JGG 2002, 29(11): 1034-1040 DOI:   ISSN: 1673-8527 CN: 11-5450/R           

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Molecular Evolution of Beta-Glucuronidase in vitro: Obtaining Thermotolerant GUS Gene

XIONG Ai-Sheng1; YAO Quan-Hong1; PENG Ri-He1; CHEN Jian-Min2

1.Shanghai Key Laboratory of Agricultural Genetics And Breeding, Agro-Biotechnology Center,Shanghai Academy of Agricultural Sciences, Shanghai 201106,China 2.College of Bioscience and Biotechnology, Yangzhou University,Yangzhou 225009, China

Abstract

The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a repoter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8kb specific product was amplified and cloned into the vector pBluescript SK.The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves Dnase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80℃ for 30min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80℃ and increased by 25℃ above GUS-ck (55℃). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.

Keywords beta-glucuronidase   molecular evolution   DNA shuffling   thermostability  
Received 1900-01-01 Revised 1900-01-01 Online 2002-11-10 
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