蛹虫草继代中菌株退化早期外部识别标志研究  

Study on Early External Identification Mark of Strain Degeneration During Successive Transfer Culture of Cordyceps militaris

作　　者：姜宛岑 王晨露 朱正州 赵英明[2] 王蒴 范文丽[1] 李天来[1] JIANG Wancen;WANG Chenlu;ZHU Zhengzhou;ZHAO Yingming;WANG Shuo;FAN Wenli;LI Tianlai(Key Laboratory of Facility Horticulture,Ministry of Education,National and Local Joint Engineering Research Center of Northern Horticulture Facility Design and Application Technology,Liaoning Key Laboratory of Functional Cordyceps militaris,College of Horticulture,Shenyang Agricultural University,Shenyang 110161,China;Liaoning Academy of Agricultural Sciences,Shenyang 110161,China;Analytical&Testing Center,Shenyang Agricultural University,Shenyang 110161,China)

机构地区：[1]沈阳农业大学园艺学院,设施园艺教育部重点实验室(沈阳农业大学),北方园艺设施设计与应用技术国家地方联合工程研究中心,辽宁省功能性蛹虫草重点实验室,沈阳110161 [2]辽宁省农业科学院,沈阳110161 [3]沈阳农业大学分析测试中心,沈阳110161

出　　处：《园艺学报》2022年第4期851-860,共10页Acta Horticulturae Sinica

基　　金：国家重点研发项目(2019YFD1001905-36);辽宁省科技攻关专项计划项目(2021JH1/10400035)。

摘　　要：以蛹虫草菌株W5继代培养(8 d转接1代)1~5代菌株(W5-1~W5-5)为研究对象,观察蛹虫草继代培养过程中菌落形态、细胞超微结构和活性氧含量的变化,以期揭示继代培养过程中蛹虫草菌株退化的早期外部识别标志。结果表明:蛹虫草W5菌株退化是个渐变的过程,继代培养至3代菌落形态、菌丝和孢子形态开始出现变化,节律环和颜色变化以及菌丝表面的缢裂和凹陷是3代的显著外部特征,可作为蛹虫草菌株退化的早期菌落形态识别标志。在蛹虫草W5菌株继代培养过程中,菌丝的细胞壁逐渐变薄,细胞核逐渐变大,线粒体逐渐肿胀变形,液泡逐渐增大,膜边体逐渐减少,圆球体逐渐溶解,形成脂滴和嗜锇颗粒。可以通过细胞中核仁是否显现、有无膜边体、有无液泡融合和自噬现象来早期判断菌株的退化程度。从3代开始菌丝细胞NBT染色颜色逐渐加深,反映细胞活性氧含量增加。细胞H_(2)O_(2)含量测定表明,3代H_(2)O_(2)含量显著高于前2代。基于线粒体与活性氧的紧密关系,认为线粒体结构和数量上的改变可能是蛹虫草退化的重要原因之一。Cordyceps militaris W5 strain was used to investigate changes of colony morphology,cell ultrastructure and reactive oxygen species(ROS)content during successive transfer culture by every eight days,aiming to reveal early external identification markers of degradation.The results showed that the degradation of C.militaris W5 strain was a gradual process.Changes of colony morphology,mycelium and spore morphology began to appear in the third generation during successive transfer culture.Rhythm rings,color changes together with constricted and sunken hyphae were the significant external characteristics of the third generation,which could be considered as early colonial morphology identification marker of degradation in C.militaris W5 strain.During the successive transfer culture of C.militaris W5 strain,the following phenomena gradually occurred:cell walls of mycelia became thinner,cell nucleus became larger,mitochondria swelled and deformed,vacuoles became bigger,lomasomes reduced,spherosome dissolved,fat droplets and osmophilic granules formed.The degradation degree of W5 strain could be estimated by whether nucleolus appeared in cell nucleus,lomasomes appeared in cell,as well as vacuolar fusion and autophagy occurred in cell.From the third generation,color of mycelial cells became deep after NBT staining,indicating the increase of reactive oxygen species content.H_(2)O_(2) content in the third generation was significantly higher than that in the first two generations.Based on the close relationship between mitochondria and reactive oxygen species,it was believed that the deformation and reduction of mitochondria might be an important reason for the degradation of C.militaris W5 strain.

关 键 词：蛹虫草 继代培养 菌株退化 细胞 超微结构 活性氧 

分 类 号：S567.3[农业科学—中草药栽培]