Lima I G P, Duarte R T D, Furlaneto L, Baroni C H, Fungaro M H P, Furlaneto M C
Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina-PR, Brazil.
Lett Appl Microbiol. 2006 Jun;42(6):631-6. doi: 10.1111/j.1472-765X.2006.01861.x.
To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies.
Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58.3 +/- 18.5, 98.3 +/- 24.8 and 169.7 +/- 35.5 (+/-SEM) transformants per 10(5), 10(6) and 10(7) target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA.
In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA.
Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.
测试根癌农杆菌介导的转化（AMT）方法对玫烟色拟青霉的适用性，玫烟色拟青霉是一种能引起包括粉虱在内的多种昆虫疾病的真菌病原体。
以大肠杆菌的潮霉素B抗性基因（hph基因）作为选择标记，成功将玫烟色拟青霉的分生孢子转化为对潮霉素B具有抗性。每10⁵、10⁶和10⁷个目标分生孢子的转化频率分别为58.3±18.5、98.3±24.8和169.7±35.5（±标准误）个转化子。经PCR确认后，对转化子进行Southern分析，结果显示45%（9个中有4个）的转化子含有单拷贝整合的T-DNA。
在我们的AMT系统中，我们有效地转化了玫烟色拟青霉的分生孢子。该方法的应用避免了耗时的原生质体制备过程，并允许分离含有单拷贝整合T-DNA的转化子。
考虑到根癌农杆菌介导转化的效率，该方法是一种用于插入诱变以鉴定对玫烟色拟青霉致病性重要基因的有用工具。
