摘要
聚十异戊烯焦磷酸合成酶催化异戊烯二磷酸与丙烯基二磷酸发生连续的缩合反应生成聚十异戊烯焦磷酸 ,构成辅酶 Q1 0 的侧链。将氧化葡糖杆菌 ( Gluconobacter oxydans)的染色体 DNA用 Eco RI酶切 ,回收 5 .0 kb左右的片段为模板 ,通过 PCR扩增得到了聚十异戊烯焦磷酸合成酶基因 ( dds A)。测序分析表明该基因有一个 95 1 bp的开放型阅读框架 ,氨基酸序列与其他聚戊烯焦磷酸合成酶具有高度同源性 ( 30 %～ 5 0 %)。将 dds A基因克隆到 p UC1 9载体上得到 p UC- GZH表达质粒 ,将其转入大肠杆菌 JM83宿主 ,经 IPTG诱导表达融合蛋白 ,在聚丙烯酰胺凝胶电泳 ( SDS-PAGE)的
Decaprenyl diphosphate (decaprenyl PP)synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to produce decaprenyl PP, which is used for the side chain of ubiquinone (Q) 10. The genomic DNA of Gluconobacter oxydans was digested with Eco RI. Eco RI fragments of approximately 5.0 kb were isolated as templates for PCR. The gene of decaprenyl diphosphate synthase was obtained after several cycles of PCR. Sequence analysis revealed the presence of an ORF of 951bp capable of encoding a polypeptide that displays high similarity(30%～50%) to other prenyl diphosphate synthase. The ddsA was cloned into a pUC19 vector to form a transcription plasmid pUC GZH.After its transformation into E.coli JM83 and induction by IPTG, the recombination strain containing pUC GZH expressed a apparent fusion protein band on SDS PAGE, whose molecular weight was 37ku.
出处
《华东理工大学学报（自然科学版）》
CAS
CSCD
北大核心
2002年第3期321-325,共5页
Journal of East China University of Science and Technology
基金
国家自然科学基金资助项目 ( 2 9776 0 1 7)
关键词
氧化葡萄糖杆菌
ddsA基因
克隆
大肠杆菌
表达
decaprenyl diphosphate synthase
Gluconobacter oxydans
ddsA
ubiquinone (Q) 10
gene expression
