摘要
1.采用Tris-HCl(pH6.9)、TBE、Tris-甘氨酸(pH8.3)、磷酸缓冲液(pH6.4)、Tris-HCl(pH8.0)、Tris-HCl(pH8.8)为提取剂,在同一条件下对桦褐孔菌菌丝体过氧化物酶同工酶进行提取,通过聚丙烯酰胺凝胶电泳技术,对桦褐孔菌菌丝体的过氧化物酶同工酶谱带进行比较研究,结果表明采用Tris-HCl(pH6.9)作为提取剂图谱条带丰富,带型清晰,提取效果最好。
2.采用聚丙烯酰胺凝胶电泳技术,对桦褐孔菌野生菌核分离得到的8个菌株菌丝过氧化物酶同工酶进行研究。结果表明,同一培养时期的各菌株间同工酶酶谱存在一定的差异,部分菌株间存在较近的亲缘关系,所有菌株酶带数目在2~8条之间,在Rf=0.75、Rf=0.98处所有菌株的酶带都较宽且着色较深,此为桦褐孔菌菌株的特征酶带。进一步采用Fuzzy聚类法进行数量分析表明,菌株间隶属度在[0.250,1.000]之间,当隶属度为0.250时将8个菌株分三类,菌株分类结果与各菌株的地理分布(纬度)差异一致。
3.采用CTAB改良法提取桦褐孔菌菌丝体基因组DNA,以其为模板对影响RAPD扩增的重要参数进行优化研究。结果表明,PCR扩增体系最佳反应条件是:在25μl体积中,模板DNA浓度为40ng/μL,10碱基引物浓度为10pmol,Mg~(2+)浓度为2mmol/L,TaqDNA聚合酶1unit,dNTPs浓度为100μmol/L。反应程序为:94℃预变性5min,94℃变性1min,40℃退火1min,72℃延伸1.5min,45个循环;72℃后延伸5min。
4.采用RAPD标记对8个桦褐孔菌菌株亲缘关系进行研究,获得了桦褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,表明RAPD标记可以用来对桦褐孔菌菌株进行鉴定;同一培养时期的各菌株间RAPD指纹图谱存在一定差异,若以遗传距离0.508为结合线,可将供试菌株划分为三大类,朝鲜菌株CX01、CX02(39~002’~40~048’)归为一类,吉林省菌株JL01、JL02、JL03、JL04、JL05(41~032’~43~035’)归为一类,黑龙江菌株HLJ01(50~013’)单独聚为一类。菌株分类结果与各菌株的地理分布差异一致,可以进一步确定各菌株间属于种内地理分布差异,部分菌株间存在较近的亲缘关系。
5.采用单因素试验设计研究了不同的碳源、氮源、无机盐和不同pH、光照、温度及水分处理对桦褐孔菌菌丝纯培养性状的影响,以菌丝体干重为指标,利用三元二次通用旋转组合设计得到了优化培养基配方。结果表明,桦褐孔菌菌丝体纯培养的最佳模式为:葡萄糖27.60g/L、黄豆粉15.60g/L、硫酸钙2.80g/L、磷酸二氢钾1.50g/L,VB_1 10mg/L、琼脂20g/L;pH值5~7、温度25~30℃、完全黑暗、适宜的二氧化碳浓度,菌丝体干重达19.02g/L。
6.以菌丝体干重为指标,采用单因素试验设计筛选出了适宜桦褐孔菌菌丝体液体培养的最佳碳源、氮源和无机盐,利用三元二次通用旋转组合设计得到了优化培养基配方,并研究了装液量、培养温度、pH值、摇床转速等对桦褐孔菌菌丝体液体培养性状的影响。结果表明,桦褐孔菌菌丝体液体培养最佳模式为:葡萄糖23.00g/L,蛋白胨1.50g/L,硫酸镁1.00g/L,磷酸二氢钾1.50g/L,VB_1 10mg/L;250ml三角瓶中装液量140ml,培养温度为25℃,培养初始pH值为7.0,摇床转速170r/min,接种量以一块为宜,振荡培养周期10~12d,菌丝体干重为1.156g/L。
7.以桦褐孔菌野生菌核为材料,采用组织分离方法获得纯菌种,并进行了桦褐孔菌各级菌种的制作研究。结果表明:桦褐孔菌母种培养基以葡萄糖20g、黄豆粉10g、KH_2PO_41g、MgSO_40.5g、水1000ml,pH自然为最佳,菌丝生长速度快、长势强、颜色正;桦褐孔菌原种培养基以玉米粒或桦木屑(粗)78%、麦麸20%、石膏1%、白糖1%、水分65%为最好;桦褐孔菌栽培种培养基以配方简单的木屑78%、麦麸20%、石膏1%、白糖1%、水分65%为最好;桦褐孔菌菌核人工代料栽培以桦木屑(细)52%、玉米芯26%、麦麸20%、白糖1%、石膏1%、水分65%为最好,生物学效率达30.8%;桦褐孔菌菌核发生适宜温度范围在22~28℃、栽培袋不开口、无光有利于菌核的形成。建议桦褐孔菌菌核人工栽培以代料栽培取代木段栽培。
8.对桦褐孔菌人工培养菌丝体、菌核与野生菌核常规营养成分、活性成分进行了分析与比较。结果表明:桦褐孔菌人工培养菌丝体的一般营养成分明显高于人工培养菌核和野生菌核,特别是粗蛋白、粗多糖、灰分含量较高;桦褐孔菌人工培养菌丝体、菌核的矿质元素含量均明显高于野生菌核;桦褐孔菌人工培养菌丝体多糖含量明显高于人工培养菌核、而人工培养菌核的多糖含量又明显高于野生菌核。
9.采用HPLC指纹图谱方法分析比较了桦褐孔菌人工培养菌丝体、菌核、野生菌核活性成分三萜类物质含量。结果表明:在相同的保留时间内,三个图谱的峰型与峰面积相似,可以判定它们均含有结构比较相似的羊毛甾烯三萜类物质;通过峰面积的分析比较可知,桦褐孔菌人工培养菌丝体中三萜类成分含量最高、其次为人工培养菌核,二者均高于野生菌核。
10.采用四氧嘧啶(200mg/kg体重)腹腔注射复制小鼠高血糖模型,分别给予小鼠桦褐孔菌人工培养菌丝体、菌核与野生菌核多糖提取物按不同剂量灌胃,葡萄糖氧化酶法测定各组的血糖水平,探讨桦褐孔菌人工培养菌丝体、菌核与野生菌核多糖的降血糖作用。结果表明:桦褐孔菌人工培养菌丝体多糖提取物不同剂量组对四氧嘧啶型高血糖模型小鼠的血糖均有抑制作用,对正常小鼠无明显降血糖作用;桦褐孔菌人工培养菌丝体、菌核与野生菌核多糖三者的降血糖作用无显著性差异。
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 Mg~(2+), 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 (39~002'~40~048'), (2)JL04 and JL05 (41~032'~43~035'), (3) HLJ01 (50~013'). 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, CaSO_42.80g/L, KH_2PO_41.50g/L, VB_110mg/L; the optimum pH is 5~7, theoptimum temperature is 25~30℃, complete darkness, appropriate concentration of CO_2,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, MgSO_41.00g/L, KH_2PO_41.5g/L, VB_210mg/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、KH_2PO_41g、MgSO_40.5g, 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.
引文
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