通过DNA改组技术获得高活性β-葡萄糖苷酸酶  

Directed Evolution of High Activity β-Glucuronidase by DNA Shuffling and Screening

作　　者：熊爱生[1] 姚泉洪[1] 彭日荷[1] 陈建民[2] 李贤[1] 范惠琴[1] 

机构地区：[1]上海市农业遗传育种重点实验室,上海市农业科学院生物技术研究中心,上海201106 [2]扬州大学生物科学与技术学院,扬州225009

出　　处：《中国生物化学与分子生物学报》2003年第2期162-167,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：上海市农业科学院青年基金项目 (No .2 0 0 1 0 9 0 1 1)~~

摘　　要：β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高The E. coli β-glucuronidase gene (GUS) developed as a reporter gene has been widely used in transgenic plants for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized for rapid nonradioactive assays. The use of the E. coli β-glucuronidase (GUS) as a reporter in gene expression studies is limited to some plants and plant-associated bacteria with endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited for high false positive rate. In order to increase the activity of GUS enzyme, molecular evolution in vitro was used. Using plamid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as that reported. Recombination in vitro (DNA shuffling) which involves DNase Ⅰ digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS24 gene was isolated after two rounds of DNA shuffling and screening. The nucleotide sequence analysis showed 99.7% homology between the GUS gene and GUS24 gene. Six base pairs were changed and the deduced amino acid sequence demonstrated that three amino acid residues were changed. Both chromogenic and fluorogenic studies indicated the activity of GUS24 was 3 times higher than the wild-type GUS.

关 键 词：β-葡萄糖苷酸酶 植物 转基因 报告基因 DNA改组技术 酶活性 体外分子进化 

分 类 号：Q943[生物学—植物学] Q785