松口蘑菌种的分离、纯化、鉴定及原生质体制备的研究
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摘要
本文依据松口蘑Tricholoma matsutake子实体及其分离物的形态特征，结合分子生物学手段，分析它们之间的遗传相似性，用以鉴别子实体和分离物之间的亲缘关系，并对松口蘑菌种的分离、培养及原生质体的制备进行了研究。
采用组织分离法对鲜松口蘑子实体菌褶、菌盖、菌盖和菌柄连接处、菌柄中部及菌柄下部5个部位进行组织分离，分别接种在PDA、PDA＋VB_1＋VB_3、PDA＋黑木耳二级种浸出液＋麦麸、滨田、滨田＋VB_1、BM及BM＋VB_3 7种培养基上培养。25d后只有菌褶部位的组织萌发出菌丝体，子实体其它部位没有萌发出菌丝体：PDA＋黑木耳二级种浸出液＋麦麸培养基上菌丝萌发率最高。
通过对松口蘑子实体和分离物的显微镜观察，比较两者的形态特征，初步确定分离物为松口蘑菌丝体；进一步利用RAPD技术分析分离物与子实体之间的亲缘关系。采用40条随机引物进行引物筛选，其中有17条引物扩增出清晰而稳定的DNA指纹图谱。以子实体和分离物的总DNA为模板，利用筛选出的17条有效引物进行亲缘关系鉴定，用NTSYS-PC软件中的SIMQUAl程序计算DNA指纹间的相似系数，按UPGMA法构建树图。结果表明：松口蘑子实体与分离物的DNA指纹相似系数为1.00，进而断定分离物为松口蘑菌丝体。
通过生长速度测定，筛选松口蘑菌丝体生长的适宜培养基、pH值和温度。结果表明：松口蘑菌丝体在PDA培养基上生长最快，其次是PDA＋VB_3培养基、磷酸二氢钾＋硫酸镁＋麦芽糖＋葡萄糖＋VB_1＋酒石酸氨培养基和滨田培养基，在PDA＋黑木耳二级种浸出液＋麦麸培养基上最慢；松口蘑较适pH为5.0～6.0，生长适宜温度为23℃。通过对菌丝产量的测定，筛选出松口蘑菌丝体生长最佳液体培养基为PDA。对液体培养基进行营养成分的优化组合、多重比较分析，适宜的组合为马铃薯400g／L，葡萄糖30g／L。
采用溶壁酶和蜗牛酶，在30℃水浴，pH为6.5条件下，以0.6mol／L MgSO_4作为渗透压稳定剂，制备松口蘑原生质体。溶壁酶的效果好于蜗牛酶，溶壁酶浓度对原生质体产量的影响在本实验范围内不显著，但酶解时间对原生质体产量的影响达到了极显著水平，酶解时间为3h时，原生质体的平均产量最高，为2.65×10~7个；0.4mol／L的甘露醇为渗透压稳定剂时，松口蘑原生质体的产量最高。
The method of combining morphological character and molecular biological techniques was developed for identifying mycelial isolates of Tricholoma matsutake with their basidiocarps in this paper. The method of isolated, cultivated and prepared protoplast of Tricholoma matsutake was also studied in this paper.
In the study, five kinds of tissues of fresh Tricholoma matsutake were isolated, which were the lamellae, the pileu, the boundary between pileu and stipe, the middle part and the below part of the stipe, then cultured separately on PDA, PDA+VB_1+VB_3, PDA+Jew's-ear-extract+wheat bran, BT, BT+VB_1, BM and BM+VB_3 mediums for germination. The result showed that only the tissue from lamellae can bourgeon mycelium after 25d. The mycelia bourgeon percent was the highest when being cultured on PDA+ Jew's-ear extract + wheat bran medium.
