Establishment And Mechanism Analysis Of Laccase Transformation Systems In Hypsizigus Marmoreus Breeding

Hypsizigus marmoreus is one of the nutritious rare fungus for edible and officinal use,and it’s one of the important species of industrialization production. However, the long planting period (120-150d), low biological efficiency, easily microbiological contaminated and other problems which restrict the scale development of Hypsizigus marmoreus. Fungal laccases have many functions such as lignin degradation, phenols oxidation, contaminated microbe inhibition and fruiting-body formation. In this study, Pleurotus sajor-caju with high activity and Hypsizigus marmoreus with no activity of laccase were selected as experimental material. The laccase transformation systems in Hypsizigus marmoreus breeding was established by the technology of enzymology combined with protoplast fusion. Protoplast of Hypsizigus marmoreus and heat-treated protoplast of Pleurotus sajor-caju were fused by the fusogen. Two fusion strains Ⅲ18C,Ⅲ2A with higher activity of laccase were screened by the RB-PDA plate. The mycelial morphology and molecular biology identification, laccase genetic stability analysis, biological characteristics, production performance testing of the two fusion strains were studied. The main results were following:1. Preparation and selection of fusion strainsConditions optimized results of protoplast preparation and fusion showed that, the quantity and quality of protoplast is higher treated by2%lywallzyme; Fusion effect is better with the reagent containing fusion agent25%PEG(4000)50mmol/L CaCl2·H2O0.6mol/L mannitol,and the osmotic stabilizer0.6mol/L mannitol, Incubated for25-30minutes at32℃.Two fusion strains Ⅲ18C,Ⅲ2A both with higher laccase activity and faster growth rate were screened by the technique of inactivated protoplast and RB-PDA plate coloration from the regeneration of fusion protoplasts.2. Genetic identification of fusion strainsThe results of antagonistic test showed that there was an obviously antagonistic line between fusion strain and parents, and also two fusion strains have antagonistic line.The results of PCR amplification using RAPD,ISSR primers showed that, two fusion strains Ⅲ18C, Ⅲ2A not only had the same bands with the parents, but also had new bands. The similarity index analysis of amplified DNA profiles showed that, The genome of the fusion strain was similarity with Hypsizigus marmoreus, and the index is more than75%. The similar indexes of fusion strain Ⅲ18C different from Ⅲ2A vice-versa different with their parents. It was suggested that the two fusion strains genomes were different, and they were different strains.3. Analysis of genetic stability of fusion strainsDetected of laccase activity and identified the laccase gene of both fusion strain’s5th generation showed that, both of them can enable the RB-PDA plates to discolor, a conserved region of laccase gene was identified by PCR and the laccase activity were3.95U/mL,3.55U/mL detected by ABTS.These results of detection and amplification of laccase of pure culture strains gained by tissue isolation dovetailed with initial test. Result of experiment indicated that the two fusion strains had a good genetic stability, and laccase genes inherited steadily during subculture.4. Biological characteristics and production performance of fusion strainsMedium optimization, biological characteristics and production performance of fusion strainsⅢ18C,Ⅲ2A were studied. The results showed two fusion strains had a highest mycelial growth rate and strongest mycelial growth vigor on the PDA carrot medium, and the optimum temperature, pH and water content were25℃, pH6.5,60%-65%;Production period shortened by3-5d,biological efficiency was72.86%,70%, Compared with Hypsizigus marmoreus increased17.15%,14.29%;commercial characteristics was much better than Hypsizigus marmoreus. Experiment results indicated that the fusion strains Ⅲ18C,Ⅲ2A had good production and application values. Main nutrient determination and toxicological test results showed that fruiting body of fusion strain Ⅲ18C has a high nutritive value and safe to eat. The results show that the fusion strain has a broad large-scale commercial production prospects.