大肠杆菌glgC基因的克隆、序列分析与定点突变
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摘要: 通过PCR,从E.coli K12 Y1090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位核苷酸由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。
关键词: AGPase; E.coli ; PCR;glgC基因;序列分析;定点突变
Cloning And Site-directed Mutagenesis of glgC Gene From E.coli Y1090
Dong Yunzhou Wang Xiaofeng Jia Shirong
(Biotechnology Research Center, Chinese Academy of Agricultural science s, Beijing 100081)
Abstract: 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 mutagenesis, and the amino acid was changed from glycine 336 to aspartic acid accordingly. The mutated gene, named glgC336 that mutated both at the position of 296(Lys→Glu) and 336(Gly →Asp), is able to increase glycogen synthesis in E.coli .
Key words: AGPase; E.coli ; glgC gene; PCR; sequence; site-directed mutagenesis
糖原是细菌细胞内碳源及能源的贮藏形式,其作用与淀粉在植物中的作用相同。糖原主要以ADPG途径合成[1]。AGPase (ADPglucose pyrophosphorylase, 腺苷酸二磷酸葡萄糖焦磷酸化酶)是糖原合成中的关键酶[1~3],它催化的反应为ATP+G-1-P ADPG+PPi。ADPG是葡萄糖的活化形式,它作为葡糖基的供体,使葡聚糖不断得以延长。大肠杆菌的AGPase是一个同型四聚体,由4个相同的亚基构成,每个亚基的分子量为50 kD,其编码基因为glgC,DNA全序列已测定[4]。Cattaneo等[5]发现了E.coli K12株系中 的一个突变体,其糖原含量比野生型高30%以上。Leung等克隆了该基因,定名为glgC16[2]。Lee(1987)和Kumar等(1989)证明第886位A→G和第1007位G→A的核苷酸双突变是导致AGPase变构调节特性的内因[6,7],这一特性为改良植物的淀粉含量或品质奠定了基础。Stark等(1992)将glgC16基因转入马铃薯,获得了淀粉含量提高的转基因植株,平均含量比对照高35 %, 最高的达60 % [8]。本研究首先对glgC基因进行克隆和定点突变,旨在通过转基因技术,改善我国马铃薯品种淀粉含量偏低(13 %~19 %)的现状, 为食品工业提供优良的原料。
1 材料与方法
1.1 材料
细菌菌株: E.coli Y1090, 来自新加坡国立大学;XLI-Blue为本室保存。质粒:pUC19, pBluescript KS为本室保存;pALTER-1购自Promega公司。工具酶多为Promega、BRL及华美公司产品。PCR引物和定点突变引物由新加坡国立大学分子和细胞生物学研究所合成。DNA手工测序,采用USB公司Sequenase version 2.0 kit。同位素α-32 P-dATP购自亚辉公司;药品、试剂为进口或国产分析纯。