Salch Y P, Beremand M N
USDA/ARS, National Center for Agricultural Utilization Research, St., Peoria, IL 61604.
Curr Genet. 1993;23(4):343-50. doi: 10.1007/BF00310897.
A genetically fertile, trichothecene-producing plant pathogen, Gibberella pulicaris (Fusarium sambucinum), was transformed with three different vectors: cosHyg1, pUCH1, and pDH25. All three vectors carry hph (encoding hygromycin B phosphotransferase) as the selectable marker. Transformation frequency was 0.03 transformants per mumg of DNA for pDH25 and 0.5 for pUCH1 or cosHyg1. The vector DNA sequences integrated at different sites into the fungal genome. Transformants were classified into three types based upon distinctive integration patterns: type A contained a single, intact copy of the vector at one site per genome; type B contained multiple tandem copies or a combination of single and multiple tandem copies at one or more sites per genome; type C contained a partial vector copy at one site per genome. While the transformants with cosHyg1 and pUCH1 were type A or B, type C was unique to pDH25 transformants. Type A and C transformants were both meiotically and mitotically stable. However, type B multiple inserts were unstable in mitosis and meiosis since: (1) multiple tandem copies were deleted; (2) rearrangements occurred during premeiosis; and (3) inserts in one of the type B transformants became methylated during premeiosis. Differential expression of transforming sequences between spore germination and mycelial growth was also observed among type B transformants. The ability to transform G. pulicaris with the resulting varied features of integration patterns and the behavior of transforming DNA during mitosis and meiosis provides a means to isolate, manipulate, and study cloned genes in this mycotoxin-producing plant pathogen.
一种具有遗传育性、能产生单端孢霉烯族毒素的植物病原菌——米根霉赤霉菌（Fusarium sambucinum），用三种不同的载体进行了转化：cosHyg1、pUCH1和pDH25。所有这三种载体都携带hph（编码潮霉素B磷酸转移酶）作为选择标记。pDH25的转化频率为每微克DNA产生0.03个转化体，pUCH1或cosHyg1的转化频率为0.5。载体DNA序列整合到真菌基因组的不同位点。根据独特的整合模式，转化体被分为三种类型：A型在每个基因组的一个位点含有单个完整的载体拷贝；B型在每个基因组的一个或多个位点含有多个串联拷贝或单个与多个串联拷贝的组合；C型在每个基因组的一个位点含有部分载体拷贝。虽然携带cosHyg1和pUCH1的转化体为A型或B型，但C型是pDH25转化体所特有的。A型和C型转化体在减数分裂和有丝分裂中都是稳定的。然而，B型多个插入片段在有丝分裂和减数分裂中是不稳定的，原因如下：（1）多个串联拷贝被删除；（2）在减数分裂前期发生重排；（3）B型转化体之一中的插入片段在减数分裂前期发生甲基化。在B型转化体中还观察到孢子萌发和菌丝生长期间转化序列的差异表达。用所得的整合模式的不同特征以及转化DNA在有丝分裂和减数分裂期间的行为来转化米根霉赤霉菌的能力，为在这种产生霉菌毒素的植物病原菌中分离、操作和研究克隆基因提供了一种手段。
