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目的:克隆贵阳腐霉类枯草菌素蛋白酶(Pr1)基因上游调控序列并进行分析,进一步了解Pr1基因的表达规律。方法:采用锅柄聚合酶链反应法(Panhandle PCR)扩增Pr1基因上游调控序列,并利用真核生物启动子分析软件对扩增序列进行启动子顺式作用元件预测分析。结果:获得864bp基因序列,分析表明,该序列包含2个TATA框,一个可能的转录起始区,并具备氮调控因子结合位点(相隔很近的GATA序列),碳调控因子结合位点(5’SYGGRG 3’)。这与Pr1的表达受碳/氮抑制的结果一致。结论:Panhandle PCR方法成功克隆贵阳腐霉Pr1基因上游调控序列,GenBank登录号为:JQ975036。
OBJECTIVE: To clone and analyze the upstream regulatory sequences of the genus Pythium subtilisin (Pr1) in Guiyang to further understand the regulation of Pr1 gene expression. Methods: Panhandle PCR was used to amplify the upstream regulatory sequence of Pr1 gene. The eukaryotic promoter analysis software was used to predict the cis-acting element of the promoter sequence. Results: The 864bp gene sequence was obtained. The analysis showed that the sequence contained two TATA boxes, one possible transcriptional initiation region, a nitrogen regulatory factor binding site (closely spaced GATA sequence), a carbon regulatory factor binding site 5’SYGGRG 3 ’). This is consistent with the result that Pr1 expression is inhibited by carbon / nitrogen. Conclusion: The Panhandle PCR method successfully cloned the upstream regulatory sequence of Pr1 gene of Pythium guiyangense, GenBank accession number is JQ975036.