基于rDNA ITS序列和ERIC-PCR研究虫草真菌无性分离物的遗传多样性

Study on relationships between isolates from Chongcao fruiting bodies based on rDNA ITS and ERIC-PCR

以24株虫草无性分离物和1个蛹虫草子实体为材料,利用ITS序列分析和ERIC-PCR方法对这些材料进行了遗传多样性分析。结果表明:ITS序列分析表明蝉花的无性分离物4645(菌生轮枝菌Lecanicillium fungicola)与蝉花的无性分离物4644和4646及蛹虫草的无性分离物亲缘关系较近,而冬虫夏草无性分离物4634及4636则为膨大弯颈霉Tolypocladium inflatum;蛹虫草Cordyceps militaris无性型与蝉花Cordyceps cicadae无性型(4644、4646)及无性分离物4645聚成一族,而冬虫夏草分离物4634及4636与冬虫夏草的关系较近,聚成另一族。ERIC-PCR分析结果表明,在相似性系数为0.76时,上述分离物分成4组,即1)L.fungicola;2)T.inflatum;3)C.cicadae;4)C.militaris。上述结果表明虫草无性分离物具有丰富的遗传多样性。同时,本研究检测了上述无性分离物胞外液中虫草素的含量,发现所有蛹虫草无性分离物(除1811之外)的胞外液中均含有虫草素,但蝉花和冬虫夏草的无性分离物(4634、4636、4644、4645和4646)胞外液中的虫草素产量极低,甚至无法检测出。

The 24 sequences of rDNA ITS and 5.8S rDNA of isolates and 1 fruiting bodies of Cordyceps and related genera were determined and compared with published sequences. And the species of the above isolates were determined. The results showed that isolates from Cordyceps militaris were identified as the anamorphic state of C. militaris, the isolates No. 4644 and 4646 from Cordyceps cicadae were identified as the anamorphic state of C. cicadae, and another isolate （4645）was Lecanicillium fungicola. The other isolates （4634 and 4636）from Ophiocordyceps sinensis were Tolypocladium inflatum. Meanwhile,based on the sequences data,the phylogenetic tree was constructed using MEGA 4.0 and NJ method. Diversity in the phylogenetic tree was found for C. militaris ,the anamorphic state of C. militaris, the anamorphic state of C. c/cadae, and isolate from C. c/cadae to be close, and for O. sinensis and two isolates from them to be close. The data of ERIC-PCR showed that the isolates were divided into 4 groups, i. e. 1 ）Lecanicillium fungicola, 2） Tolypocladium inflatum,3） the anamorphic state of C. c/cadae, and 4）the anamorphic state of C. militaris at the similarity level of 0. 76, and also showed that rich genetic diversity was present in the isolates, and this was very conducive to attain new improved breeds. Meanwhile, cordycepin contents of the above isolates were detected. And the data showed that all isolates from C. militaris could secrete cordycepin with the exception of No. 1811 ,but those isolates from C. c/cadae and O. sinensis hardly secrete cordycepin.