金龟子绿僵菌羧基转运蛋白基因MaJEN1及其启动子的克隆与分析

Isolation and Characterization of a Carboxylic Transport Protein JEN1 and Its Promoter from Metarhizium anisopliae

用YADE法扩增了球孢白僵菌T DNA插入突变体T1 2 中与T DNA左边界相连的基因组序列。在此基础上得到了金龟子绿僵菌的羧基转运蛋白的全长cDNA ,MaJEN1。MaJEN1全长 16 95bp ,其中含有长为 15 2 4bp的开放阅读框 (ORF) ,编码 5 0 8个氨基酸的蛋白。氨基酸序列与粗糙脉孢霉和啤酒酵母菌的羧基转运蛋白JEN1相似性分别为 6 9%和 31%。采用PCR扩增得到了MaJEN1的基因组序列GMaJEN1,序列分析发现 ,GMaJEN1含有两个内含子。Southern杂交发现GMaJEN1在金龟子绿僵菌基因组上为单拷贝。利用RT PCR法对MaJEN1的表达特性进行了分析 ,结果表明MaJEN1在蟑螂壳诱导培养基中表达 ,在该培养基中的表达受葡糖糖抑制。进一步采用YADE法得到了长为 16 2 6bp的GMaJEN1上游序列 ,其中含有可能的葡萄糖抑制调控序列。

Based on the flanking sequence of T-DNA of a T-DNA insertion mutant of Beauveria bassiana,T12,the full length cDNA of carboxylic transport protein,designated MaJen1,was cloned from Metarhizium anisopliae.MaJen1 is 1 695 bp long and contained a 1 524 bp ORF which predicted a protein of 508 amino acid.The amino acid sequence of the gene showed 69% and 31% identity to the carboxylic transport protein of Neurospore crassa and Saccharomyces cerevisiae,respectively.The genome sequence,GMaJen1,was amplified by PCR,indicating that there were two introns in GMaJen1.Southern analysis indicated that GMaJen1 was present as a singl copy in Metarhizium anisopliae.The result of RT-PCR showed that expression of MaJen1 was induced by the cuticle of cockroach and repressed by glucose.A 1 626 bp upstream sequence of GMaJen1 was amplified by YADE method,which contained several putative binding domains of glucose repressor.