基因组重排技术在草菇耐低温菌株选育上的应用-生物化学与分子生物学专业论文.docxVIP

上海海 上海海洋大学硕士学位论文 PAGE PAGE VI 由此可见，将基因组重排技术用于草菇的耐低温选育是可行的。本文首次将 基因组重排技术用于食用菌方面，为食用菌的选育提供了新思路。 关键词 草菇， 原生质体，诱变，突变体库，双亲灭活，递归式融合，基因组重 排 Selection of Volvariella volvacea with Low Temperature Tolerance by Genome Shuffling ABSTRACT The straw mushroom, Volvariella volvacea, is one of edible mushrooms cultivated in large areas, but it has lower antifreeze capacity. Low temperature (4℃) induced Volvariella volvacea mycelium autolyzed and fruiting body liquefied, which brings great difficulty to the preservation of strains and storage of fruiting body. Our study was the first to screen low-temperature-resistant strains of Volvariella volvacea using genome shuffling technology. Three low-temperature-resistant strains were obtained by parents inactivated mark and low-temperature screening condition. In this study, the conditions of preparation and regeneration of protoplast were optimizated and the mutant library was constructed. Main results were as follows: The optimum conditions of protoplast preparation of Volvariella volvacea were mycelium cultured for 60h, 0.6mol/L mannitol as osmotic stabilizer, 1.5% lywallzyme enzymolysis for 3h at 32℃. The optimum conditions of protoplast regeneration were mycelium cultured for 84h, 0.6mol/L mannitol as osmotic stabilizer, 1.5 % lywallzyme enzymolysis for 2h at 32℃, then cultured in constant temperature incubator at 32℃. The protoplasts of 16 strains were mutagenized with ultraviolet, chemical, electron beam and 60Co-?. 194 mutant strains were obtained through low temperature (0 ℃) selection and stability testing. These mutant strains could survive about 24h in 0℃. Their antifreeze capacity of 0 ℃ increased 1.4 times compare to original strains. The construction of mutant libraries laid a good foundation for genome shuffling. Mutants were chemical (PEG) fusion by ultraviolet-inactivated (20W, 30cm, 110s) and heat inactivated (50℃, 3min) parents inactivated notation. The results showed that the highest fusion rate was 0.517% at the