Lu S, Lyngholm L, Yang G, Bronson C, Yoder O C, Turgeon B G
Department of Plant Pathology, Cornell University, Ithaca, NY 14853.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12649-53. doi: 10.1073/pnas.91.26.12649.
We have used the restriction enzyme-mediated integration insertional mutagenesis procedure to tag the Tox1 locus in the filamentous Ascomycete Cochliobolus heterostrophus. Mutations at other, unselected, loci were also identified and a high proportion (30-50%) of them were tagged. This procedure may be of general utility for simultaneously mutating and tagging genes in fungi and in other eukaryotes. The Tox1 locus of C. heterostrophus has been defined by Mendelian analysis as a single genetic element that controls production of T toxin, a linear polyketide involved in virulence of the fungus to its host plant, corn. To tag Tox1, protoplasts of a Tox1+ (T-toxin producing) strain were transformed with a linearized, nonhomologous plasmid along with an excess of the restriction enzyme used to linearize the plasmid. Of 1310 transformants recovered, two produced no detectable T toxin in culture or on corn plants. In each of these transformants, the Tox- mutation mapped at Tox1, was tagged with the selectable marker (hygB) on the transforming plasmid, and was tightly linked to the other tagged Tox- mutation. The two mutations, however, represent two different points of plasmid insertion at the Tox1 locus.
我们使用了限制性内切酶介导的整合插入诱变程序，对丝状子囊菌异旋孢腔菌（Cochliobolus heterostrophus）中的Tox1基因座进行标记。还鉴定出了其他未选择基因座处的突变，其中很大一部分（30%-50%）被标记。该程序可能普遍适用于同时对真菌和其他真核生物中的基因进行突变和标记。通过孟德尔分析，异旋孢腔菌的Tox1基因座被定义为一个单一的遗传元件，它控制T毒素的产生，T毒素是一种线性聚酮化合物，与真菌对其寄主植物玉米的致病性有关。为了标记Tox1，用线性化的非同源质粒以及过量用于线性化质粒的限制性内切酶转化Tox1+（产生T毒素）菌株的原生质体。在回收的1310个转化体中，有两个在培养物或玉米植株上均未检测到T毒素。在这些转化体中，每个Tox-突变都定位在Tox1处，被转化质粒上的选择标记（hygB）标记，并且与另一个标记的Tox-突变紧密连锁。然而，这两个突变代表了质粒在Tox1基因座处的两个不同插入点。
