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.
