White David, Chen Weidong
USDA-ARS, Grain Legume Genetics and Physiology Research Unit, Washington State University, 303 Johnson Hall, Pullman, WA 99164-6434, USA.
Curr Genet. 2006 Apr;49(4):272-80. doi: 10.1007/s00294-005-0048-8. Epub 2005 Dec 21.
In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.
为了研究植物病原菌菜豆壳二孢（Ascochyta rabiei）的致病机制，对利用农杆菌介导转化的高效转化条件进行了研究。在选择转化体方面，潮霉素B抗性（hph）优于遗传霉素抗性（nptII），并且构巢曲霉trpC启动子比花椰菜花叶病毒35S启动子（CaMV35S）能更有效地表达hph基因。在固体培养基上共培养72小时是产生转化体的最佳条件，但增加细菌细胞与分生孢子的比例并不影响转化效率。通过聚合酶链反应（PCR）确定，所有潮霉素B抗性转化体均携带转移DNA（T-DNA），并且通过Southern杂交检测发现T-DNA整合似乎是随机的且为单拷贝。在没有选择压力的情况下，转化体对潮霉素B仍保持抗性。在不同培养条件下，在潮霉素B存在的情况下观察到菌落形态的变化，并且在550个转化体中观察到包括毒力降低在内的多种改变的表型。在从T-DNA边界鉴定侧翼基因组序列方面，反向PCR比热不对称交错PCR（TAIL-PCR）更有效，并对可能的原因进行了讨论。这种转化技术以及使用反向PCR回收侧翼DNA将为菜豆壳二孢的遗传学研究提供一个有用的工具。
