Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Genetic Engineering: Methodology
Application of Reporter Genes in Plant Cell Transformation

To identify transformed Cells, it is necessary to detect foreign DNA integrated into the plant's genomic DNA. Moreover, when studying Transcriptional Regulation signals and their Functions in specific plant Tissues (leaves, roots, or flowers), it is often crucial to quantify the expression level of a Gene encoding an easily identifiable product. This requires The Use of reporter genes, which allow either the Selection of transformed cells or the Assessment of the activity of the encoded enzyme. Several different genes have been tested for use as dominant selectable markers, as well as genes whose protein products can be detected using specific assays (Table 17.4). Since many of these reporter genes are of bacterial origin, they have been equipped with regulatory sequences that drive their expression in plant cells. Selection using a dominant marker allows for the generation of a culture containing only transformed cells. For instance, in the presence of kanamycin, only plant cells synthesizing active neomycin phosphotransferase survive. The choice of a particular reporter gene is dictated by The Nature of the specific experiment. If Gene Expression interferes with normal plant growth, it cannot be used as a reporter. Furthermore, biotechnology experts suggest that the presence of certain genes and their products could contaminate commercial products. Consequently, it is preferable to avoid introducing Antibiotic Resistance genes into crop plants.

Class="center">Table 17.4. Reporter and selectable marker gene systems of plant cells¹⁾

¹⁾ From Walden, Schell, Eur. J. Biochem. 192: 563—576; Gruber, Crosby, p. 80—119, in B. R. Glick, J. E. Thompson (ed.), Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla.

Some reporter gene products (such as ß-D-glucuronidase, as well as luciferase synthesized by Bacteria and fireflies) can be detected in intact plant tissues. In transformation systems, the E. coli ß-D-glucuronidase gene (GUS gene) is most commonly used. It encodes a stable enzyme, typically absent in plants, which catalyzes the Cleavage of ß-D-glucuronides. Its activity in transformed plant tissues can be detected by the appearance of a blue color resulting from the Hydrolysis of the colorless substrate, 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid. An alternative, more sensitive method for quantifying GUS gene activity in plant extracts is based on measuring the fluorescence intensity of the hydrolysis product, 4-methylumbelliferyl-β-D-glucuronide.