Expression and Transformation Studies of Antifreeze Protein Gene of Carrot in Potato

Abstract

The Antifreeze protein gene (1.022Kb) from local of D.carrota cultivar T29 was amplified from four weeks old seedling leave tissue genomic DNA. The PCR amplified gene was cloned in TA-Cloning vector pCR2.1. The cloned DcAFP gene was then sequenced, the DNA BLAST homology with wild carrot AFP was 96.1%. The 999bp gene of DcAFP from T29 cultivar was translated into amino acid sequence, the translated amino acid sequence when aligned with wild carrot AFP gene of amino acid sequence. The gene has 13 amino acid variability. The Asparagine amino acid residues which play an important role as an ice interacting/ice binding sites were intact as in wild species of carrot amino acid sequence, but extra three Asparagine amino acid were noted in 332 amino acid gene sequence as in the case of DcAFP (T29). The 944bp DNA sequence that code the mature peptide of DcAFP was cloned in pET15b E.coli expression vector. The IPTG induced 36kDa protein detected from the BL21 expression host transformed with expression plasmid. The 36kDa recombinant protein inclusion refolded and purified for polyclonal antibody production. Purified IgG of DcAFP was used in western blot, protein dot blot and ELISA analysis of transgenic potato lines. The 999bp full length gene was ligated in pCAMBIA-1301 without selection marker. Plant expression construct pCAMBIA1301hph - DcAFP (T29) was transformed in Agrobacterium host strain LBA4404. Agrobacterium with pCAMBIA construct was transformed in potato nodal explant. The transgenic plant first selected by PCR, DNA dot blot and GUS reporter assay. Stable integration confirmed by southern blot analysis. The DcAFP mRNA detected by Northern blotting after cold induction of transgenic potato plants. DcAFP protein detected by protein dot blot and western blotting methodologies. The sandwich ELISA was successfully done for expressed protein estimation in transgenic potato lines. The stable potato transgenic lines were tested with ILA (Ion Leakage Assay), which results are significant when compared with the control plants. It was noted that due to DcAFP protein expression in transgenic potato lines demonstrating that transgenic plants are tolerating the frost temperature.