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

A b-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 b-glucuronidase activity after exposure to 60uC for 10 min, while those hosting the gus-tr3337 gene retained 70 % or 50 % activity after exposure to 80uC for 10 min or 30 min, respectively. Similarly, in vivo b-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 80uC for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic b-glucuronidase activity, which is a welcome improvement in its use as a reporter