甲基化相关基因和交配型基因在蛹虫草退化过程中的作用

Role of Methylation-Related Genes and Mating-Type Genes in Degeneration of Cordyceps militaris

通过继代培养正常蛹虫草(Cordyceps militaris)菌株CmHY-1(双交配型)获得不同继代次数菌株CmHY-2~CmHY-9并栽培,测定菌株CmHY-1、CmHY-7和CmHY-9液体培养时的腺苷和虫草素含量;制备菌株CmHY-1、CmHY-5、CmHY-7和CmHY-9分生孢子单孢分离菌株并鉴定交配型;检测菌株CmHY-1、CmHY-7和CmHY-9甲基化修饰相关基因表达量;构建菌株CmHY-7甲基化相关基因过表达菌株,测定其菌丝生长速度和分生孢子产量并观察菌丝、分生孢子形态和菌落颜色并进行栽培,研究甲基化相关基因对蛹虫草生长发育的影响。结果表明:继代培养过程中蛹虫草菌株发生退化,与菌株CmHY-1相比,菌株CmHY-9的腺苷和虫草素含量极显著下降;菌株CmHY-1及其继代培养菌株的分生孢子单孢分离菌株只含有一个交配型,菌株交配型发生明显偏分离,菌株CmHY-1、CmHY-5、CmHY-7和CmHY-9单孢分离菌株中交配型为MAT1-1的占比分别为51.25%、40.0%、6.25%和0,交配型为MAT1-2的占比相应增加;与菌株CmHY-1相比,菌株CmHY-7的CmDmta基因表达量显著降低,CmDim-2、CmHh3、CmHmd、CmMft、CmHn、CmMfl基因表达量显著上升;菌株CmHY-9的CmHln、CmDmta和CmSam基因表达量显著降低,CmDim-2、CmHh3、CmHmd、CmM16、CmLsd、CmHn和CmMfl基因表达量极显著上升。菌株CmHY-7的11个甲基化相关基因过表达菌株的基因表达量提高4.1~5.6倍。与菌株CmHY-7相比,其过表达菌株CmMFT的菌落直径极显著增大,过表达菌株CmLSD、CmMFL、CmHN、CmHMD、CmM16、CmDMTA和CmHH3的菌落直径显著减小;过表达菌株CmMFT、CmLSD、CmHMD、CmDMTA和CmSAM分生孢子产量显著上升,过表达菌株CmMFL、CmHN和CmHH3分生孢子产量极显著下降。菌株CmHY-7不能形成子实体但对蚕蛹具有侵染性,其多数甲基化相关基因过表达菌株不能形成子实体;然而在和菌株CmHY-1分生孢子单孢分离菌株(交配型为MAT1-1)共同栽培后,多数过表达菌株可以形成小子实体,过表达菌株CmHN可以形成完整的子实体(具有子囊壳)。研究结果表明交配型基因与甲基化相关基因在蛹虫草退化过程中发挥协同作用。

The Cordyceps militaris strain CmHY-1 was successively subcultured to generate strains of different passages CmHY-2 to CmHY-9, respectively. The subcultured strain of each passage was then cultivated on both rice medium and silkworm chrysalis to be observed for strain degeneration with subculture. Contents of cordycepin and adenosine in CmHY-1,CmHY-7 and CmHY-9 during liquid fermentation were measured, respectively. Conidia of CmHY-1,CmHY-5, CmHY-7 and CmHY-9 were used to prepare their monospora strains and then determined for mating type, respectively. Expression of methylation-related genes in CmHY-1, CmHY-7 and CmHY-9 were measured, respectively. Overexpression strains of 11 methylation-related genes were constructed in CmHY-7 respectively, and then the strains were evaluated for mycelial growth rate, conidium production, conidium morphology and colony color. The results showed that CmHY-1 degenerated with subculture. Compared with CmHY-1, the 8 th passage CmHY-9 showed an extremely significant decrease in both cordycepin and adenosine contents. The proportion of MAT1-1 in monospora strains of CmHY-1, CmHY-5, CmHY-7 and CmHY-9 were 51.25%, 40.0%, 6.25% and 0, respectively. Compared with CmHY-1, CmHY-7 had a significant decrease in CmDmta expression and a significant increase in CmDim-2, CmHh3, CmHmd, CmMft, CmHn and CmMfl expression. CmHY-9 had a significant decrease in CmHln, CmDmta and CmSam expression, and a significant increase in CmDim-2, CmHh3, CmHmd, CmM16, CmLsd, CmHn and CmMfl expression. The expression levels of the 11 methylation-related genes in the overexpression strains were increased by 4.1-5.6 times. Compared with CmHY-7, CmMFT had an extremely significant increase in colony diameter, whereas CmLSD, CmMFL, CmHN, CmHMD, CmM16, CmDMTA and CmHH3 had a significant decrease in colony diameter. Conidium production was significantly increased in CmMFT, CmLSD, CmHMD, CmDMTA and CmSAM, but extremely significantly decreased in CmMFL, CmHN and CmHH3. Regardless of failing to develop fruiting bodies, CmHY-7 remained infectious to silkworm chrysalis. Most methylation-related gene overexpression strains of CmHY-7 failed to develop fruiting bodies. In contrast, most of the overexpression strains developed small fruiting bodies when co-cultured with the MAT1-1 monospore strain of CmHY-1, i.e CmHN developed intact fruiting bodies with perithecium. These findings suggested that mating type genes act synergistically with methylation-related genes during degeneration of C. militaris.