Liu J, Sun S Y, Wang T H
State Key Laboratory of Microbial Technology, Shandong University, Shanda Nanlu, Jinan, China.
Lett Appl Microbiol. 2004;38(4):277-82. doi: 10.1111/j.1472-765x.2004.01497.x.
To construct a yeast one-hybrid system and isolate transcriptional activators.
A 1.1-kb promoter region of xylanase2 from Trichoderma reesei was cloned by PCR and sequenced (GenBank accession number: AY263380). Sequence analysis revealed that typical binding sites for several transcription factors in filamentous fungi, such as CREI, XLNR, ALCR, AREA and CCAAT enhancer, are located in the promoter. To isolate xyn2 transcription factors, the reporter plasmid of a yeast one-hybrid system was constructed on the backbone of the plasmid pRS415 containing the leu2 selective marker, with the xyn2 promoter region and Saccharomyces cerevisiae his4 as a reporter gene. The reporter gene contained 123-bp minimal promoter region. The S. cerevisiae H158 strain containing the reporter plasmid was transformed with a T. reesei expression cDNA library, and 34 transformants were collected from SC-Leu-His-Ura plates. The isolation of the gene ace2 from several transformants showed that the one-hybrid system approach was successful. Then, approx. 59 mg l(-1) of ace2 was overexpressed in Escherichia coli BL21.
The yeast one-hybrid system is suitable for isolating transcription factors of filamentous fungi. ACE II is a main and universal transcriptional activator that controls cellulase and hemicellulase transcription regulation in T. reesei.
构建酵母单杂交系统并分离转录激活因子。
通过PCR克隆了里氏木霉木聚糖酶2的1.1 kb启动子区域并进行测序（GenBank登录号：AY263380）。序列分析表明，丝状真菌中几种转录因子的典型结合位点，如CREI、XLNR、ALCR、AREA和CCAAT增强子，位于该启动子中。为了分离xyn2转录因子，在含有亮氨酸2选择标记的质粒pRS415骨架上构建了酵母单杂交系统的报告质粒，以xyn2启动子区域和酿酒酵母his4作为报告基因。报告基因包含123 bp的最小启动子区域。用里氏木霉表达cDNA文库转化含有报告质粒的酿酒酵母H158菌株，并从SC-Leu-His-Ura平板上收集了34个转化子。从几个转化子中分离出ace2基因表明单杂交系统方法是成功的。然后，在大肠杆菌BL21中过量表达了约59 mg l(-1)的ace2。
酵母单杂交系统适用于分离丝状真菌的转录因子。ACE II是控制里氏木霉纤维素酶和半纤维素酶转录调控的主要且通用的转录激活因子。
