野生虫草无性型分离株分子鉴定及遗传多样性的研究

Molecular Identification of Wild Cordyceps Asexual Isolates and Investigation of Their Genetic Diversity

摘要:

虫草具有较高的食用与药用价值，本文以19株野生虫草分离株为研究材料，利用ITS引物进行PCR扩增、测序，并从Genbank上下载虫草属相关序列，构建系统发育树，并对19株虫草无性型分离物进行ERIC-PCR扩增，同时还检测了上述无性分离物发酵液中虫草素的含量。系统发育分析表明在分子水平上19株虫草的无性型分离菌株，都为蛹虫草（Cordyceps militaris）。与高雄山虫草(C.takaomontana)、蝉花(C.cicadae)、古尼虫草(C.gunnii)以及冬虫夏草(C.sinensis)平均遗传距离分别为0.1269、0.1228、0.2251和0.2354，在遗传距离上，与高雄山虫草和蝉花较近，与冬虫夏草较远。ERIC-PCR相似系数在0.8水平上，可以将19个蛹虫草菌株分为3组，相似系数在0.9水平上19株蛹虫草菌种可以分为8组。对蛹虫草发酵液中虫草素含量检测发现，所有蛹虫草无性型分离物发酵液都含有虫草素，最高的可达到126.33 mg/L，为现有的蛹虫草生产菌株JD的119倍。

Abstract:

Cordyceps has high edible and medicinal values. Using nineteen wild Cordyceps isolates as the experimental material, internal transcribed spacer (ITS) universal primers were used for polymerase chain reaction (PCR) amplification and the products were sequenced. Cordyceps-related sequences were downloaded from GenBank to construct a phylogenetic tree, the enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification of 19 Cordyceps asexual isolates was conducted, and the cordycepin content of fermentation broths of the isolates was determined. Phylogenetic analysis showed that all 19 Cordyceps asexual isolates were C. militaris. The average genetic distances of C. militaris from C. takaomontana, C. cicadae, C. gunnii, and C. sinensis were 0.1269, 0.1228, 0.2251, and 0.2354, respectively. In terms of genetic distance, C.militaris was relatively close to C. takaomontana, and far from C. sinensis. ERIC-PCR results showed that the 19 C. militaris strains could be divided into three distinct groups with a similarity coefficient of 0.8, and could also be divided into eight groups with a similarity coefficient of 0.9. Measurement of the cordycepin contents of the fermentation broths of the isolates showed that cordycepin could be detected in all broths, and the highest content reached 126.33 mg/L, which was 119 times as much as that of the existing production strain JD.