白地霉2.498与白腐真菌5.776原生质体融合
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摘要
本文采用近年兴起的微生物育种的一种有效手段—原生质体融合技术，用PEG6000作为助融剂使对数生长中期的白腐真菌(Phanerochaetechrysosporium 5.776)原生质体与白地霉(Geotrichum candidium 2.498)原生质体融合，结合抗药性及灭活单一亲本的方法在选择性培养基上筛选出产单细胞蛋白能力强又能有效分解木质素的融合菌株。本研究着重探讨了影响白地霉(Geotrichum candidium 2.498)和白腐真菌(Phanerochaete chrysosporium5.776)原生质体形成和再生的各种因素，并采用多种方法对融合子进行了鉴定。试验的具体方法及研究结果如下：
1．白地霉和白腐真菌分别采用平皿菌落计数法和菌丝干重法测定生长曲线，结果显示：白地霉和白腐真菌对数生长中期分别为35小时和70小时。
2．选用11种药物分别对白地霉和白腐真菌进行抗药性测定，最终选择放线菌酮作为抗性鉴定用药品。
3．利用单因素实验探讨影响白地霉和白腐真菌原生质体形成率和再生率的各种因素，结果是：菌龄为对数生长中期、酶浓度为2％纤维素酶+2％蜗牛酶、酶解时间为2小时、酶解温度为31℃、脱壁促进剂为0.2％L-半胱氨酸、渗透压稳定剂为0.8MNaCL。
4．选用正交实验探讨影响白地霉和白腐真菌原生质体形成率和再生率的各种因素的最佳水平组合，结果与单因素实验结果相吻合。
5．选用巯基酶的专一性抑制剂—碘乙酰氨对白地霉原生质体进行灭活处理。
6．用PEG6000作为助融剂，在20℃水浴条件下使二者融合。在选择性培养基上获得72株融合子。连续传10代，获得12株稳定的融合子。
7．比较亲本及融合子菌落表型、显微形态、拮抗试验、对融合子初步鉴定。
摘要
8.用全细胞可溶性蛋白电泳及同工酶电泳进一步对融合子进行鉴定，
去伪存真获得3株遗传稳定的融合子。
9.对亲本及融合子木素过氧化物酶、锰过氧化物酶及漆酶活性进行测
定。结果证明融合子Rl三种酶活都不同程度的高于亲本白腐真菌，将Rl
确立为进一步发酵秸杆的菌种。
In this paper, we used the useful means in microorganism breeding in recent years which was called protoplast fusion, we used PEG 6000 as a reapent in the process of protoplast fusion of geotrichum candidium and planerochaete chrysosporium grown in the logarithmic metaphase of growth. In the meanwhile, the resistance of action of drugs and the destruction of a single parent were also used in order to select out the crasis which could decompose lignin in straw cellular effectively and also produce excellent SCP in the selecting cultural medium. In this paper, we mainly discuss all kinds of factors which affect the formation and regeneration, of phanerochaete chrysosporium and geotrichum candidium, and use various kinds of measures to identify the crasis. Specitic measures and research results were showed as follows:
1. We measured the growth curve of geotichum candidium and phanerochaete chrysosporium by means of caculating the colong in plants and weighing the dry hypha respectively. The results showed that the logarithmic metaphase of growth of the twofurgus were 35 hours and 70 hours respectively.
2. choosing 11 kinds of drugs to measure the resistence to dction of drugs of geotrichum candidium and planerochaete chrysosporium, at last choosing actidione acting as the medicine to identify the resistence
3. Discussing all kinds of factors which affect the formation rate and regeneration rate of geotrichum candidium and phanerochaete chrysosporium by means of one-way experiment. The results showed that: the age of the fungus was the logarithmic metaphase of growth.
4. Discussing the prime level combination of all kinds of factors which affect the formation rate and regeneration rate by means of intercross experiment.
5. Choosing the specific depressor of mercapto-iodoacetamide (IAM) to destroy the geotrichum candidium of protoplast.
6. using PEG6000 acting as reapent to help fuse at 35# in water bath getting 72 individual plants erases on selecting culture medium and make them exist from the first generation to the 10th generation, getting 12 indiviolual plants of steady erases.
7. Compaing the appearance of colony, micrososmic configuration, antagonistic experiment of the parent and crasis to identity the crasis preliminarily
8. Identifying the crasis further by means of full-cell solubility protein electrophoresis and isoenzyme to get 3 indiviolual plants of steady genetic erases.
