共生菌Hebeloma sp.的线形质粒DNA的分子克隆及测序的研究

Cloning and Sequencing of a Linear Plasmid in the Ectomycorrhizal Basidiomycete Hebeloma circinas

共生担子菌滑菇 H ebeloma circinas携带两个线形的染色体外 DNA分子 .全部的菌丝 DNA经蛋白酶 K处理后 ,通过琼脂凝胶电泳观察到 :在染色体 DNA旁有两个大小不等的 DNA带 ,命名为 p HCl和 p HC2 ,其分子量分别为 1 0 .3kb和 9.1 kb.用核酸外切酶处理 p HC DNA,确认其 5′端被保护 .对 p HC2用不同的内切酶处理并确定其内切酶图谱 ,对其 3.2 kb H ind 片段进行克隆 ,亚克隆和测序 .结果表明 :其片段有两个开读框 ( open reading frames) ,携带类似于病毒 B型的DNA和 RNA多聚酶基因编码 .p HC2为该菌携带的一个典型的线形质粒 ,这也是首次在共生担子菌中发现的线形质粒 .

In the basidiomycete Hebeloma circinas two linear extrachromosomal DNA molecules were detected.Bulk DNA was treated with proteinase K and submitted to agarose gel electrophoresis.Two minor bands as well as high molecular weight DNA were observed.The two bands,termed pHC1 and pHC2,were shown to be dsDNA molecules with molecular weights of 10.3 kb and 9.1 kb,respectively.By using different restriction endonucleases,a physical map of pHC2 was cnstructed.Accoding to exonuclease treatment,the element had proteins covalently attrached to its 5′ ends.A 3.2 kb fragment of pHC2 was cloned E.coli and sequenced,two open reading frames ORF1 and ORF2 was decided in this sequenced part.By analysis of amino acid sequence from both ORF using computer,one encoded a viral B type DNA polymerase and the other a RNA polymerase.Thus,this fungus harbors a typical linear plasmid,so far rarely described for basidiomycetes and hitherto unknown for mycorrhizal species.