Production and characterization of polyclonal antibody against Arabidopsis GIGANTEA, a circadian clock controlled flowering time regulator

Abstract

Arabidopsis GIGANTEA (GI) is encoded by a single gene and highly conserved among vascular plants and its mutants display pleiotropic phenotypes involved in diverse biological processes such as light signaling, circadian clock, and sucrose metabolism as well as abiotic stress responses. However, molecular mechanisms of GI are largely unknown due to the lack of useful antibody. To date, the epitope tags have been widely used to detect GI in plants, but it needs to generate the transgenic plants which take a few months. Here, we produced polyclonal alpha-GI antibody using truncated variants of GI having amino-terminal (1-858 aa) and carboxyl-terminal (920-1173) regions as antigens. Both recombinant His-GI(1-858) and His-GI(920-1173) proteins were individually and successfully expressed in E. coli and immunized into rabbit. Anti-serum was purified by antigenspecific affinity purification method using both recombinant His-GI(1-858) and His-GI(920-1173) proteins. Purified polyclonal alpha-GI antibody not only detected endogenous GI proteins in wild-type Arabidopsis plants, but also reenacted its diel oscillations. Furthermore, the antibody showed cross-reactivity with the GI orthologs in other plants such as Chinese cabbage, rape and tomato. Our polyclonal GI antibody could help to determine the molecular mechanisms of GI involved in largely unknown pleiotropic responses in plants.