Whitehead M P, Gurr S J, Grieve C, Unkles S E, Spence D, Ramsden M, Kinghorn J R
Plant Molecular Genetics Unit, University of St. Andrews, Fife, U.K.
Gene. 1990 Jun 15;90(2):193-8. doi: 10.1016/0378-1119(90)90179-u.
We report the development of a homologous transformation system for Cephalosporium acremonium using the niaD gene of the nitrate assimilation (NA) pathway. Mutants in the NA pathway were selected on the basis of chlorate resistance by conventional means. Screening procedures were developed to differentiate between nitrate reductase apoprotein structural gene mutants (niaD) and molybdenum cofactor gene mutants (cnx) as wt C. acremonium, unlike most filamentous fungi, fails to grow on minimal medium with hypoxanthine as a sole source of nitrogen. Phage clones carrying the niaD gene were isolated from a C. acremonium library constructed in lambda EMBL3 using the A. nidulans niaD gene as a heterologous probe. An 8.6-kb EcoRI fragment was subcloned into pUC18, and designated pSTA700. pSTA700 was able to transform stable niaD mutants to NA at a frequency of up to 40 transformants per microgram DNA. Transformants were easily visible since the background growth was low and no abortives were observed. Gene replacements, single copy homologous integration and complex multiple integrations were observed. The niaD system was used to introduce unselected markers for hygromycin B resistance and benomyl resistance into C. acremonium by cotransformation.
我们报道了利用硝酸盐同化（NA）途径的niaD基因开发顶头孢霉同源转化系统的过程。通过常规方法，基于对氯酸盐的抗性筛选NA途径中的突变体。由于野生型顶头孢霉与大多数丝状真菌不同，不能在以次黄嘌呤作为唯一氮源的基本培养基上生长，因此开发了筛选程序以区分硝酸还原酶脱辅基蛋白结构基因突变体（niaD）和钼辅因子基因突变体（cnx）。使用构巢曲霉niaD基因作为异源探针，从构建于λEMBL3的顶头孢霉文库中分离出携带niaD基因的噬菌体克隆。将一个8.6 kb的EcoRI片段亚克隆到pUC18中，并命名为pSTA700。pSTA700能够以每微克DNA高达40个转化体的频率将稳定的niaD突变体转化为具有硝酸盐同化能力的菌株。由于背景生长低且未观察到流产转化体，转化体很容易被看到。观察到基因替换、单拷贝同源整合和复杂的多拷贝整合。通过共转化，利用niaD系统将潮霉素B抗性和苯菌灵抗性等未选择的标记引入顶头孢霉。
