TRANSFORMATION OF PENICILLIUM-CHRYSOGENUM USING DOMINANT SELECTION MARKERS AND EXPRESSION OF AN ESCHERICHIA-COLI LACZ FUSION GENE
TRANSFORMATION OF PENICILLIUM-CHRYSOGENUM USING DOMINANT SELECTION MARKERS AND EXPRESSION OF AN ESCHERICHIA-COLI LACZ FUSION GENE
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DOI：
10.1016/0378-1119(88)90586-0
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发表时间：
1988-01-01
期刊：
影响因子：
3.5
通讯作者：
SCHWAB, H
中科院分区：
文献类型：
--
作者：
KOLAR, M;PUNT, PJ;SCHWAB, H
使用两个显性选择标记转化工业产黄青霉菌株，即与真菌启动子融合的腐草霉素抗性细菌基因 (ble) 和来自构巢曲霉的乙酰胺酶 (amdS) 基因。使用ble系统获得每μg DNA多达20个转化体的转化频率。使用 amdS 标记，频率高达 120 个转化子。当使用 amdS 作为选择标记时，共转化非常有效。 pAN5-41B（一种携带大肠杆菌 lacZ 基因的质粒，与来自构巢曲霉的强甘油醛-3-磷酸脱氢酶基因 (gpd) 启动子融合）的引入导致在 XGal 平板上形成蓝色菌落，表明 lacZ 融合基因在 P. chrysogenum 中表达。对几个转化体中表达水平的更详细分析表明，高达6％的可溶性蛋白总量由β-半乳糖苷酶融合蛋白组成。
An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phleomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans. Transformation frequencies of up to 20 transformants per .mu.g of DNA were obtained with the ble system. With the amdS marker the frequency was up to 120 tranformants. Cotransformation was very efficient when using amdS as a selection marker. The introduction of pAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A. nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P. chrysogenum. A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the .beta.-galactosidase fusion protein.