【作者】 郑亚凤；

【导师】 郑金贵； 谢宝贵；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 收集了全国26份姬松茸和29份灰树花茵种,筛选出高多糖的姬松茸和灰树花品种,并研究其多糖的生物活性,主要结果如下:1.通过RAPD、SRAP、ISSR分子标记和ITS序列扩增对26（A01～A26）份姬松茸菌种过行多态性分析,综合四种分子标记的结果,采用类平均法（UPGMA）进行聚类分析,姬松茸可分为4大类.其中第2类的A08和A10茵种间的相异系数为0;第3类的A15、A16、A17菌种间的相异系数为0;第4类的A20、A21、A22菌种间的相异系数为0;这些菌种是同种异名,为同一个栽培种.通过RAPD、SRAP和ISSR的综合聚类分析,29份灰树花菌种（G01～G29）可分为8大类.其中第3类的G15与G16菌种间的相异系数为0;第8类的G07和G10菌种间的相异系数为0;第8类的G14、G18、G21、G26、G27菌种间的相异系数为0;这些菌种是同种异名,为同一个栽培种.2.采用正交法,对热水浸提法,超声辅助热水浸提法及微波辅助热水漫提法的姬松茸、灰树花多糖提取工艺进行了优化.姬松茸多糖热水浸提法的最佳工艺为:pH值7.5,料水比为1∶20,提取温度为100℃,提取时间为4 h,醇沉浓度为100%,多糖提取率为12.17%,多糖含量为64.84%.超声辅助热水浸提法的最佳条件为:超声功率600W,超声时间4min,漫提时间2h,多糖提取率为13.76%,多糖含量为54.97%;应用超声波可提高姬松茸多糖提取率13.06%,但是多糖含量下降了9.87%.微波辅助热水浸提法的最佳条件为:中功率,微波时间4min,水提2h,多糖提取率为14.13%,多糖含量为66.29%;应用微波可提高姬松茸多糖提取率16.10%,对多糖含量没有显著影响.灰树花多糖热水浸提法的最佳工艺为:pH值7.5,料水比为1∶20,提取温度为100℃,提取时间为4 h,醇沉浓度为95%,多糖提取率为7.08%,多糖含量为73.49%.超声辅助热水浸提法的最佳条件为:超声功率400W,超声时间6min,浸提时间2h,多糖提取率为8.04%,多糖含量为58.60%;应用超声波可提高灰树花多糖提取率13.56%。但与热水浸提法相比,多糖含量下降了14.89%.微波辅助热水浸提法的最佳条件为:高功率,微波时间4min,水提2h,多糖提取率为9.17%,多糖含量为70.41%,应用微波可提高灰树花多糖提取率29.52%,对多糖含量没有显著影响.3.对4类姬松茸品种和8类灰树花品种的多糖进行提取测定.筛选出高多糖姬松茸品种“AB01”,其发酵胶多糖含量达2.05mg/ml,是应用面积最广品种“N-2”（1.44mg/ml）的1.42倍,是平均值（1.27mg/ml）的1.61倍,是多糖含量最低品种“AB03”（0.61mg/ml）的3.36倍.该品种的深层发酵产量高,胞内多糖含量为12.04%,胞外多糖含量为1.54mg/ml.筛选出高多糖灰树花品种“GF06”,其发酵胶多糖含量达1.65mg/ml,是应用面积最广品种“52”（0.65mg/ml）的2.53倍,是平均值（0.59mg/ml）的2.79倍,是多糖含量最低品种“GF03”（0.22mg/ml）的7.50倍.该品种胞内多糖含量为7.22%,胞外多糖含量为0.98 mg/ml.4.通过构建小鼠S₁₈₀荷瘤模型对姬松茸和灰树花及复合多糖组分进行抗肿瘤作用研究.姬松茸组中:“AB01”的发酵胶多糖（AB01-P）对小鼠S₁₈₀肿瘤有极显著的抑制活性,与剂量呈正相关,高剂量组的抑瘤率为45.36%;高于“AB03”的发酵胶多糖（AB03-P）的最佳抑瘤率（25.14%）、“AB01”的胞内多糖（AB01-P-1）的最佳抑瘤率（36.61%）、“AB01”的胞外多糖（AB01-P-2）的最佳抑瘤率（30.05%）.灰树花组中:“GF06”的发酵胶多糖（GF06-P）有极显著的抑制S₁₈₀肿瘤活性,与剂量呈正相关,高剂量组的抑瘤率为42.08%;高于“GF03”的发酵胶多糖（GF03-P）的最佳抑瘤率（26.78%）、“GF06”的胞内多糖（GF06-P-1）的最佳抑瘤率（38.25%）、“GF06”的胞外多糖（GF06-P-2）的最佳抑瘤率（39.89%）.“AB01”和“GF06”发酵胶复合多糖（AG-P）低、中剂量组的抑瘤率呈剂量效应,高剂量组的抑瘤率较中剂量组没有提高,中剂量组的抑瘤率为49.18%.结果表明复合多糖组的抑瘤率较单个菌种多糖给药组略有提高.与对照组相比,姬松茸、灰树花及复合多糖各组分均可显著或极显著地提高S₁₈₀荷瘤小鼠的胸腺指数和脾指数（免疫指标）.5.采用Annexin V/PI双染色流式细胞仪研究了高多糖姬松茸品种“AB01”发酵胶多糖（AB01-P）、高多糖灰树花品种“GF06”发酵胶多糖（GF06-P）和复合多糖（AG-P）对人骨肉瘤细胞株（MG-63）早期凋亡的影响.结果表明AB01-P可诱导MG-63细胞凋亡,且具有量效关系,2.0mg/ml剂量组的凋亡率为30.98%;GF06-P对MG-63细胞凋亡没有明显作用.AG-P对MG-63细胞有一定的凋亡作用,凋亡率为16.35%,不具剂量效应.6.应用RT-PCR技术,研究了AB01-P和GF06-P对人脐血粒-巨噬细胞集落刺激因子（GM-CSF）基因表达的影响。AB01-P有诱导人脐血单个核细胞表达GM-CSF的作用,最佳浓度为2.0μg/ml,从而证实了姬松茸多糖促进粒-巨噬细胞生成和调节动物机体免疫功能的分子机理.GF06-P对人脐血单个核细胞GM-CSF基因表达的诱导作用不明显.7.通过果蝇生存试验计数果蝇半数死亡天数、平均寿命、最高平均寿命;测定果蝇组织匀浆中的SOD（超氧化物歧化酶）值、MDA（丙二醛）含量和总抗氧化能力值.姬松茸组中:AB01-P对果蝇平均寿命的延长率最高,与对照组相比,对雄果蝇寿命的延长率达17.32%,对雌果蝇的寿命延长率为11.40%;对果蝇半数死亡的延长率为10.81%（雄）,10.21%（雌）;对果蝇平均最高寿命的延长率为15.91%（雄）,13.11%（雌）.灰树花组中:GF06-P能显著延长雌果蝇的平均寿命,延长率为为22.00%,对雄果蝇寿命的延长率为17.67%;对果蝇半数死亡的延长率为13.31%（雄）,19.71%（雌）;对果蝇平均最高寿命的延长率为12.74%（雄）,28.32%（雌）.复合多糖AG-P对雄果蝇的寿命延长率达23.78%;对雌果蝇寿命延长率为13.06%;显著延长雄果蝇平均最高寿命,延长率为20.77%,对雌果蝇平均最高寿命的延长率为19.01%.AB01-P能极显著提高雄果蝇的总抗氧化能力,提高率为28.89%,对雌果蝇没有影响.AB01-P-2能极显著提高雄性果蝇的SOD值,提高率为42.77%,但对雌果蝇没有影响;极显著提高雄雌果蝇的总抗氧化能力,提高率分别为68.89%、40.54%.GF06-P能显著提高雄果蝇的总抗氧化能力,提高率为17.78%;极显著提高雌果蝇的总抗氧化能力,提高率为48.64%.GF06-P-1可以显著降低雌性果蝇的MDA含量,极显著降低雄性果蝇的MDA含量;显著提高雌果蝇的总抗氧化能力,提高率为18.92%。GF06-P-2可以显著提高雄果蝇的SOD值,提高率为12.26%;极显著提高雌果蝇SOD值,提高率为38.41%.复合多糖（AG-P）可以显著降低雌性果蝇的MDA值,但对雄性果蝇没有显著影响.更多还原

