Armillariella tabescens阿拉伯糖苷酶基因全长cDNA的克隆与表达

通过对假密环菌(Arm illariella tabescens)的表达cDNA克隆文库进行随机测序,获得一段与多形拟杆菌的阿拉伯糖苷酶序列同源性很高的EST序列(AJ620046).根据获得的EST序列用SMART-RACE技术成功从假密环菌中克隆出了阿拉伯糖苷酶基因的全长cDNA,用生物信息学的方法对所获的序列进行信息学分析.分析结果显示其全长cDNA为1 016 bp,其最长的开放阅读框架为924 bp,编码307个氨基酸,该全长cDNA序列与多形拟杆菌的阿拉伯糖苷酶序列具有较高同源性,80~279位氨基酸序列属于阿拉伯糖苷酶超家族,58~184位氨基酸是一个典型的结构功能域,蛋白分子质量预测为32 881 u,等电点为4.59.构建重组质粒pPIC9-AF,并在毕赤酵母菌GS115中对所获得的基因进行了表达,相对酶活达到了81.25%.

Cloning and expression of arabinosidase from Armillariella tabescens

Through random sequencing of an expression cDNA library of Armillariella tabescens,AJ620046 was detected that it has significant similarity to the arabinosidase of Bacteroides thetaiotaomicron.By using SMART-RACE technology,the full length cDNA of AJ620046 was obtained successfully.Analyzing with bioinformatics methods.Results showed the full-length cDNA has an open reading frame of 924 bp encoding a 307 amino acid protein.The full-length cDNA has significant similarity to the arabinosidase of Bacteroides thetaiotaomicron.It belongs to Arabinanase/levansucrase/invertase superfamily in 80~279 amino acid.From 58 to 184 amino acid is a typical functional domain.DNAMAN software predicted its relative molecular weight is(32 881) and pI is 4.59.The coding sequence of AJ620046 was cloned into the pPIC9 vector to create a fusion with the yeast.The pPIC9-AF recon was integrated into P.pastoris strain GS115 and expressed.The relative activity is 81.25 %.