Establishment And Mechanism Analysis Of Lignin Oxidation Enzymatic System Of Transformation Systems Were Used In Fuse Breeding Of Hypsizigus Marmoreus

Hypsizigus marmoreus, also named as Hypsizigus or mushroom crab, belongs to Agaricales, Tricholomataceae and Hypsizigus. However, the development of Hypsizigus marmoreus is restricted due to long fruiting period, low biological efficiency, easy contamination with mold and other problems. In this study, we established the technology of enzymology combined with protoplast fusion to enhance the biological efficiency, strengthen the ability of antibiosis and shorten the fruiting period of Hypsizigus marmoreus.Firstly, Clitocybe maxima and Hypsizigus marmoreus were selected as experimental materials. Protoplast of Hypsizigus marmoreus and heat-treated protoplast of Clitocybe maxima were fused by the protoplast fusion technology. Four fusion strains ⅠM1, ⅡM1, IIIM3, and ⅢM5 with higher activity of lignin oxidase were screened by RB-PDA plate and aniline blue plate.Secondly, compared the mycelial morphology of fusion strains ⅠM1, ⅡM1, ⅢM3 and ⅢM5, we found fusion strains ⅠM1 and ⅢM5 have the obvious lock shape union, the typical binuclear cells and the thin hypha diameter as it is in Hypsizigus marmoreus. The results of antagonistic test showed that there was an obvious antagonistic action between fusion strain and parents, and four fusion strains also have antagonistic action among them. Analysis of PCR amplification using RAPD, ISSR primers showed that two fusion strains ⅠM1,ⅢM5 had some new bands except the same bands as the parents. In addition, there are the different bands between the two fusion strains. The bands of DNA amplification demonstrated that there was higher similarity between Hypsizigus marmoreus and ⅠM1,ⅢM5 than Clitocybe maxima, which indicated that fusion strain’s genomes were similary to Hypsizigus marmoreus. Whatmore, the differences also appeared between two fusion strains genome. However, the bands of DNA of fusion strains ⅡM1,ⅢM3 are identical with Hypsizigus marmoreus. Therefore, fusion strains ⅡM1, ⅢM3 might be Hypsizigus marmoreus. Ultimately, fusion strains ⅠM1,ⅢM5 with heterologous traits were confirmed furtherly.Lastly, biological characteristics of fusion strains IM1, IIIM5 were investigated. The results showed the optimum temerature of two fusion strains was 24 ℃, the optimum pH of IM1 and IIIM5 were 8.0 which was the same as Clitocybe maxima in PDA plate, and the optimum lime content was 2% in the cultivation of compound. While the optimum pH of Hypsizigus marmoreus was 7.0 in PDA plate and the optimum lime content was 1% in the cultivation of compound. We also discovered that Ca2+ may promote the growth of hypha. Addition of carbon source, nitrogen source and ion had great effects on the growth speed of hypha and the shape of hypha. The test indicated that the hypha growths of fusion strains and Hypsizigus marmoreus were very fast, and hypha was pure white and thick. Edible fungin with hight growth ability and excellent hypha shape is useful in future study.The optimum water content for fusion strains is 60%-65%. The result of fruiting test showed that the biological efficiencies of fusion strain IM1 and the IIIM5 were increased 14.58% and 4.67% respectively, compared with the Hypsizigus marmoreus. And the fruiting period of fusion strain IM1 was shortened by 4-6 days. Experiment results indicated that the fusion strains IM1 had good production and application values. However, the biological efficiency of fusion strain IIM1 was only 17.28% which was lower than Hypsizigus marmoreus. It is indicated that there are both advantage and disadvantage in fusion strains due to randomness of fusion.