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.
Highlights
There are about fifty reporter genes that have proved useful in transgenic plant research, based on their efficiency, scientific applications and commercialization
There were no noticeable differences in the morphology or growth among the above transgenic plants and the parental Arabidopsis plants
Seedlings containing the wild-type gus-wt gene or the mutant gus-tr3337 gene exhibited stable GUS activity when they were not subjected to high temperature treatments (Figures 2A and 3A)
Summary
There are about fifty reporter genes that have proved useful in transgenic plant research, based on their efficiency, scientific applications and commercialization. The advantage of GFP and LUC as reporters includes the possibility of monitoring in live tissues and in real time [1,4,8]. These two reporters allow continuous monitoring of the gene activity even through the developmental stages of the transgenic plants [1,5,9]. In case of GFP, there may be interference due to a high background of auto-fluorescence Another major drawback in the use GFP as a reporter is that its detection requires relatively expensive instrumentation, namely the fluorescence microscope
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