| 1,By using Tris-HCl(pH6.9), TBE, Tris- glycine(pH 8.3),PBS (pH6.4), Tris-HCl (pH8.0), Tris-HCl(pH 8.8) as extracting agent to extract peroxidase isozyme of Inonotusobliquus under the same condition, applying polyacrylamide gel electrophoresis to conductcomparative study of peroxidase isozyme bands of Inonotus obliquus mycelia. The resultsshowed that the sample applied the extracting agent with Tris-HC1 (pH 6.9) throughelectrophoresis showed the best extracting effect, it was affluent in bands and the bandswere also clearly; the worst extracting effect was extracting agent with Tris- glycine(pH8.3).2,By means of polyacrylamide gel electrophoresis, analysing peroxidase isozymes ofInonotus obliquus species. All the peroxidase isozyme of Inonotus obliquus strain shows2-8 enzyme zone, enzyme zone of all the strains are wider and deeper color at Rf=0.75 andRf=0.98, which is the characteristic enzyme zone of Inonotus obliquus. The Fuzzy clustermethod was used for the further study. The results indicate that the membership grade ofstrains is between 0.250 and 1.000. When homologyλ=0.250, the writer can take 3taxonomy on 8 strains. The results indicate among isozyme bands of each strain which is inthe same cultural stage, the consanguinity of part strains is close. The clusters results ofstrains is same with geographic distribution difference.3,An improved CTAB method is applied to obtaining genomic DNA. And it was used fortemplate then to optimize the reaction system of Amygdalus. The result indicated that thereaction mixture (25μL) for PCR amplication consisted of 40 ng template DNA,10 pmol10 base primer,2mmol/L Mg2+, 1unit TaqDNA polymerase, 100μmol/L dNTPs. The thermalprogramme for ampliication was predegeneration at 94℃for 5 min, denaturation at 94℃for 1 min, annealing at 40℃for 1 min, extention at 72℃for 1.5 min and 45 of cyclenumber, then the final extention at 72℃for 5 min.4,RAPD profiling of a collection of Inonotus obliquus strains isolated from sclerotia indifferent areas was performed in order to analyze the possible genetic variability The DNAfingerprints of different strains showed that there was genetic diversity among the strainstested. The results indicated that 12 random primers were used to generated 167 bandsRAPD fragment data by PCR. of which 101 bands showed polymorphism and the averagepolymorphism rate was 60.5％. This shows that strains can be analysis by RAPD. There is a little intraspecific difference among fingerprints of each strains. At the level of 0.508(genetic distance), UPGMA analysis of genetic distances calculated from RAPD fragmentdata produced a phyllogram that classified the entires into three main clusters: (1) CX01and CX02 (39002'～40048'), (2)JL04 and JL05 (41032'～43035'), (3) HLJ01 (50013'). Theclusters results of strains is same with geographic distribution difference, indicate that thereis intraspecies and geographic distribution difference among each strains, theconsanguinity of part strains is close.5,The effects of different carbon source, nitrogen source, pH value, illumination,temperature and water treatment on mycelium culture character of Inonotus obliquus werestudied. Taking the dry weight of mycelia as the major index, the optimized combinedmedium was obtained by quadratic general rotational combination design with three factors.The result showed that the best pure cultivation model for Inonotus obliquus is following:the optimum nutrition substance and the best dosage is glucose27.60g/L, soybean power15.60g/L, CaSO42.80g/L, KH2PO41.50g/L, VB110mg/L; the optimum pH is 5～7, theoptimum temperature is 25～30℃, complete darkness, appropriate concentration of CO2,the heaviest dry weight of mycelium is 19.02g/L.6,Taking the dry weight of mycelia as major evaluation index, at first to select the optimalcarbon source, nitrogen source, inorganic compound for the growth of Inonotus obliquus.The optimized combined medium was obtained by quadratic general rotationalcombination design with three factors. And study the effect of the medium capacity,culture temperature,pH,the spawning rate to the effect on mycelium culture character ofInonotus obliquus liquid cultivation on this basis. The experiment results showed that theoptimum formula for Inonotus obliguus liquid cultivation medium was as follows: glucose23.00g/L, peptone1.50g/L, MgSO41.00g/L, KH2PO41.5g/L, VB210mg/L. While theoptimum medium capacity,the culture temperature,the culture optimum pH,the spawningrate and the culture period were 140 ml, 25℃, 7.0, 170r/min, 10～12d, and the biggest dryweight of mycelia was 1.156g/L.7,Choosing wild sclerotium of Inonotus obliquus as material,by applying tissue isolatemethod to obtain the pure strain.The optimum mother medium was: glucose 20g,soybeanpowder 10g,KH2PO41g,MgSO40.5g, water1000ml, pHnature; the optimum originalspawn substrate was: corn or birch sawdust (crude) 78％,wheat bran 20％,casts 1％, white sugar 1％,water content 65％; the cultispecies substrate was:corn or birch sawdust(crude) 78％,wheat bran 20％,casts 1％,white sugar 1％,water content 65％;theoptimum formulae of compost of sclerotia artificial cultivation was birch sawdust(subtle)52％, corn-core 26％,wheat bran 20％, white sugar1％, casts1％and the water content65％whose biological efficiency reached to 30.8％; to obtain the sclerotium of Inonotusobliquus,the optimum condition was:temperature 22℃～28℃,the bag is astomatous andnonluminous.We suggested that log should be replaced with sawdust medium in artificialcultivation of Inonotus obliquus.8,The general nutrition component and active component among artificial cultivationmycelium of Inonotus obliquus, sclerotium and wild sclerotium were analysed andcompared. The general nutrition component of artificial cultivation mycelium ofInonotus obliquus is significantly higher than sclerotium and wild sclerotium, especiallythe content of crude protein, crude polysaccharide and ash. The content of mineralelements of artificial cultivation mycelium and sclerotium are significantly higher thanwild sclerotium;the polysaccharide content of artificial cultivation mycelium of Inonotusobliquus is obviously higher than fungus nucleus, the polysaccharide content ofsclerotiumis obviously higher than wild sclerotium.9,The triterpenoids among artificial cultivation mycelium, sclerotium and wildsclerotiumof Inonotus obliquus were analysed and compared by using HPLC. The resultsshowed that: in the same retention time,peak shape and peak area of three figs are similar,from which can conclude all these substances contain lanostane type triterpenoids withrelatively similar structure; the content of triterpenoids in artificial cultivation mycelium ofInonotus obliquus is the highest through analyzing and comparing peak area, secondly issclerotium.Artificial cultivation mycelium and sclerotium are both higher than wildsclerotium.10,Adopting alloxan (200mg/kg body-weight)antrum injection to copy the hyperglycemiamodels of mice. 3 different dosages of crude polysaccharide extracts from artificialcultivation mycelium, sclerotium and wild sclerotiumof Inonotus obliquus were used onmice through stomach feeding. Serum Blood glucose level was determined by glucoseoxidase method. The results showed that 3 different dosages of crude polysaccharideextracts from artificial cultivation mycelium have no inhibitory effect on blood glucose of alloxan-induced hyperglycemia rat model.The effect of crude polysaccharide extracts fromartificial cultivation mycelium, sclerotiumand and wild sclerotium of Inonotus obliquus onlowering blood glucose level had no significant difference.
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