酵母菌色氨酸合成酶基因的克隆与表达

CLONING AND EXPRESSION OF TRYPTOPHAN SYNTHETASE GENE (TRPS) IN YEAST

用BamHI酶切酿酒酵母(Saccharomyces cerevisiae)1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿梭质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(α,trp5,adel,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3.2kb的TRP5 DNA片段,分别命名为pCN60(trp5)_(1-9)。转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。

TRP5, one of five genes required for tryptophan synthetase in S. cerevisiae, has been isolated on recombinant plasmids. A genomic DNA bank, containing the entire yeast genome was constructed by complete digestion of yeast 1412-4D DNA with restriction endonuclease BamHI, size fractionated by sucrose gradient (2-4kb), and insertion of the fragments into the yeast shuttle vector pCN60.9 recombinants plasmids capable of complementing trp5 mutations were isolated by transformation of yeast cell C9 (α, trp5, adel, ade6). The recombinant plasmids, containing 3.2 kb DNA fragments located TRP5 gene, were named pCN60 (TRP5)_(1-9).Tryptophan synthetase activity of transforments was 3-fold higher than that of original strain 1412-4D.