地中海拟无枝菌酸菌U32中生物素羧基载体蛋白结构基因的克隆、表达及转录  

Cloning, Expression and Transcriptional Analysis of Biotin Carboxyl Carrier Protein Gene (accA) from Amycolatopsis mediterranei U32

作　　者：卢捷[1] 姚玉峰[1] 姜卫红[1] 焦瑞身[1] 

机构地区：[1]中国科学院上海生命科学研究院植物生理生态研究所微生物次生代谢分子调控开放实验室,上海200032

出　　处：《微生物学报》2003年第1期56-64,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金重点项目资助 ( 1 0 0 39630 0 1 0 )

摘　　要：乙酰辅酶A羧化酶 (AcetylCoACarboxylaseEC 6.4.1 .2 ,ACC)催化依赖于ATP的乙酰辅酶A羧化形成丙二酸单酰辅酶A ,该反应是脂肪酸生物合成途径中的第一步 ,也是受到调控的关键一步。根据结核分枝杆菌 (M .tuberculosis)和天蓝色链霉菌 (S .coelicolor)中ACC -α亚基的氨基酸保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好 ,设计简并引物以U32基因组DNA为模板扩增出一条约 2 5 0bp的片段 ,并以此片段作探针成功地从U32基因组cosmid文库中克隆到相应的ACC -α亚基的编码基因accA。该基因对应的ORF长1 797bp,编码一个 5 98个氨基酸的蛋白 ,推算出的分子量是 63,71 4Da ;基因G +Cmol%含量为70 .1 % ,符合U32基因结构特征 ,距起始密码子GTG上游 6个碱基处有链霉菌典型的RBS序列AGGAGG ,并有生物素羧化酶特征的ATP结合区。利用pET2 8(b)系统构建表达载体 ,在E .coliBL2 1 (DE3)中实现了accA的诱导表达 ,产物大部分以可溶形式存在 ,并通过WesternBlot证明该蛋白上确有共价结合的生物素。NorthernBlot分析了各种氮源对accA基因转录水平的不同影响。Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G+C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the -6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28(b) vector and expressed solubly in E. coli BL21(DE3) by 0.1mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources. =

关 键 词：地中海拟无枝菌酸菌U32 生物素羧基载体蛋白 结构基因 克隆 表达 转录 

分 类 号：Q933[生物学—微生物学]