用YADE法克隆球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1的启动子及启动子序列分析

扩增与已知序列相连的未知序列是一项常用的分子生物学技术。与其它方法相比,目前在棉花上应用的YADE法具有效率高,成本低和假阳性低等特点。因此,作者尝试将该法引入到昆虫病原真菌的分子生物学研究,并取得了成功,建立了适合于球孢白僵菌和金龟子绿僵菌的YADE的技术体系。类枯草杆菌蛋白酶在昆虫病原真菌穿透寄主体壁时起重要作用,作者在已克隆的球孢白僵菌类蛋白酶基因CDEP-1的基础上,利用YADE法,克隆类球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1的启动子CDEPP。序列分析发现:CDEPP中没有明显的TATA和CAAT框,含有8个可能的碳调控因子的结合位点5'>(g/c)YggRg<3'和2个氮调控因子的结合位点——相隔很近的GATA。这与CDEP-1的表达受碳/氮抑制的结果一致。本文的结果将会为研究CDEP-1的表达规律奠定基础。

CLONING AND ANALYSIS OF THE PROMOTER OF SUBTILISIN-LIKE PROTEASE GENE CDEP-1 FROM BEAUVERIA BASSIANA BY YADE METHOD

Amplification of the unknown sequence adjacent to known sequence is a n important molecular biology technique. Compared with other methods employed in unknown sequence amplification, YADE, which has successfully used in cotton genomic walking, is characterized by high efficacy and less false positive. In this paper, YADE was successfully introduced into entopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae was constructed. Using YADE, a 1705bp upstream sequence, designated CDEPP, of subtilisin-like protease gene CDEP-1 was cloned. Seven putative binding site with consensus sequence 5'>(g/c)YggRg<3'of carbon regulator protein and two closely spaced GATA, which was putative binding site of nitrogen regulator protein, were detected in CDEPP, which was in agreement with the results that CDEP-1 was repressed by glucose and organic nitrogen.