关键词: 玉米大斑病菌, 蛋白激酶C, 启动子, 半定量RT-PCR, 基因表达

Abstract:

【Objective】 The aim of this research was to clone the gene encoding the protein kinase C (PKC) and its promoter in Setosphaeria turcica, analyze the gene copy number in the genome of S. turcica and study the effects of different conditions on expression of PKC.【Method】 The homologous fragment of PKC 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. Then the gene’s expression was analyzed by semi-quantitative RT-PCR, when Setosphaeria turcica was cultured on different carbon/nitrogen media, and different abiological stresses.【Result】 PKC, encoding a protein of 1 171 amino acid residues, including 7 extons and 6 introns, was 3 837 bp of DNA and 3 516 bp of cDNA. There were CAAT-box and TATA-box in its upstream sequence, while the transcriptional initiator such as HSF, AP1 and Sp1-binding site, was observed. Southern blotting analysis indicated that there was a single copy of the PKC in the genome of S. turcica. By semi-quantitative RT-PCR analysis, it was found that the expression of PKC was the highest on saccharose as the carbon source, while it was distinctly inhibited by NH4+ and heavy metal ions such as Mn2+, Cu2+, and Zn2+. After addition of different concentrations of sorbitol, it has a positive correlation with the inhibition and concentrations. However, the highest expression was observed in response to isotonic NaCl. 【Conclusion】 Cloning of PKC and its promoter region in S. turcica have enriched the biological information resource of filamentous fungi, and thus laying a foundation for the functional analysis of the signal transduction pathway in phytopathogenic fungi.

Key words: Setosphaeria turcica, protein kinase C, promoter, semi-quantitative RT-PCR, gene expression