Food Microbiology

Cotton is a cash crop of Pakistan and Gossypium arboreum is a locally cultivated variety, which has considerable resistance against various biotic and abiotic stresses. This variety of cotton is considered as good reservoir of stress tolerance genes, while based on EST data mostly of its genes are uncharacterized. Universal stress protein-2 (USP-2) gene was identified in 15 days drought stressed leaves of G.arboreum-FDH-171. Full length of this gene was mutated at three different (M1usp-2, M2usp-2, M3usp-2) positions (fig: 1) in three separate clones in E. coli-uspABC-mutant and Pichia pastoris-gs115 strains for its functional validation under various abiotic stress treatments (NaCl 800mM, PEG 8%, Heat 37-45°C, Cold 4°C). The expression of 1st mutant (M1usp-2) was noted as 8.3 fold under NaCl stress and 9.7 fold under PEG stress treatments, recombinant cells showed higher growth up to 10-5 dilution in spot assay as compared to control and other genes. The 2nd mutant form of USP-2 was expressed on induction but it was failed to initiate stress tolerant mechanism in both organisms. No significant difference was noted in between 3rd mutant form and wild type USP-2. However, all mutant forms showed little tolerance against heat and cold stresses. The results of this study showed that activity of USP-2 was enhanced in M1usp-2 by enhancing its ATP binding capacity at 2X but wipe out in M2usp-2 with zero ATP-binding ability and 4X enhanced CMP capacity has no effect on activity of M3-usp-2. In silico analysis showed that 1st and 3rd mutant forms of USP-2 may directly involve in stress adaptive mechanism or it might be functional as a signalling molecule to initiate stress mechanism.