摘要
当酵母细胞处于高渗压环境时，甘油被诱导合成以提高其胞内渗透压，这一过程受ＨＯＧ途径的调控。ＧＰＤ１基因为ＨＯＧ途径的重要靶基因，高效表达使胞内３磷酸甘油脱氢酶酶活水平提高可极大地提高甘油的产量。本研究将产甘油假丝酵母（Ｃａｎｄｉｄａｇｌｙｃｅｒｏｌｏｇｅｎｅｓｉｓ）染色体ＤＮＡ经Ｓａｕ３ＡＩ部分酶解后的５～１０ｋｂＤＮＡ片段与经ＢａｍＨＩ线性化及ＣＩＰ处理过的酵母大肠杆菌穿梭质粒ＹＥｐ５１连接，以大肠杆菌ＤＨ５α为受体，构建产甘油假丝酵母的染色体基因文库。通过遗传互补法，在含５０ｇ／Ｌ氯化钠的培养基上筛选出１５个转化子，对转化子０６０１进行了进一步鉴定，转化子０６０１所含质粒ＹＥｐ０６０１带有ＹＥｐ５１的标记并可以消除Ｓａｃｃｂａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ６４２菌株由于其ＧＰＤ１，ＧＰＤ２两基因的缺失突变而表现出的渗透压敏感性。
The response of the yeast Saccharomyces cerevisiae t o osmotic stress is to synthesis and accumulate the glycerol in order to increase the internal osmolarity and this response is controlled by the high osmolarity glycerol (HOG) response pathway, whose important target gene is GPD1 . The increase of the activity of glycerol 3 phosphate dehydrogenase by over expression of GPD1 gene can increase the glycerol yield greatly. In this study, a gene encoding cytoplasmic glycerol 3 phosphate dehydrogenase of Candida glycerolgenesis was cloned out by inserting Sau 3AI generated chromosomal DNA fragments into the Bam HI site of a yeast E. coli shuttle vector, YEp51. Fifteen transformants were isolated on a supplemented minimal medium containing 50g/L of sodium chloride from the constructed C.glycerolgenesis genomic library by using genetic complement approach. The recombinant plasmid, YEp0601, from transformant 0601, possessed the genetic markers of YEp51 and was able to restore the osmotolerance of S.cerevisiae 642( gpd1Δ,gpd2Δ ). These indicated that a gene coding for cytoplasmic glycerol 3 phosphate dehydrogenase of C.glycerolgenesis was successfully cloned out.
出处
《微生物学报》
CAS
CSCD
北大核心
1999年第4期321-326,共6页
Acta Microbiologica Sinica
基金
国家"九五"攻关项目
关键词
产甘油假丝酵母
磷酸甘油脱氢酶
基因
克隆
Candida glycerolgenesis , Glycerol 3 phosphate deh ydrogenase, Cloning
