The Study Of Early Screening Techonology By Measurment Of Laccase Activity On Lentinula Edodes Breeding

Lentinula edodes(Berk.)Pegle has a delicious taste,tender meat and rich nutrients.In the past 10 years,the production of L.edodes in China has increased by 70%,and its proportion in the total edible fungus production has also increased from 17% to 26%.Half of the cultivated varieties of L.edodes used in China originated abroad.Monospore hybridization is the main method for the production of L.edodes.About 60% of cultivars are obtained through selection and breeding of this method.However,the biggest obstacle in the hybridization of L.edodes is the low proportion of strains that can normally form fruiting body in the hybrid population.Many strains were removed during the mycelium culture and the shapeless primordia stage,and only 40% had the ability to form normal fruiting body.After comprehensively considering other agronomic traits,the proportion of excellent strains was less than 1%.At present,the selection method of progeny in L.edodes breeding is mainly to observe agronomic traits and select excellent individuals through 2-3 rounds of cultivation tests.The purpose of this study is to obtain an early screening technique for the viable strains or excellent strains in the breeding population by detecting the early physiological,biochemical or molecular biological characteristics of the strains in the laboratory stage.Laccase is the main enzyme in the lignin-degrading enzyme system,widely distributed in plants,microorganisms and edible fungi.Laccase activity was detected at the mycelium,brown mycelial mat,primordium,young fruiting body and mature fruiting body stages of L.edodes,and the laccase activity was different at different stages,and there were great differences among different strains(such as wide-range-weather?cold-weather).In this study,laccase was used as the research object to establish an early screening technology for L.edodes breeding,and to analyze the mechanism of the fruiting ability of L.edodes.As a qualitative detection method,the plate color method cannot obtain accurate values.The spectrophotometric method is simple,low cost,and has high accuracy,but it is not suitable for large numble of strains.Therefore,we combined the advantages and disadvantages of the plate color method and spectrophotometry to develop a set of high-throughput quantitative methods for the detection of laccase activity.Using the one-way agar diffusion principle,a 1%low-melting agarose medium containing 2 mmol / L of guaiacol solution was added to a 24-well cell culture plate.The agar disk or crude enzyme solution was placed in the detection medium,and then placed in a 28? constant temperature incubator for diffusion reaction.After incubation,and the absorbance value of the each cell was read at 465 nm by microplate reader,with three repeats,one contrast.Through the single factor test on the diffusion reaction time,the optimal reaction time was determined to be 8-12 h.The established laccase activity detection method was used to detect the dynamic enzyme activity of 36 known phenotypes of L.edodes strains on the PDA,PDA plus vanillic acid(VC)and sawdust-based medium,and the strains were classified and analyzed according to the law of enzyme activity change.A bivariate correlation analysis was performed on the laccase activity and average growth rate,mycelial mat,the brown film formatted on the artificial logs,and the fruiting ability on these three media.Only the fruiting ability had a significant positive correlation with the laccase activity on non-induced PDA medium.On the PDA medium,the pearson correlation coefficient between the laccase activity and the fruiting ability on 4th day and 5th day reached 0.5,showing a very significant correlation.In order to verify the correlation between laccase activity and fruiting ability,a total of 127 hybrid strains were constructed,which was divided five hybrid populations,such as QL8,L86,L62,SQ,and SZ,and then correlation analysis was performed between the laccase activity measured on the two media(PDA andsawdust-based medium)and the fruiting ability of the strains.The analysis results verified that there was a very significant correlation between laccaseactivity on the PDA with fruiting ability.Two of these groups,L86 and L62,were not related,and the two groups contained a common parent,L60.According to the correlation of the verification population,different thresholds of laccase activity were set,and this threshold was used as a screening criterion.When THR = 1,41.73% of the strains can be eliminated,of which only13% have the fruiting ability.After eliminating these strains,the fruitng rate of the remaining population increased to 32.43%;as the threshold continues to expand,the The fruiting rate has increased from the initial 24% to 53.85%,which has more than doubled,but correspondingly,the probability of mis-screening the mushrooms has also increased to 16.83%.In order to further understand the relationship between the fruiting ability and laccase gene function of L.edodes hybrid strains,163 strains were divided into three groups accroding according to the different laccase activities and the fruiting abilities,and then sequencing of mixed pool transcriptome was performed from PDA and sawdust-based medium Upsampling.Through GO analysis and comparison of different genes,five candidate genes related to laccase activity and fruiting ability,such as Lac1,Lac6,Lac14,hydrophobin SC3,and alternative oxidase,were finally selected.Except Lac14,the expression level of several other genes were up-regulated on the PDA.Sequencing results of transcriptomes with different laccase activity and fruiting ability provided a lot of data support for laccase functional genes and the fruiting mechanism of L.edodes.