Selection For Cold-Resistent Strain By Mutagenesis Of Volvariella Volvacea And Molecular Biological Identification

Volvariella volvacea commonly known as the Chinese Straw Mushroom is an important edible fungus of the tropics and subtropics. Chilling injury also has been found in this mushroom. Low temperature induced V.volvacea cell protein degradation and resulted in mycelium autolyzed, which brings great difficulty to the cultivation and fruiting body storage. Fixing low temperature as selection index, cold resistant strain VH3 was obtained through protoplast complex mutagenesis, the mutant mechanism was also studied. The results indicated:1. The optimum conditions of protoplast preparation, regeneration of V. volvacea were mycelium cultured for 3 days,0.8mol/Lmammitol as osmotic stabilizer,2% lywallzyme enzymolysis for 2h at 32C with pH natural(tested ,repeated Wang Fude' experiment).2. The protoplast was mutagenized with UV, 60Co- y and DES, four mutants which could survival about 10 days in 4C were obtained from 1500 mutants. The mycelia growth rate of the four mutants was higher than the control except VH1 at comparatively low temperature(18, 22, 26, 28, 30C).3.There was antagonism reaction between the mutants and control. The mean mutation coefficient was 0.213 by RAPD analysis. The biological efficiency of VH at 26C, 28C, 30C was higher than the control strain at significant difference level.The optimum cultivation temperature of VH3 was 28C, VH3 can also be cultivated at 26C. The strain VH3 was genetically stable, and can be cultivated at comparatively low temperature. 4.The storage experiment at 4C , 15C , 25C showed that ,all the mutants were higher than the control,at 4C VH1,VH3 were 50% higher than the control.At 15C the storage life was the best,then was at 25C,at 4C was the worst.The amino acid of the fruiting body analysis between the mutant and control showed, there was no apparent difference of thetotal amino acid, the Glu of VH3 was higher than control.5. Isozymes analysis showed that variation existed between VH3 and control strain on bothzymogram and activity of peroxidase isoenzyme and esterase isoenzyme.Cold shockprotein PZ1 was found in soluble proteins of V. volvacea mycelium when treated with lowtemperature(4C) for two hours, but the protein didn't found in VH3,protein PZ2 was foundin VH3. The result of SDS-PAGE indicated that PZ2 was composed of one polypeptidewith the molecular mass of 75KD,PZ1 is composed of two subunits, which molecular massare 42KD,21KD. The isoelectric point of PZ1 and PZ2 are 5.75, 4.8.6 Northern hybridization showed that, a small quantity of the cold induced gene fragmentexpressed in VH3.Analysis showed that VH3 was a good strain which could be cultivated at comparatively low temperature(26C,28C). A small quantity of the cold induced gene fragment expressed in VH3. There was some common ground of the cold resistant mechanism between V. volvacea in low temperature and mutant VH3 .