球孢白僵菌羧基转运蛋白基因BbJEN1及其上游序列的克隆与分析  

Cloning and Characterization of a Carboxylic Transport Protein JEN1 and Its Upstream Sequence from Beauveria bassiana

作　　者：张永军[1] 方卫国[1] 肖月华[1] 金凯[1] 裴炎[1] 马金成 罗志兵 

机构地区：[1]农业部生物技术与作物品质改良重点实验室西南农业大学生物技术研究中心,重庆400716

出　　处：《微生物学报》2004年第2期198-201,共4页Acta Microbiologica Sinica

基　　金：国家"8 63计划"( 2 0 0 2AA2 45 0 2 1) ;国家自然科学基金资助项目 ( 3 0 3 0 0 2 3 5 )~~

摘　　要：在分析一株球孢白僵菌T DNA插入突变体T12的Tagging序列的基础上 ,根据与其具有高度同源性的一条金龟子绿僵菌EST序列 (编号为AJ2 732 2 6 )设计简并引物 ,用YADE法从球孢白僵菌中扩增出该EST的同源序列及其延伸序列。序列分析表明 ,该片段与粗糙脉孢霉的羧基转运蛋白JEN1具有高度同源性 ,由此确定该序列为球孢白僵菌羧基转运蛋白JEN1基因的部分序列。然后利用YADE法延伸扩增该序列的上、下游序列 ,获得球孢白僵菌羧基转运蛋白JEN1的全长DNA序列 ,命名为GBbJEN1。利用 3′ RACE扩增出球孢白僵菌羧基转运蛋白JEN1的cDNA序列 ,命名为BbJEN1。BbJEN1全长 16 5 6bp ,编码 5 14个氨基酸的蛋白。推测蛋白分子量为 5 5 975 .37Da,等电点 9.32。氨基酸序列与金龟子绿僵菌、粗糙脉孢霉和酿酒酵母羧基转运蛋白JEN1的同源性分别为 77%、6 6 %和30 %。序列分析表明 ,GBbJEN1含有 2个内含子。Southern杂交表明 ,GBbJEN1基因在球孢白僵菌基因组中为单拷贝。利用RT PCR法对BbJEN1的表达特性进行了分析 ,结果发现BbJEN1基因的转录受蟑螂壳、蝉蜕等昆虫体壁的诱导 ,受葡萄糖的抑制。进一步利用YADE法获得了长为 977bp的GBbJEN1上游序列 ,其中含有可能的葡萄糖抑制调控序列和压力反应元件。The degenerate primers were designed based on an EST(AJ273226) of Metarhizium anisopliae, which was highly homologous with a DNA tagging from a T-DNA insert mutant of Beauveria bassiana. And homologous DNA fragment of the EST and it's extending sequence were amplified from B. bassiana using YADE method. The sequence analysis showed that the amplified DNA fragment was a partial fragment of gene encoding carboxylic transport protein JEN1, because the putative amino acid sequence was similar to carboxylic transport protein JEN1 from Neurospora crassca. Thereafter, the whole DNA sequence of GBbJEN1 encoding JEN1 from B. bassiana was obtained by extending upstream and downstream sequence of the amplified fragment using YADE method. The cDNA of JEN1,designated BbJEN1, was cloned from B. bassiana by 3′RACE according to the sequence of GBbJEN1,which is 1656bp long and encoded a protein of 514 amino acid with Mr=55975.37 Da and PI=9.32. The amino acid sequence of the gene showed 77%, 66% and 30% similarity to MaJEN1 of M. ansopliae, JEN1 of Neurospora crassca and JEN1 of Saccharomyces cerevisiae, respectively. Sequence analysis indicated that GBbJEN1 contained two introns. Southern analysis indicated that GBbJEN1 was present as a single copy in B. bassiana. The result of RT-PCR showed that transcription of BbJEN1 was induced by the cuticle of cockroach or cicada, and repressed by glucose. A 977bp upstream sequence of GBbJEN1 was amplified using YADE method, which contain several putative binding domains of glucose repressor and one stress response element (STREs).

关 键 词：球孢白僵菌 羧基转运蛋白 基因 突变体 病原真菌 产孢机制 

分 类 号：Q785[生物学—分子生物学]