【Abstract】 This research collected 26 Agaricus blazei Murill varieties and 29 Grifola frondosa varieties,screened all the varieties to select high polysaccharide content varieties,and studies the biological activities of the polysaccharides,the main results are as follows:1.Using RAPD,SRAP,ISSR molecular markers and ITS-PCR,the polymorphism analysis was done to 26 Agaricus blazei Murill varieties（A01～A29）. Based on the results of four kinds of molecular markers,using UPGMA for the clustering analysis,Agaricus blazei Murill varieties were temporarily clustered into 4 sorts.The difference coefficient among A20,A21 and A22 was 0;The difference coefficient among A15,A16 and A17 was 0;The difference coefficient between A08 and A10 was 0.These mushroom varieties could be the same variety with different names.Using RAPD,SRAP and ISSR molecular markers,we clustered 29 Grifola frondosa varieties（G01～G29）were clustered into 8 sorts.The difference coefficient between G15 and G16 was 0;The difference coefficient between G07 and G10 was 0; The difference coefficient among G14、G18、G21、G26 and G27 was 0.These mushroom varieties could be the same variety with different names.2.Using the orthogonal test,we carried out the research on Agaricus blazei Murill and Grifola frondosa polysaccharide extraction by the methods including hot water method,ultrasonic and hot water method,microwave and hot water method. The suitable conditions for hot water extraction of Agaricus blazei Murill were pH 7.5, the ratio between material and water 1:20,extraction temperature 100℃,extraction time 4h,ethyl alcohol concentration 100%.The suitable conditions for ultrasonic and hot water extraction method were ultrasonic power 600W,4min for ultrasonic,2h for extraction,which improved 13.06%of extraction rate of Agaricus blazei Murill polysaccharide,while the polysaccharide content was decreased 9.87%.The suitable conditions for microwave and hot water extraction method were medium power,4min for microwave,2h for water extraction,which improved 16.10%of Agaricus blazei Murill polysaccharide extraction,while little influence on polysaccharide content.The suitable conditions for hot water extraction of Grifola frondosa were pH 7.5, the ratio between material and water 1:20,extraction temperature 100℃,extraction time 4h,ethyl alcohol concentration 95%.The suitable conditions for ultrasonic and hot water extraction method were ultrasonic power 400W,6min for ultrasonic,2h for extraction,which improved 13.56%of extraction rate of Grifola frondosa polysaccharide,while the polysaccharide content was decreased 14.89%.The suitable conditions for microwave and hot water extraction method