title: A thermostable β-glucuronidase obtained by directed evolution as a reporter gene in transgenic plants.
abstract: A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.
authors.affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, China. xiongaisheng@njau.edu.cn
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Jing
authors.initials: J
authors.lastname: Zhuang
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics*
chemical_list: D005966:Glucuronidase
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