A polyclonal antibody against a recombinantly expressed Triticum aestivum RHT-D1A protein

Abstract

Background: Reduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein.

Results: To generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A-agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid.

Conclusion: The purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

Keywords: DELLA; Norin 10; Polyclonal antibody; RHT-D1A; Saratovskaya 29; Triticum aestivum.

Fig. 1

PCR analysis of wheat varieties Norin 10 (a) and Saratovskaya 29 (b). PCR products were separated on 2% agarose gels after amplification with the following primer sets: BF-MR1, BF-WR1, DF-MR2, and DF2-WR2. The expected product sizes are 237 bp for BF-MR1 and BF-WR1, 254 bp for DF-MR2, and 264 bp for DF2-WR2

Fig. 2

Purification of the rRHT-D1A protein of wheat variety Saratovskaya 29. Lane M: standard protein molecular weight markers; lane 1: a protein lysate from untransformed E. coli; lane 2: an E. coli protein lysate prior to induction; lane 3: the lysate of the same cells with IPTG induction; lane 4: the purified rRHT-D1A protein

Fig. 3

Purification of the rabbit anti-RHT-D1A polyclonal antibody and a western blot assay. a Purification of the rabbit anti-RHT-D1A polyclonal antibody. M: standard protein molecular weight markers; lane 1: immune serum (3 μg of protein); lane 2: the eluate from protein A–agarose (3 μg of protein). b SDS-PAGE of affinity-purified recombinant protein 6xHis-RHT-D1A. c The western blot assay. Lane 1: 3 μg of protein; lane 2: 1 μg of protein

Fig. 4

The titer of antiserum according to the ELISA. The purified antibody was subjected to serial dilution (from 1000- to 128000-fold) and reacted with the purified rRHT-D1A protein. Preimmunization rabbit serum served as a negative control. The antibody titer is defined as the highest dilution of serum at which the A₄₀₅ ratio (A₄₀₅ of postimmunization serum/A₄₀₅ of preimmunization serum) is greater than 2:1. Data are presented as the mean + standard deviation

Fig. 5

SDS-PAGE and immunoblot analysis of the polyclonal anti-RHT-D1A antibody by means of native and denatured rRHT-D1A proteins. a Five hundred nanograms of each protein was subjected to SDS-PAGE in a 10% gel and visualized by Coomassie brilliant blue staining. M: standard protein molecular weight markers. b Dot blot analysis of the anti-6xHis-RHT-D1A antibody against the native 6xHis-RHT-D1A recombinant protein. The anti-RHT-D1A, anti-APE1L, and anti-His antibodies were evaluated by the dot blot assay of purified 6xHis-RHT-D1A, a lysate of E. coli cells expressing 6xHis-RHT-D1A, and purified 6xHis-APE1L, which were spotted on PVDF membranes. c Western blot analysis of the polyclonal anti-RHT-D1A antibody against denatured rRHT-D1A proteins. The anti-RHT-D1A, anti-APE1L, and anti-His antibodies were evaluated by western blot analysis of 6xHis-RHT-D1A and 6xHis-APE1L. In all three western blots, 1 μg of purified 6xHis-RHT-D1A was loaded in the first lane and 0.5 μg of purified 6xHis-APE1L in the second lane

Fig. 6

IP of the 6xHis-RHT-D1A recombinant protein by the polyclonal anti-RHT-D1A antibody. The immunoprecipitate was tested for 6xHis-RHT-D1A in a lysate of the E. coli strain expressing the recombinant protein. a Identification of 6xHis-RHT-D1A by SDS-PAGE in a 10% gel. The gel was stained with Coomassie brilliant blue. M: standard protein molecular weight markers; lane 1: the cell lysate; lane 2: the immunoprecipitate of a preparation not incubated with the antibody (control); and lane 3: the immunoprecipitate of the lysate treated with the polyclonal anti-RHT-D1A antibody. b Identification of 6xHis-RHT-D1A by western blot analysis with the polyclonal anti-RHT-D1A antibody. Lanes are the same as in Fig. 6a

Fig. 7

The effect of the GA biosynthesis inhibitor on the stability of RHT-D1A and on α-amylase production. a Western blotting of RHT-D1A and α-amylase. b Activity staining of α-amylases with native PAGE. Extracts were prepared from 4-day-old endosperm of wheat seedlings treated with 10 μM GA with or without 100 μM PBZ