Application Of Genetic Transformation System Based On Lignin Degrading Enzyme On Species Breeding Of Edible Fungi

The main purpose of breeding was to improve the quality and production of edible fungi. The traditional breeding method required much time and work because of lacking convenient markers for screening. Ligninases can degrade lignin which was the most important carbon source during the growth of edible fungi. New species were breeded by protoplast fusion technology based on enzymology properties of ligninases.The fusion strains were screened color changes on RB-PDA medium and aniline blue medium.The genetic transformation system of lignin degrading enzyme was established. Fusion strains I3CF1,14CF1,15CF1,17 with high ligninase activities were screened and their biological characteristics were analyzed. The results as followed:(1)Determination and comparation of lignin degrading enzymes of different edible fungi.The results showed 16 normal edible fungi were selected and their ability to produce ligninases were analyzed. The laccase activities of Pleurotus geesteranus, Pleurotus eryngii, Pleurotus pulmonarius were high and were respectively 1.4 U/mL, 4.1 U/mL and3.4 U/mL; The Lignin Peroxidase activities of Hypsizigus marmoreus, Auricularia auricular, Clitocybe maxima were high and were respectively 6.2 U/mL, 6.3 U/mL and 5.4 U/mL; The manganese peroxidase activities of Pleurotus ostreatus,Pleurotus citrinipileatus, Lepista sordida were high and were respectively 17.9 U/mL、23.5 U/mL,19.8 U/mL; The ligin degrading enzymes activities of Crifola frondosa were not detected activities. By analysis the results of growth rate of mycelium experiment and their abilities to produce ligninases, Pleurotus geesteranus and Pleurotus pulmonarius was selected to breed new species as metrical because of fast growth rates and high ligninases activities, Hypsizigus marmoreus 515 was selected to breed new species as metrical because of its long cultivation cycle, Crifola frondosa was selected to breed new species as metrical because of good nutritive value.(2)Protoplast fusion of different edible fungi strains. Hypsizigus marmoreus 515 Protoplasts and heat-treated Pleurotus geesteranus protoplast were fused with PEG mediator. Pleurotus pulmonarius Protoplasts and heat-treated Crifola frondosa protoplast were also fused with PEG. Fusion strains I3CF1,I4CF1,I5CF1 and 17 were selected on color changes by RB-PDA plate and Aniline Blue plate.The results of the antagonistic test showed that a dense antagonism line was formed between two strains.Fusion strains were identified with RAPD, ISSR and SRAP markers at molecular lever. The result showed these fusion strains had genetic differenties with their parental strains.The result indicted showed that fusion strains were new fusion strains.(3) Determination of biological characterstics and polysaccharide of fusion strains. The results showed the optional temperature and pH of fusion strain I5CF1 were respectively 24℃ and 6.0 and were higher than parental strain Hypsizigus marmoreus 515.The biology efficiency of I3CF1,14CF1 and I5CF1 were respectively 60.04%, 61.49% and 70.09%,and increased by 9.45%,10.05% and 11.50% compared to Hypsizigus marmoreu. The biological characterstics of fusion strain 17 was similar to these of Pleurotus pulmonarius.The result of polysaccharius content determination showed that polysaccharius content of fusion strain 17 was higher than Pleurotus pulmonarius that of and lower than Crifola frondosa. The polysaccharius content of fusion strain 17 was improved.