A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium.
A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium.
复制标题
一种用于木质素降解担子菌 Phanerochaete chrysosporium 的新型染色体外维持转化载体。
DOI：
10.1128/jb.173.2.776-782.1991
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发表时间：
1991
影响因子：
3.2
通讯作者：
Boominathan,K
中科院分区：
文献类型：
--
作者：
Randall,T;Reddy,CA;Boominathan,K
报道了一种稳定的木质素降解丝状真菌黄孢原毛平革菌染色体外保持的转化载体pG12-1。该载体全长6.3kb，含有一个KANR标记pBR322 Ori和一个2.2kb的片段(ME-1)，该片段来源于黄孢原毛平革菌的内源染色体外DNA元件。载体pG12-1能够将黄孢霉转化为G418抗性菌株，并可通过大肠杆菌转化从转化子的总DNA中快速、稳定地回收。Southern杂交分析表明，pG12-1在真菌转化子中保持在较低的拷贝数。经聚合酶链式反应扩增后，在转化子的G418抗性担孢子代的总DNA中才能检测到该载体。核酸外切酶III和DAM甲基化分析分别表明，pG12-I在黄孢原毛平革菌中经历了复制，并以环状形式存在于染色体外。即使在长期的非选择性生长之后，载体也能稳定地保持在转化子中。没有证据表明该载体在任何阶段都整合到了染色体中。
A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading filamentous fungus Phanerochaete chrysosporium is described. The vector is 6.3 kb and contains a Kanr marker, pBR322 ori, and a 2.2-kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of P. chrysosporium. Vector pG12-1 was able to transform P. chrysosporium to G418 resistance and was readily and consistently recoverable from the total DNA of transformants via Escherichia coli transformation. Southern blot analyses indicated that pG12-1 is maintained at a low copy number in the fungal transformants. The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction. Exonuclease III and dam methylation analyses, respectively, indicated that pG12-I undergoes replication in P. chrysosporium and that it is maintained extrachromosomally in a circular form. The vector is stably maintained in the transformants even after long-term nonselective growth. There is no evidence for integration of the vector into the chromosome at any stage.