摘要
应用蓝藻类金属硫蛋白基因启动子（ｓｍｔＯＰ）的金属诱导性，在单细胞的聚胞藻ＰＣＣ６８０３中表达小鼠金属硫蛋白结构基因（ｍＭＴ１ｃＤＮＡ）。在大肠杆菌ＨＢ１０１中构建含有ｓｍｔＯＰ和ｍＭＴ１ｃＤＮＡ的穿梭表达载体ｐＫＴＭＲＥ，经质粒转移，链霉素筛选，Ｓｏｕｔｈｅｒｎ和Ｗｅｓｔｅｒｎ杂交分析鉴定得稳定的转基因工程藻落。同时，做小批量锌诱导表达，并纯化了外源蛋白，５Ｌ培养液含鲜藻重５０ｇ，得到３５ｍｇｍＭＴ１；转基因藻在高金属浓度下的耐受性测定表明，外源基因的表达提高了蓝藻对金属离子的抗性，约为野生藻的２倍。
Cyanobacterium MT like promoter( smt O P) was used in expression of mMT 1 cDNA in Synechocystis sp.PCC 6803 to enhance metalbinding ability and specialty.Shuttle expression vector pKT MRE was constructed in E.coli (HB101),and through triparental conjugated transfer,Streptomycin screening,Southern and Western blotting analysis,transgenic cyanobacterium clone was acquired.The cultivation and Zn 2+ inducing of transgenic cyanobacterium were carried on ,and the foreign mMT 1 was purified through the improved gel filtration and ion exchange chromatography.About 3.5mg mMT 1 were obtained from 5g wet cyanobacterial cells in 5L culture scale.Cyanobacterial metal resistance measurement indicated that the expression of foreign mMT 1 enhanced the transgenic cyanobacterial metalresistance to two times than the wild Synechocystis sp.PCC 6803
出处
《生物工程学报》
CAS
CSCD
北大核心
1998年第4期405-411,共7页
Chinese Journal of Biotechnology
基金
国家863生物高技术项目基金
关键词
聚胞藻PCC6803
smtO-P区域
金属硫蛋白
蓝藻
Transgenic cyanobacterium, Synechocystis sp.PCC 6803, mouse metallothionein(mMT) 1, smt O P,metal induced expression
