a thermostable β-glucuronidase obtained by directed evolution as a reporter gene in transgenic plants一种耐热性的β-glucuronidase通过定向进化作为报告基因在转基因植物.pdfVIP

A Thermostable b-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants 1 2 3 4 2 3 Ai-Sheng Xiong *, Ri-He Peng , Jing Zhuang , Jian-Min Chen , Bin Zhang , Jian Zhang *, Quan-Hong Yao2* 1 State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, China, 2 Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China, 3 Alberta Innovates-Technology Futures, Vegreville, Alberta, Canada, 4 College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China Abstract 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. Citation: Xiong A-S, Peng R-H, Zhuang J, Chen J-M, Zhang B, et al. (2011) A Thermos