2×Phanta Max Master Mix),and their identification by agarose gel electrophoresis and sequencing indicated successful cloning.Primers were designed based on data from the shiitake genome(G408PP-4).6.The overexpression recombinant vectors OE-hsp70,OE-α-amy4,and OE-psd for three candidate genes were successfully built and confirmed by twofold digestion utilizing Bgl II and Spe I without mistakes using plasmid p CAMBIA1303-gpd A-GFPTrpC-Hygro as the original framework.7.The mycelium and juvenile hyphae of Le.M-sd2 were successfully infected using the Agrobacterium tumefaciens transformation method,and it was determined that the overexpression strain of the candidate gene differs from the wild-type Le.Msd2 in terms of the change in opening degree.