拟康氏木霉egl基因的克隆及在酿酒酵母中的分泌表达  

Cloning of egl Gene from Trichoderma pseudokoningii and Its Secretory Expression in Saccharomyces cerevisiae

作　　者：陶芳[1] 王旭[1] 江海洋[1] 范军[1] 朱苏文[1] 程备久[1] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036

出　　处：《激光生物学报》2011年第6期809-814,801,共7页Acta Laser Biology Sinica

基　　金：安徽省高等学校省级自然科学研究重点项目(KJ2009A75)

摘　　要：从拟康氏木霉3.3002基因组中克隆了内切葡聚糖酶EGI基因,该基因全长1566bp,由3个外显子2个内含子组成,编码461个氨基酸。编码蛋白EGI的N端为22aa组成的信号肽,其后依次为催化结构域、连接肽和结合结构域。采用重叠PCR法获得无内含子的内切葡聚糖酶基因egI,并将其成熟肽编码序列插入酿酒酵母分泌型表达载体pYEα中,构建成pYEα-Peg1重组质粒,转化酿酒酵母。重组转化子经β-半乳糖诱导,检测表达产物的分子大小以及酶活,结果表明,转化子在刚果红平板上可产生明显的水解圈;酶活检测显示该基因能在酿酒酵母中表达有生物活性的EG I并分泌到胞外;SDS-PAGE电泳显示EGI蛋白分子量比预期目的蛋白稍偏大。A gene（eg1） for an endoglucanaseⅡ（EGI） was cloned from Trichoderma pseudokoningii strain 3. 3002.The gene was composed of 1566 bp,interrupted by 2 introns,and coding for 461 amino acid residues.The deduced amino acid sequence of EGI revealed a multi-domain structure composed of a 22aa signal peptide,a catalytic domain, a linker,and a cellulose-binding domain from the N-terminus.The eg1 gene with no introns was obtained by overlap PCR,and the sequence encoding the mature peptide was inserted into the Saccharomyces cerevisiae secretion vector pYEa.The recombinant expression plasmid pYEα-Pegl was constructed and then transformed into S.cerevisiae,along with the yeasts transformed with vector pYEa as controls.After induction of galactose,the resulting S.cerevisiae transformant secreted a recombinant ECI that had enzymatic properties detected by congo red assay and enzyme activity assay. The expression was confirmed by a protein band in the SDS-PAGE,which is a little larger than predicted ECI.

关 键 词：拟康氏木霉 eg1 酿酒酵母 分泌表达 

分 类 号：Q78[生物学—分子生物学]