人工栽培蛹虫草（Cordyceps militaris）性状变异的遗传学分析

【作者】 李美娜；

【导师】 冯伯森； 李庆伟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2003， 硕士

【摘要】 蛹虫草Cordyceps militaris（L.）Link为虫草属食、药用真菌，它可以代替冬虫夏草入药。而在人工驯化栽培过程中，蛹虫草菌种的退化现象比较严重，目前对虫草属真菌的研究只限于菌种的鉴定、营养成分分析、药用价值研究及人工栽培等。用常规选育方法虽然能减少一定的损失，但不能从根本上解决菌种退化这一长期困扰企业生产的问题。针对菌种退化问题，主要是利用有性循环、原生质体融合和遗传工程等途径进行菌株改良，应用分子生物学手段只是研究种内、种间的系统关系等，而关于菌种退化遗传机理的研究尚未见报道。菌种退化是菌类栽培过程中普遍存在的，也是生产当中迫切希望得到解决的问题。本实验中，运用RFLP和RAPD两种分子标记技术及同工酶分析技术对正常菌株和其退化菌株进行了DNA和蛋白质水平的分析，以期获得表型性状的变化所涉及的DNA和蛋白质水平的标记。PCR-RFLP实验，采用真菌通用引物ITS1和ITS4，扩增出5.8S和其两端的两个转录间隔区（ITS），选用5种识别四个碱基的内切酶（HaeⅢ、AfaⅠ、TagⅠ、AluⅠ和XspⅠ）进行酶切，其中只有XspⅠ酶切结果在两个菌种中存在差异，进一步测序表明，整个片段全长534bp，共有13个位点发生碱基突变，且都为c转换为t。野生驯化菌株的测序结果与 Genbank中检索到的序列顺序一致。RAPD实验，选用Operon公司的4组（G组、H组、L组、M组）计80个随机引物进行PCR扩增，共筛选出9个对所有供试个体均存在明显差异的引物。由这9种引物计算所得的菌株间的相似系数为：OPG9:28.6%;OPH4：44.4%；OPH12：33.3%；OPL1：50%；OPL9：40%；OPM2：33.3%；OPM17：57.1%；OPM19：72.7%；OPG13为0，其中最大值为72.7% ，平均值为39.9%，结果表明了退化菌株较野生驯化菌株在DNA水平上发生了频率较高的突变，这些突变可能与菌种退化的基因相关联。采用聚丙烯酰胺凝胶电泳技术对菌丝体阶段的蛹虫草Cordyceps militaris (L.)Link及其退化菌株进行了同工酶分析。共检测了酯酶（EST）、过氧化物酶(PER)、超氧化物歧化酶(SOD)、苹果酸酶(ME)、苹果酸脱氢酶(MDH)、谷氨酸脱氢酶(GDH)、乙醇脱氢酶(ADH)、细胞色素氧化酶共计8种同工酶，其中酯酶、超氧化物歧化酶、苹果酸酶、苹果酸脱氢酶在正常菌株和退化菌株中出现了明显的差异。而其余四种酶在正常菌株和退化菌株中酶谱一致。本研究不仅可以作为菌株退化早期鉴别的手段之一，而且为进一步研究菌种退化遗传机理提供了蛋白质水平上的一些理论依据。更多还原

【Abstract】 Cordyceps militaris(L.)Link is one of the most important fungus in cordyceps genus.It can replace the Cordyceps sinensis in diet and traditional Chinese medicine. In the artificial-planting,the strain of Cordyceps militaris is easy to degenerate.At present,the study about the fungus in Cordyceps Genus are just in the identification of fungi、the nutrition analysis、the using in medicine and artificial-planting.Although,it could reduce the lost by the usual breeding way,we could not solve the problem at basic.About the degeneration of strain,people mostly use the gender-cycling、the fusing-cytoplasmic technology and the genetic-engineering method to reform the strain.Most of people use moleculor method in the study of relationships among the species and between the species. We did not find none of the articles report about the genetic and degenerative mechanism.The degeneration of strain is common in artificial-planting,it is one of the most urgent problems that need to solve.In this experiment, PCR-RFLP and RAPD , were used to study the level of differences in gene of naturalized Cordyceps militaris and its degenerative strain.By using PCR-RFLP,5.8S and its two ITS regions were amplified and digested with five restriction enzymes (HaeⅢ、AfaⅠ、TagⅠ、AluⅠand XspⅠ).The sequencing results showed the length of 5.8S and ITS region is534bp and there are 13mutant bases.The sequence ofnaturalized strain is the same with the sequence searching in the Genbank.Of 80primers,9ones generated reproducible RAPDprofiles.From these 9primers we getthesimilar-ratio: betweenthenaturalizedCordycepsmilitarisanditsdegenerativestrain:OPG9:28.6%;OPH4:44.4%;OPH12:33.3%;OPL1:50%;OPL9:40%;OPM2:33.3%;OPM17:57.1%;OPM19:72.7%;OPG13:0. The maxim is 72.7% ,the average is 39.9%.The results showed the degenerative strain gene rated high frequency mutation. The mutation may associate with the gene which resulted in degeneration of the strain .The isozymes of the Cordyceps militaris and its degenerative strain on the stage of mycelia were analysed by polycrylancide gel electrophoresis. The tested isozymes included esterase(EST)、superoxide dismutase(SOD)、Malate.dehydrogenase(MDH)、malic enzyme(ME)、Glutamate dehydrogenase(GDH)、peroxidase(PER) and alcohol dehydrogenase(ADH).There were obvious differences between the normal strain and its degenerative strain in EST、MDH、ME、SOD.The zymotype were the same in the other isozymes.The results were not only used to test the degenerative strain at young stage ,but also as the theoretical base to study the genetic mechanism of the degenerative strain.更多还原