To gain insight into the roles of specific genes, one can use gene "knock outs" to reveal gene function and reporter gene fusions to reveal gene expression patterns. We will identify mutant plants and generate reporter genes to determine the functions and expression patterns of genes encoding cell wall modifying enzymes and calcium binding proteins whose roles are currently unknown.

I. Genes can be mutated by the random insertion of foreign DNA. For plants, this is accomplished by the random insertion of a fragment of DNA, called the T-DNA, from the natural pathogen Agrobacterium. Agrobacterium has the capability of causing insertion of DNA into plant genomes.
T-DNA insertion mutants were generated at the Salk Institute. To identify plants that have T-DNA insertions within genes of interest (in this case, genes encoding cell wall modigying enzymes, called XTHs, or calcium-binding proteins, called CMLs), one uses polymerase chain reaction (PCR):

• With one PCR primer specific to the gene of interest and another primer specific for the inserted T-DNA, one generates a PCR product ONLYwhen the T-DNA is inserted within the region of interest.
• DNA from homozygous mutant plants will generate ONLY the T-DNA insert specific product; NO product will result using primers that recognize only the uninterrupted gene locus.
• To verify the homozygosity of mutants, progeny resulting from self fertilization are again checked by PCR. All progeny should be homozygous for the insertion mutation.
• Identified mutants are examined for physiological defects to gain insight into the normal function of the gene.

II. Reporter genes are an easy way to determine where a gene is expressed, that is actively being transcribed. For plants, the GUS gene, encoding the enzyme beta-glucuronidase, is used because it generates a blue precipitate when provided with substrate. The sites of active gene expression are thus revealed by blue coloration in the plant.

• To generate reporter gene fusions, we PCR amplify the potential regulatory region of the gene, which is generally within 1 kilobase of sequences found 5-prime to the start of transcription.
• This PCR product is ligated to the GUS reporter gene.
• After ligation and transformation, verification of the novel recombinant DNA product is revealed by restriction endonuclease digestion and agarose gel electrophoresis.
• The successful DNA products are transformed into Agrobacteria; the Agrobacteria are used to introduce the reporter gene into plants.
• The resulting transgenic plants are stained with the GUS substrate (X-gluc) to reveal sites of gene expression (i.e., GUS activity) and examined by microscopy.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 23 July 2003
Updated 7 August 2008