Overexpression of indole-3-acetic acid-amido synthetases GH3 in Arabidopsis thaliana

The GH3 family of acyl-acid-amidosynthetases catalyze the formation of amino acid conjugates to modulate levels of active auxins in plants. The aim of this study was to regenerate plants with overexpression of different GH3 genes. Wild type Arabidopsis thaliana plants, ecotypes WS and Col-0, were previously transformed via floral dip with Agrobacterium tumefaciens (strain GV3101, pMP90) to obtain AtGH3.3, PbGH3 and PpGH3.2 overexpressing plants. Primary transformants (T1) were analyzed for the presence of transgenes by DNA isolation, PCR amplification and gel electrophoresis. Because PpGH3.2 transgenic plants could not be obtained, floral dip transformation was conducted with new plant material. Secondary transformants (T2) were grown on hygromicin selective plates and lines with one T-DNA insertion were selected. AtGH3.3-His6 and PbGH3-His6 transgenic proteins were extracted from T2 plants and purified on Ni-NTA agarose. Western-blotting and immunodetection showed successful purification of transgenic PbGH3-His6, while AtGH3.3-His6 was degraded. T3 seedlings were grown on selective plates and homozygous lines were determined. These lines were used for RNA isolation and qPCR for measuring transgene overexpression. Seeds with overexpression of GH3 genes, regenerated in this research, will be further used to study the auxin metabolism in plants.