The morphological character was compared by observing the mycelia of Tricholoma matsutake and the isolated with microscope, and it was primary confirmed that the isolated is the mycelia of Tricholoma matsutake; then the relations between Tricholoma matsutake and the isolated were analyzed by the RAPD technology. The RAPD-PCR conditions were optimized, and 17 of 40 arbitrary decamer nucleotide primers were also screened with good implication and repetition. The PCR reaction adopted the 17 kinds of random primers. The clear and steady DNA fingerprints were obtained. The SIMQUAL process of the NTSYS-PC software was used to count DNA fingerprints similarity. The result showed that the DNA fingerprints similarity coefficient between basidiocarps and their mycelia were 1.00 by constructing the UPGMA tree chart. So it was concluded the isolated mycelia are the mycelia of Tricholoma matsutake.
By mensurating the rate, of growth, the better medium, pH and temperature for the mycelia of Tricholoma matsutake were filtered. The results showed that PDA medium is the best for culturing mycelia, the second is PDA+VB_3 medium, KH_2PO_4+ MgSO_4+ maltose+glucose +VB_1+ ammonia tartaric acid medium and BT medium, the last is PDA+ Jew's-ear extract +wheat bran medium; The best pH for culturing Tricholoma matsutake is 5.5～6.0 and the best temperature is 23℃. By mensurating the output of mycelia of Tricholoma matsutake, the best liquid culture medium for culturing Tricholoma matsutake was screened out. The result showed that the mycelia on PDA medium without agar medium grow the best. The data were analyzed by the Multiple Comparisons. The best compound of liquid-medium is potato 400g/L and dextrose 30g/L.
Lywallzyme and snail enzyme were adopted to prepare the protoplast of Tricholoma matsutake in the condition of 30℃, pH 6.5 and 0.6 mol/L MgSO_4 qua osmotic pressure stabilize. The result indicated that the affection of lywallzyme enzyme is better than that of snail enzyme. When the time of lywallzyme enzyme is 3 hours, the output of Tricholoma matsutake protoplast is 2.65×10~7 which is the biggest. Under the same condition, different kinds and concentration of osmotic pressure stabilizer to prepare the protoplast of Tricholoma matsutake was screened. The result shown: 0.4mol/L mannitol is the best.
采用组织分离法对鲜松口蘑子实体菌褶、菌盖、菌盖和菌柄连接处、菌柄中部及菌柄下部5个部位进行组织分离，分别接种在PDA、PDA＋VB_1＋VB_3、PDA＋黑木耳二级种浸出液＋麦麸、滨田、滨田＋VB_1、BM及BM＋VB_3 7种培养基上培养。25d后只有菌褶部位的组织萌发出菌丝体，子实体其它部位没有萌发出菌丝体：PDA＋黑木耳二级种浸出液＋麦麸培养基上菌丝萌发率最高。
通过对松口蘑子实体和分离物的显微镜观察，比较两者的形态特征，初步确定分离物为松口蘑菌丝体；进一步利用RAPD技术分析分离物与子实体之间的亲缘关系。采用40条随机引物进行引物筛选，其中有17条引物扩增出清晰而稳定的DNA指纹图谱。以子实体和分离物的总DNA为模板，利用筛选出的17条有效引物进行亲缘关系鉴定，用NTSYS-PC软件中的SIMQUAl程序计算DNA指纹间的相似系数，按UPGMA法构建树图。结果表明：松口蘑子实体与分离物的DNA指纹相似系数为1.00，进而断定分离物为松口蘑菌丝体。
通过生长速度测定，筛选松口蘑菌丝体生长的适宜培养基、pH值和温度。结果表明：松口蘑菌丝体在PDA培养基上生长最快，其次是PDA＋VB_3培养基、磷酸二氢钾＋硫酸镁＋麦芽糖＋葡萄糖＋VB_1＋酒石酸氨培养基和滨田培养基，在PDA＋黑木耳二级种浸出液＋麦麸培养基上最慢；松口蘑较适pH为5.0～6.0，生长适宜温度为23℃。通过对菌丝产量的测定，筛选出松口蘑菌丝体生长最佳液体培养基为PDA。对液体培养基进行营养成分的优化组合、多重比较分析，适宜的组合为马铃薯400g／L，葡萄糖30g／L。
采用溶壁酶和蜗牛酶，在30℃水浴，pH为6.5条件下，以0.6mol／L MgSO_4作为渗透压稳定剂，制备松口蘑原生质体。溶壁酶的效果好于蜗牛酶，溶壁酶浓度对原生质体产量的影响在本实验范围内不显著，但酶解时间对原生质体产量的影响达到了极显著水平，酶解时间为3h时，原生质体的平均产量最高，为2.65×10~7个；0.4mol／L的甘露醇为渗透压稳定剂时，松口蘑原生质体的产量最高。
The method of combining morphological character and molecular biological techniques was developed for identifying mycelial isolates of Tricholoma matsutake with their basidiocarps in this paper. The method of isolated, cultivated and prepared protoplast of Tricholoma matsutake was also studied in this paper.
