黑曲霉糖化酶cDNA的改造及其在酿酒酵母中的表达  

MODIFICATION OF GLUCOAMYLASE GAI cDNA OF A. NIGERAND IT'S EXPRESSION IN S. CEREVISIAE

作　　者：李文清[1] 罗进贤[1] 叶若邻[1] 徐柏年[1] 

机构地区：[1]中山大学生命科学学院,广州510275

出　　处：《微生物学通报》1998年第4期205-209,共5页Microbiology China

基　　金：广东省自然科学基金

摘　　要：应用PCR技术扩增黑曲霉糖化酶cDNA不含非编码区50bp的5’端740bp的序列与该cDNA3’端1400bp的序列连接，获得切除了5’端非编码的糖化酶cDNA。将改造后的cDNA插到质粒pMA91的酵母PGK基因的启动子和转录终止信号之间，构建了含黑曲霉糖化酶基因的表达载体pMAG17。用原生质体转化法将重组质粒pMAG17引入酿酒酵母GRF18。酿酒酵母GRF18转化子在淀粉平板上产生水解透明圈，表明糖化酶已在酵母中表达并分泌至培养基中。测定转化子的胞外酶活力及淀粉水解率。结果表明：改造后的糖化酶基因在酵母中的表达、分泌水平及水解淀粉的能力都高于未改造的基因。The 0.74kb 5' end fragment without noncoding region from A. niger glucoamylaseGAI cDNA was amplified by PCR tecnique. The 1.4kb 3'end sequence of that cDNA wasseparated and ligated to the 0.74kb PCR fragment resulting in intact GAI cDNA with the 5' endnoncoding region being deleted. The modified cDNA was then insected between the PGKPromoter and terminator on plasmid pMA91, and the recombinant plashed pMAG17 wastransformed to S. cerevisiae GRF18. Sharch-degrading halos were formed around the yeastbosformants, indicating that glucoamylase cDNA was expresed in S cerevisiae and the enzymesecreted into culture medium. The glucoamylase activity and amylolytic were determined. Theresults show that the expression and secretion level of the modified cDNA and the amylolyticcapability of the engineered yeast strain are higher than those of the stain with cDNA.

关 键 词：黑曲霉糖化酶 基因改造 表达 酿酒酵母 CDNA 

分 类 号：Q78[生物学—分子生物学] Q949.326.1