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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.

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