In the study, five kinds of tissues of fresh Tricholoma matsutake were isolated, which were the lamellae, the pileu, the boundary between pileu and stipe, the middle part and the below part of the stipe, then cultured separately on PDA, PDA+VB_1+VB_3, PDA+Jew's-ear-extract+wheat bran, BT, BT+VB_1, BM and BM+VB_3 mediums for germination. The result showed that only the tissue from lamellae can bourgeon mycelium after 25d. The mycelia bourgeon percent was the highest when being cultured on PDA+ Jew's-ear extract + wheat bran medium.
The morphological character was compared by observing the mycelia of Tricholoma matsutake and the isolated with microscope, and it was primary confirmed that the isolated is the mycelia of Tricholoma matsutake; then the relations between Tricholoma matsutake and the isolated were analyzed by the RAPD technology. The RAPD-PCR conditions were optimized, and 17 of 40 arbitrary decamer nucleotide primers were also screened with good implication and repetition. The PCR reaction adopted the 17 kinds of random primers. The clear and steady DNA fingerprints were obtained. The SIMQUAL process of the NTSYS-PC software was used to count DNA fingerprints similarity. The result showed that the DNA fingerprints similarity coefficient between basidiocarps and their mycelia were 1.00 by constructing the UPGMA tree chart. So it was concluded the isolated mycelia are the mycelia of Tricholoma matsutake.
By mensurating the rate, of growth, the better medium, pH and temperature for the mycelia of Tricholoma matsutake were filtered. The results showed that PDA medium is the best for culturing mycelia, the second is PDA+VB_3 medium, KH_2PO_4+ MgSO_4+ maltose+glucose +VB_1+ ammonia tartaric acid medium and BT medium, the last is PDA+ Jew's-ear extract +wheat bran medium; The best pH for culturing Tricholoma matsutake is 5.5～6.0 and the best temperature is 23℃. By mensurating the output of mycelia of Tricholoma matsutake, the best liquid culture medium for culturing Tricholoma matsutake was screened out. The result showed that the mycelia on PDA medium without agar medium grow the best. The data were analyzed by the Multiple Comparisons. The best compound of liquid-medium is potato 400g/L and dextrose 30g/L.
Lywallzyme and snail enzyme were adopted to prepare the protoplast of Tricholoma matsutake in the condition of 30℃, pH 6.5 and 0.6 mol/L MgSO_4 qua osmotic pressure stabilize. The result indicated that the affection of lywallzyme enzyme is better than that of snail enzyme. When the time of lywallzyme enzyme is 3 hours, the output of Tricholoma matsutake protoplast is 2.65×10~7 which is the biggest. Under the same condition, different kinds and concentration of osmotic pressure stabilizer to prepare the protoplast of Tricholoma matsutake was screened. The result shown: 0.4mol/L mannitol is the best.
引文
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