Yang Jin-Ling, Xiao Wei, He Hui-Xia, Zhu Hui-Xin, Wang Shu-Fang, Cheng Ke-Di, Zhu Ping
Key Laboratory of Biosynthesis of Natural Products, Ministry of Health, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Yao Xue Xue Bao. 2008 Apr;43(4):421-6.
Phylogenetic relationship between Paecilomyces hepiali and Cordyceps sinensis was studied by analyzing the sequence of rDNA-ITS. The samples of C. sinensis were collected from Hualong County in Qinghai Province and Kangding County in Sichuan Province in May and June, respectively. The rDNA-ITS fragments were obtained by PCR amplification with the template genomic DNA of the fresh stroma or caterpillar body of the collected samples and the cultured mycelium of P. hepiali, with the universal fungal primers ITS1/ITS4. The amplified fragments were cloned into pMD18-T Vector and sequenced. Phylogenetic analysis was performed with these sequences and those from GenBank. The result showed that all of the 46 clones randomly chosen from the amplification of C. sinensis shared identical or almost identical rDNA-ITS regions and had over 99% identity with some rDNA-ITS sequences of Hirsutella sinensis and C. sinensis registered in GenBank, but all of them had only about 72% identity with that of P. hepiali. Two pairs of specific primers were designed based on the rDNA-ITS sequence of P. hepiali, then PCR and Nest-PCR were performed with the template genomic DNA of the stroma or caterpillar body of C. sinensis samples mentioned above. The apparent bands amplified by Nest-PCR were obtained from all of the samples, and the sequences showed 100% identity with the rDNA-ITS sequence of P. hepiali. In addition, another pair of specific primers were designed based on the rDNA-ITS sequence registered in GenBank as the marker of C. sinensis (accession no. AB067740) but the latter only shared 87.3% identity with that of H. sinensis (accession no. AJ309353). This pair of primers was used to amplify the C. sinensis samples by PCR, and the amplified sequence showed 100% identity with that of AB067740. The result indicated that H. sinensis is the main body of C. sinensis, while some other endoparasitic fungi such as P. hepiali commonly exist in the natural C. sinensis.
通过分析rDNA-ITS序列，研究了蝙蝠蛾拟青霉与冬虫夏草之间的系统发育关系。冬虫夏草样本分别于5月和6月采自青海省化隆县和四川省康定县。以采集样本的新鲜子座或虫体以及蝙蝠蛾拟青霉培养菌丝体的基因组DNA为模板，使用通用真菌引物ITS1/ITS4通过PCR扩增获得rDNA-ITS片段。将扩增片段克隆到pMD18-T载体中并进行测序。用这些序列和来自GenBank的序列进行系统发育分析。结果表明，从冬虫夏草扩增产物中随机选取的46个克隆，其rDNA-ITS区域均相同或几乎相同，与GenBank中登记的中华被毛孢和冬虫夏草的一些rDNA-ITS序列具有99%以上的同一性，但与蝙蝠蛾拟青霉的rDNA-ITS序列均只有约72%的同一性。根据蝙蝠蛾拟青霉的rDNA-ITS序列设计了两对特异性引物，然后以上述冬虫夏草样本的子座或虫体的基因组DNA为模板进行PCR和巢式PCR。巢式PCR扩增出的明显条带在所有样本中均能获得，且序列与蝙蝠蛾拟青霉的rDNA-ITS序列显示100%的同一性。此外，基于GenBank中登记的作为冬虫夏草标记的rDNA-ITS序列（登录号AB067740）设计了另一对特异性引物，但该序列与中华被毛孢（登录号AJ309353）的序列仅具有87.3%的同一性。用这对引物通过PCR扩增冬虫夏草样本，扩增序列与AB067740的序列显示100%的同一性。结果表明，中华被毛孢是冬虫夏草的主体，而一些其他内生真菌如蝙蝠蛾拟青霉在天然冬虫夏草中普遍存在。
