为探明玉米黑粉菌cyp51基因的表达调控机制,根据玉米黑粉菌cyp51基因cDNA的5’-序列,采用染色体步移技术,获得其5’-上游调控区序列,总长为490bp。利用NNPP分析软件预测转录起始位点,并采用TFSEARCH1.3软件分析转录因子结合位点。结果显示:转录起始位点位于上游134bp处;上游调控区不仅包含启动子的核心结构序列TATA盒(分别位于-30、-58、-318和-348bp处)和CAAT盒(分别位于-150、-161和-191bp处),亦包含多个转录因子结合位点,如AP-4、GATA-1、CdxA、Dfd和Oct-1等;在上游调控序列中嘌呤含量高,而且从-222bp处开始存在4个连续高嘌呤含量的热激转录因子特异性结合位点(HSE)。
Based on the 5 '-end nucleotide sequence of cyp51, the upstream region of cyp51 was obtained by genome walking technique to investigate the regulatory mechanism of cyp51 gene expression. The upstream fragment of 490 bp was successfully cloned and sequenced. The transcription initiation site of cyp51 predicted by the NNPP software is located at 134 bp in front of ATG. Several c/s-acting elements analyzed by TFSEARCH 1.3 software were in the upstream region of cyp51, including not only the core promoter sequences TATA-box ( - 30, - 58, - 318, - 348 bp) and CAAT-box ( - 150, - 161, - 191 bp), but also several transcriptional element binding sites (AP4, GATA-1, CdxA, Dfd, and Oct-l, etc). The current study was benefit to further investigating the regulation of cyp51 over-expression and molecular mechanism involved in fungicides resistance of Ustilago mayd/s in future. In addition, there are four consecutive high-purine heat shock elements (HSE) located from -222 to -195 bp.