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Identification, Characterization and Expression of Drought Related alpha-Crystalline Heat Shock Protein Gene (GHSP26) from Desi Cotton

Center of Excellence in Molecular Biology, University of the Punjab, 87-Canal Bank Road, Thokar Niaz Baig Lahore (53700) Pakistan

View larger version (32K): [in this window] [in a new window]   Figure 1. Differential display profile of Gossypium arboreum with different set of primers. Result are distributed in three lines, each line representing different cDNA fragments amplified in water stressed and control plants. cDNA fragment A shows high homology to SHSP.

View larger version (8K): [in this window] [in a new window]   Figure 2. (a) Schematic representation of three steps to obtain full length gene. Primer pairs used to obtain gene specific fragments are indicated. (b) Complete gene sequence. Solid black bars indicate exons; gray bar indicates intron while black lines indicate untranslated regions. The number of nucleotides in each intron and UTRs are indicated.

View larger version (95K): [in this window] [in a new window]   Figure 3. Alignment of GHSP26 with related HSPs from other plant species. The deduced amino acid sequence of GHSP26 was aligned using clustalW with default parameters. Black and gray shadings indicate conserved amino acid residues.

View larger version (8K): [in this window] [in a new window]   Figure 4. A distance-based, neighbor-joining tree relating complete G. arboreum GHSP26 amino acid sequence to full length HSP amino acid sequences from plants. Sequences were aligned with the CLUSTALW. A neighbor-joining tree was constructed with the MEGA program. A bootstrap analysis (1050 replicates) was performed Nitrosococcus oceani (SHSP) was used as an outgroup.

View larger version (26K): [in this window] [in a new window]   Figure 5. RT-PCR Expression analysis of cotton GHSP26 in stressed and control leaf, stem and roots. S: Stressed, C: Control; cotton GAP gene was used as housekeeping control; product for each sample was separated on a 1.8% (w/v) agarose gel.

View larger version (13K): [in this window] [in a new window]   Figure 6. Relative fold expression of GHSP26 in root, stem and leaves of well watered and stressed cotton plants through real-time PCR. Solid bars represent FAM (carboxyfluorescein) signals during the reaction.