Abstract
Gossypium arboreum is considered a rich source of stress-responsive genes and the EST database revealed that most of its genes are uncharacterized. The full-length Gossypium universal stress protein-2 (GUSP-2) gene (510 bp) was cloned in E. coli and Gossypium hirsutum, characterized and point mutated at three positions, 352–354, Lysine to proline (M1-usp-2) & 214–216, aspartic acid to serine (M2-usp-2) & 145–147, Lysine to Threonine (M3-usp-2) to study its role in abiotic stress tolerance. It was found that heterologous expression of one mutant (M1-usp-2) provided enhanced tolerance against salt and osmotic stresses, recombinant cells have higher growth up to 10-5dilution in spot assay as compared to cells expressing W-usp-2 (wild type GUSP-2), M2-usp-2 and M3-usp-2 genes. M1-usp-2 gene transcript profiling exhibited significant expression (8.7 fold) in CIM-496-Gossypium hirsutum transgenic plants and enhance drought tolerance. However, little tolerance against heat and cold stresses in bacterial cells was observed. The results from our study concluded that the activity of GUSP-2 was enhanced in M1-usp-2 but wipe out in M2-usp-2 and M3-usp-2 response remained almost parallel to W-usp-2. Further, it was predicted through in silico analysis that M1-usp-2, W-usp-2 and M3-usp-2 may be directly involved in stress tolerance or function as a signaling molecule to activate the stress adaptive mechanism. However, further investigation will be required to ascertain its role in the adaptive mechanism of stress tolerance.
Highlights
Abiotic stresses are major threat to environment and agriculture
Total RNA was extracted from leaves of FDH-171-Gossypium arboreum plants after drought stress of 15d and Gossypium Universal stress Protein-2 (GUSP-2) gene (Fig. 2) was amplified which showed clone number 3 and 5 generated desired fragments of 510 bp and 3.8 kb size (Fig. S1A,B, Supplementary data) and were proceed for Sanger sequencing (Fig. S2A,B, Supplementary data)
GUSP-2-protein sequence was aligned with 2gm[3].A protein sequence, template protein which was used to predict GUSP-2-protein 3D-Model, to verify the mismatched residues in pocket region
Summary
Abiotic stresses are major threat to environment and agriculture. Salinity, heat cold and drought are the abiotic environmental factors that adversely affect growth, limit productivity and geographic distribution of plants[1]. G. arboreum has many remarkable benefits over G. hirsutum, and has significant resistance against biotic and abiotic stresses especially drought and salinity, which makes it valuable gene pool for improving modern cotton c ultivars[7,8,9]. Adenosine-triphosphate-binding USP protein, which has been proposed to function as molecular switch in dehydration stress adaptation[12]. Nucleotide sequence of this gene showed 81% sequence similarity while it’s encoded protein share 77% amino acid homology. The expression and cellular function of newly identified GUSP-2 gene (mutated and wild type) were analyzed to ascertain its possible role in abiotic stress tolerance mechanism. The study was carried out to understand the major metabolic pathways in connection with abiotic tolerance, which will be helpful in providing direction for future metabolic engineering for abiotic-stress tolerance
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