1．白地霉和白腐真菌分别采用平皿菌落计数法和菌丝干重法测定生长曲线，结果显示：白地霉和白腐真菌对数生长中期分别为35小时和70小时。
2．选用11种药物分别对白地霉和白腐真菌进行抗药性测定，最终选择放线菌酮作为抗性鉴定用药品。
3．利用单因素实验探讨影响白地霉和白腐真菌原生质体形成率和再生率的各种因素，结果是：菌龄为对数生长中期、酶浓度为2％纤维素酶+2％蜗牛酶、酶解时间为2小时、酶解温度为31℃、脱壁促进剂为0.2％L-半胱氨酸、渗透压稳定剂为0.8MNaCL。
4．选用正交实验探讨影响白地霉和白腐真菌原生质体形成率和再生率的各种因素的最佳水平组合，结果与单因素实验结果相吻合。
5．选用巯基酶的专一性抑制剂—碘乙酰氨对白地霉原生质体进行灭活处理。
6．用PEG6000作为助融剂，在20℃水浴条件下使二者融合。在选择性培养基上获得72株融合子。连续传10代，获得12株稳定的融合子。
7．比较亲本及融合子菌落表型、显微形态、拮抗试验、对融合子初步鉴定。
摘要
8.用全细胞可溶性蛋白电泳及同工酶电泳进一步对融合子进行鉴定，
去伪存真获得3株遗传稳定的融合子。
9.对亲本及融合子木素过氧化物酶、锰过氧化物酶及漆酶活性进行测
定。结果证明融合子Rl三种酶活都不同程度的高于亲本白腐真菌，将Rl
确立为进一步发酵秸杆的菌种。
In this paper, we used the useful means in microorganism breeding in recent years which was called protoplast fusion, we used PEG 6000 as a reapent in the process of protoplast fusion of geotrichum candidium and planerochaete chrysosporium grown in the logarithmic metaphase of growth. In the meanwhile, the resistance of action of drugs and the destruction of a single parent were also used in order to select out the crasis which could decompose lignin in straw cellular effectively and also produce excellent SCP in the selecting cultural medium. In this paper, we mainly discuss all kinds of factors which affect the formation and regeneration, of phanerochaete chrysosporium and geotrichum candidium, and use various kinds of measures to identify the crasis. Specitic measures and research results were showed as follows:
1. We measured the growth curve of geotichum candidium and phanerochaete chrysosporium by means of caculating the colong in plants and weighing the dry hypha respectively. The results showed that the logarithmic metaphase of growth of the twofurgus were 35 hours and 70 hours respectively.
2. choosing 11 kinds of drugs to measure the resistence to dction of drugs of geotrichum candidium and planerochaete chrysosporium, at last choosing actidione acting as the medicine to identify the resistence
3. Discussing all kinds of factors which affect the formation rate and regeneration rate of geotrichum candidium and phanerochaete chrysosporium by means of one-way experiment. The results showed that: the age of the fungus was the logarithmic metaphase of growth.
4. Discussing the prime level combination of all kinds of factors which affect the formation rate and regeneration rate by means of intercross experiment.
5. Choosing the specific depressor of mercapto-iodoacetamide (IAM) to destroy the geotrichum candidium of protoplast.
6. using PEG6000 acting as reapent to help fuse at 35# in water bath getting 72 individual plants erases on selecting culture medium and make them exist from the first generation to the 10th generation, getting 12 indiviolual plants of steady erases.
7. Compaing the appearance of colony, micrososmic configuration, antagonistic experiment of the parent and crasis to identity the crasis preliminarily
8. Identifying the crasis further by means of full-cell solubility protein electrophoresis and isoenzyme to get 3 indiviolual plants of steady genetic erases.
引文
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2．丁左龙，1997，碳元组合方式对粉刺侧孢霉产木素过氧化物的影响。生物学杂志。14(5)23-25．
3．甘志波 1993 华中农业大学学报。12(5)：502-509。
4．甘志波，1996．微生物原生质体融合：问题与对策．微生物学杂志。46-50
5．高培基，1980．译自Enzyme Microb．Technol．11-12；
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7．韩建林等 1997．利用白腐真菌提高秸杆利用率的研究．饲料与营养．(1)19-20
8．贺淹才等．1997．启地霉和产爰假丝酵母．中国饲料．(7)38-40
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11．康从宝等，2002．白腐真菌产漆酶的纯化及部分酶学性质．中国生物化学和分子生物学报．18(5)638-642
12．李慧蓉，1996．白腐真菌的研究进展．环境科学进展．(6)69-75
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2．丁左龙，1997，碳元组合方式对粉刺侧孢霉产木素过氧化物的影响。生物学杂志。14(5)23-25．
3．甘志波 1993 华中农业大学学报。12(5)：502-509。
4．甘志波，1996．微生物原生质体融合：问题与对策．微生物学杂志。46-50
5．高培基，1980．译自Enzyme Microb．Technol．11-12；
6．韩建林等，1996．白腐真菌可提高秸杆利用率．中国饲料．(24)28-29
7．韩建林等 1997．利用白腐真菌提高秸杆利用率的研究．饲料与营养．(1)19-20
8．贺淹才等．1997．启地霉和产爰假丝酵母．中国饲料．(7)38-40
9．胡能书，1985．同工酶技术及其应用．湖南科学技术出版社．
10．江慧修等，1993．啤酒酿造永凝集性酵母融合与中的研究．微生物学保．33(1)：22-31．
11．康从宝等，2002．白腐真菌产漆酶的纯化及部分酶学性质．中国生物化学和分子生物学报．18(5)638-642
12．李慧蓉，1996．白腐真菌的研究进展．环境科学进展．(6)69-75
13．刘大军主编．生物技术．江苏科学技术出版社．1992：65—138
14．刘小牧等，2000．白腐真菌与秸杆饲料的有效利用．饲料研究(1)42-43
15．闵小梅，孟庆翔．2000．白腐真菌处理秸杆的研究．饲料研究．(9)7-9
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