were high power,4min for microwave,2h for water extraction,which improved 29.52%of Grifola frondosa polysaccharide extraction,while little influence on polysaccharide content.3.The polysaccharides from 4 Agaricus blazei Murill varieties and 8 Grifola frondosa varieties were extracted and analyzed.We selected high polysaccharide Agaricus blazei Murill "AB01",whose polysaccharide content in submerged fermentation liquid was 2.05mg/ml,1.61 times to the average value（1.27mg/ml）,3.36 times to the lowest polysaccharide content variety "AB03"（0.61mg/ml）.The output of this variety’s submerged fermentation was high,the intracellular polysaccharide content was 12.04%,and the extracellular polysaccharide content was 1.54 mg/ml.We selected high polysaccharide Grifola frondosa "GF06",whose polysaccharide content in submerged fermentation liquid was 1.65mg/ml,2.79 times to the average value（0.59mg/ml）,7.50 times to the lowest polysaccharide content variety "GF03"（0.22mg/ml）.This variety’s intracellular polysaccharide content was 7.22%and the extracellular polysaccharide content was 0.98 mg/ml.4.Through setting up mouse S₁₈₀tumor-bearing models,we carded out the research on the anti-tumor functions of the polysaccharide（AB01-P）from fermentation liquid of "AB01","AB03-P" from fermentation liquid of "AB03", "AB01-P-1" from intracellular polysaccharide,"AB01-P-2" from extracellular polysaccharide,and polysaccharide "GF06-P" from the fermentation liquid of "GF06", "GF06-F-1" from intracellular polysaccharide,"GF06-P-2" from etracellular polysaccharide and compound polysaccharide "AG-P" containing "AB01" and "GF06".In the group of Agaricus blazei Murill:AB01-P has obvious function on the suppression of the S180 tumor’s activity,relating with the dosage present.High dosage group’s tumor suppression rate was 45.36%,which was higher than AB03-P’s highest rate 25.14%,AB01-P-1’s highest rate 36.61%,AB01-P-2’s highest rate 30.05%.In the group of Grifola frondosa,GF06-P had obvious function on the suppression of the S₁₈₀tumor’s activity,relating with the dosage present.High dosage group’s tumor suppression rate was 42.08%,which was higher than GF03-P’s highest rate 26.78%,GF06-P-1’s highest rate 38.25%,GF01-P-2’s highest rate 39.89%. Compound polysaccharide（AG-P）’s tumor suppression was related with low and medium dosage,high dosage didn’t bring high tumor suppression rate comparing to medium dosage,medium dosage group’s tumor suppression rate was 49.18%.The results indicated that compound polysaccharide had a little higher tumor suppression rate comparing to single polysaccharide.Comparing with the control group,all of Agaricus blazei Murill、Grifola frondosa and compound polysaccharide could obviously or extremely obviously enhanced the S₁₈₀tumor-bearing mouse’s thymus index and the spleen index.5.We used Annexin the V/PI double dyeing flow cell sorter to study the influence on the early death of human usteosarcoma