title: Molecular cloning, characterization, heterologous expression and in-silico analysis of disordered boiling soluble stress-responsive wBsSRP protein from drought tolerant wheat cv.PBW 175.
abstract: The structural and physico-chemical properties that account for the multi-functionality of dehydrins remain largely unknown. In this study, we identified, sequenced and cloned a stress regulated cDNA encoding a dehydrin-like boiling stable protein (designated as wBsSRP; wheat boiling stable stress responsive protein) from drought stressed seedlings of drought tolerant cultivar of wheat (PBW 175). qRT-PCR analysis documented high transcripts levels of wBsSRP during drought and cold conditions in the tolerant cv. PBW 175 as a part of adaptive response to stress while the levels were significantly lower in the sensitive cv. PBW 343. We also describe in-silico characterization and molecular modeling of wBsSRP through homology search, motif analysis, secondary structure prediction, active site prediction and 3D structure analysis. The physico-chemical properties and theoretical data of wBsSRP depicts that it is a canonical group 2 LEA protein. The recombinant wBsSRP protein when expressed in E. coli detected a specific differential band (∼11 kDa) on SDS- PAGE after IPTG induction. The functional analysis of wBsSRP in E. coli revealed that wBsSRP is essential for the survival of E. coli as well as for maintaining bacterial growth under various stress conditions. In vitro peroxidase protection assay during heat stress (50 and 100 °C) showed that in the presence of wBsSRP, peroxidase activity was significantly retained and/or increased. Based upon the findings, it is suggested that wBsSRP accentuated the effects of stress by acting as a protectant and by the stabilization of membranes, thereby contributing to the improved stress tolerance of the recombinant E. coli under various abiotic stress conditions. We suggest that these findings might provide the rationale for the mechanism of how these proteins obviate the adverse effects of dehydration stress.
authors.affiliation: PG Department of Biotechnology, Lyallpur Khalsa College, G.T. Road, Jalandhar 144001, Punjab, India.
authors.forename: Gurmeen
authors.initials: G
authors.lastname: Rakhra
authors.affiliation: Biodiversity and Applied Botany Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India.
authors.forename: Tarandeep
authors.initials: T
authors.lastname: Kaur
authors.affiliation: Biodiversity and Applied Botany Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India.
authors.forename: Dhiraj
authors.initials: D
authors.lastname: Vyas
authors.affiliation: PG Department of Biotechnology, Lyallpur Khalsa College, G.T. Road, Jalandhar 144001, Punjab, India. Electronic address: arundevsharma47@gmail.com.
authors.forename: Arun Dev
authors.initials: AD
authors.lastname: Sharma
authors.affiliation: Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, India.
authors.forename: Jatinder
authors.initials: J
authors.lastname: Singh
authors.affiliation: PG Department of Biotechnology, Lyallpur Khalsa College, G.T. Road, Jalandhar 144001, Punjab, India.
authors.forename: Gobind
authors.initials: G
authors.lastname: Ram
mesh_terms: D000222:Adaptation, Physiological; D000465:Algorithms; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003198:Computer Simulation; D055864:Droughts; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006358:Hot Temperature; D008958:Models, Molecular; D009195:Peroxidase / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping; D060888:Real-Time Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013312:Stress, Physiological; D014908:Triticum / Q000502:physiology*
chemical_list: D010940:