关键词: 玉米大斑病菌, 异三聚体G蛋白, Genome walking, 启动子, 原核表达

Abstract:

【Objective】 The aim of this research was to clone heterotrimeric G protein alpha subunit gene in Setosphaeria turcica and express it in vitro. 【Method】 The homologous fragment of Gα gene was isolated by degenerate PCR, the 5′and 3′ flanking sequences were cloned using Genome walking, and the full length cDNA was obtained by RT-PCR. Gene structure and putative cis-acting elements were analyzed by bioinformatics methods and then the gene was expressed in vitro by pET expression system. 【Result】 The gene named as Stga-2, which encoded a protein of 356 amino acid residues, was 1 318 bp and interrupted by four introns. There wasn’t typical TATA-box and CAAT-box in its upstream sequence, while the transcriptional initiator and C+T rich region were observed. Other cis-acting elements, such as Sp1-binding site, CCA motif and CCG repeats, were also found here. At last, Stga-2 was expressed in vitro by pET system. 【Conclusion】 Cloning of Stga-2 gene and its promoter region in S. turcica enriched the biological information resource of filamentous fungi, and laid a foundation for the functional analysis about signal transduction pathway in phytopathogenic fungi.

Key words: Setosphaeria turcica, heterotrimeric G protein, Genome walking, promoter, prokaryotic expression