Research Article: A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants

Date Published: November 9, 2011

Publisher: Public Library of Science

Author(s): Ai-Sheng Xiong, Ri-He Peng, Jing Zhuang, Jian-Min Chen, Bin Zhang, Jian Zhang, Quan-Hong Yao, Annalisa Pastore.

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.

Partial Text: There are about fifty reporter genes that have proved useful in transgenic plant research, based on their efficiency, scientific applications and commercialization. Reporter genes serve as indicators to study transgenic events, for example by facilitating visual identification of successfully transformed entities in a large background of non-transformed material. Although many reporter genes have been proposed, only a few of them have been commercialized [1]. β-glucuronidase (GUS) [2], [3], green fluorescent protein (GFP) [4], [5], luciferase (LUC) [6], [7], [8] are three important reporters that have a proven track record in transgenic plant research.

Various reporter genes encoding GFP, GAL, LUC, GUS, or oxalate oxidase (OxO) have been used in transgenic plant research or crop development, and have been assessed for the efficiency, scientific application and commercialization. Many of these genes have specific limitations or have not been sufficiently tested to merit their widespread use [1]. There are some reports on improved reporter genes that allow easy detection or a wide range of applications and that are generated by directing their evolution in the laboratory. Crameri and his colleagues used DNA shuffling to obtain GFP variants with 40 times increased fluorescence intensity [42]. A mutant GFP with stronger fluorescence intensity was selected by directed evolution in conjunction with a functional salvage screen [43].