大肠杆菌glgC基因的克隆、序列分析与定点突变  

Cloning And Site-directed Mutagenesis of glgC Gene From E. coil Y1090

作　　者：董云洲[1] 王晓峰[1] 贾士荣[1] 

机构地区：[1]中国农业科学院生物技术研究中心,北京100081

出　　处：《农业生物技术学报》1999年第2期173-178,共6页Journal of Agricultural Biotechnology

基　　金：863青年基金

摘　　要：通过PCR，从E．coliK12Y1090株系中扩增获得glgC基因，插入pUC19的PstI和SacI位点之间，得到重组质粒pAGP。对所克隆的glgC基因进行全序列测定，结果表明，与已发表的序列相比，有7个核苷酸发生了改变，核苷酸顺序的同源性为99．4％；推导的氨基酸序列的同源性为99．2％，其中，第296位由赖氨酸变为谷氨酸。通过寡核苷酸介导的定点诱变，将第1007位核苦酸由G变为A，从而使第336位氨基酸由甘氨酸变为天门冬氨酸，将此glgC的突变基因定名为glgC336。经I2-KI染色法鉴定，突变基因的表达可提高细菌中的糖原含量。ADPglucose pyrophosphorylase (AGPase) is the key enzyme catalyzing the rate-limiting reaction of starch biosynthesis in bacteria and plants. By using genomic DNA of E. coli K12 strain Y 1090 as a template, glgC gene was amplified by polymerase chain reaCtion (PCR) and cloned intO pUC19. The full length of the glgC gene (1296 bp) was sequenced. The nucleotide sequence is 99.4 % homologous to the published glgC gene. The deduced amico acid sequence is 99.2 % homologous to the known sequence. It is noticed that amino acid was changed at the POsition 296 from lysine tO glutamic acid. The nucleotide 1066 was changed from G to A via site-directed mutagcnesis, and the amino acid was changed from glycine 336 to aspartic acid accordingly. The mutated gene, named glgC336 that mutated bath at the POsition of 296 (Lys→Gin) and 336 (Gly →Asp), is able to increase glycogen synthesis in E. Coli.

关 键 词：AGPASE E.COLI PCR glgC基因 序列分析 定点突变 

分 类 号：Q939.121[生物学—微生物学]