一种酿酒酵母表达cDNA文库分离纤维质降解酶系基因的方法

Method of isolating fiber degrading enzymes gene by S.cerevisiae expression cDNA library

目的:建立一种筛选自然界产纤维质降解酶系基因的新方法。方法:对酿酒酵母表达载体pYES2多克隆位点加入真核生物稀有限制性酶切位点SfiI进行改造,利用SMART技术,以大肠杆菌文库为转导,构建了拟青霉(Paecilomyces sp.H28)的酿酒酵母全长cDNA表达文库。结果:利用纤维质-刚果红染色法从文库筛选到多种纤维素和半纤维素降解酶系基因。结论:成功构建了拟青霉的酿酒酵母表达cDNA文库,加快了纤维质降解酶系基因的快速分离,也为其它相关基因的快速分离提供了有益的借鉴。

Object：Establish a method of isolating fiber degrading enzymes gene.Methods：pYES2 expression vector of Saccharomyces cerevisiae was reconstructed by adding the rare SfiI restriction site in eukaryotes into multiple cloning site（MCS）.Using SMART technology,through Escherichia coli cDNA library,constructed the Saccharomyces cerevisiae full-length cDNA express library of Paecilomyces sp.H28.Results：Using the method of fiber-Congo red stain,screened a variety of cellulose and hemicellulose degrading enzymes genes from the library.Coclusion：Constructed S.cerevisiae cDNA expression library of Paecilomyces successfully.The method speeded up the rate of isolation of fiber-degrading enzymes genes,and provided the beneficial reference for other related genes isolation.