cell line（MG-63）by AB01-P, GF06-P and AG-P.The results indicated that AB01-P induced MG-63 cell death, relating to the dosage present.The group of 2.0mg/ml had the death rate 30.98%. GF06-P had no influence on MG-63 cell death.AG-P induced some MG-63 cell death, and the rate was 16.35%,not relating to the dosage present.6.Using the RT-PCR technology,we studied the influence on granulocyte-macrophage colony-stimulating factor（GM-CSF）gene expression by "AB01-P" and "GF06"."AB01-P" induced granulocyte mononuclear cell to express GM-CSF,the most suitable concentration was 2.0μg/ml,and thus confirmed its molecular mechanism of promotion of granulocyte-macrophage cells and regulation of animals’ immune system.GF06-P could not obviously induce the mononuclear cell GM-CSF gene expression.7.We carried out the fruit fly survival experiments to test the days of half death, average lifetime and the longest average lifetime,and also SOD value,MDA total content and total anti-oxidation value in fruit fly homogenate.We studied the anti-senile function and its mechanism in fruit fly by AB01-P,AB03-P,AB01-P-1, AB01-P-1 and GF06-P,GF03-P,GF06-P-1,GF06-P-2 and compound polysaccharide AG-P.AB01-P was the best polysaccharide elongating the fruit fly’s average lifetime, elongating male fruit fly’s lifetime 17.32%,and female fruit fly’s lifetime 11.40%;the elongation of half death time were 10.81%（male）and 10.21%（female）;The elongation of fruit fly’s the longest average lifetime were 15.91%（male）and 13.11% （female）.GF06-P obviously elongated the average lifetime of female fruit fly,which was 22.00%,and 17.67%for male.The elongation of half death time were 13.31% （male）and 19.71%（female）;The elongation of fruit fly’s the longest average lifetime were 12.74%（male）and 28.32%（female）.Compound polysaccharide AG-P elongated the lifetime 23.78%for male and 13.06%for female;AG-P also obviously elongated the longest average lifetime 20.77%fro male and 19.01%for female.AB01-P extremely obviously enhanced the male fruit fly’s total anti-oxidation ability 28.89%（p＜0.01）,while no influence on female fruit fly.AB01-P-2 extremely obviously enhanced SOD value 42.77%（p＜0.01）to male fruit fly,while no influence on female fruit fly;it also extremely obviously enhanced total anti-oxidation 68.89% （male）and 40.54%（female）（p＜0.01）.GF06-P obviously enhanced male fruit fly’s total anti-oxidation 17.78%（p＜0.05）;it extremely obviously enhanced the female fruit fly’s total anti-oxidation 48.64%（p＜0.01）.GF06-P-1 obviously decreased MDA content in female fruit fly and extremely obviously decreased MDA content in male fruit fly.GF06-P-1 obviously enhanced female fruit fly’s total anti-oxidation 18.92% （p＜0.05）.GF06-P-2 obviously enhance male fruit fly’s SOD value 12.26%,and 38.41%for female fruit fly（p＜0.01）.Compound polysaccharide AG-P obviously decreased female fruit fly’s MDA value,while no obvious influence on male fruit fly.更多还原