Ilmén M, Onnela M L, Klemsdal S, Keränen S, Penttilä M
VTT Biotechnology and Food Research, Espoo, Finland.
Mol Gen Genet. 1996 Dec 13;253(3):303-14. doi: 10.1007/pl00008597.
Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.
以大肠杆菌lacZ基因作为报告基因，对丝状真菌里氏木霉的纤维素酶启动子cbh1进行了功能分析。开发了一种基于在微量滴定板中的固体培养基上培养的检测方法，该方法能够对大量转化体的β-半乳糖苷酶表达进行快速且可靠的半定量分析。对cbh1启动子的2.2 kb区域进行了一系列缺失和专门设计的改变。相对于起始ATG，去除核苷酸-500上游的序列消除了葡萄糖阻遏作用。在核苷酸-720处的单个六核苷酸序列5'GTGGGG发生突变就足以实现去阻遏。该位点类似于酿酒酵母的葡萄糖阻遏物MIG1和丝状真菌的CREA/CREI的结合位点。去除葡萄糖阻遏物位点并不影响槐糖诱导。即使在缺失-161位置上游序列的缺失衍生物中，启动子的槐糖诱导作用仍然保留，这些衍生物在转录起始点上游保留了约70 bp，在TATA框上游仅保留了30 bp。
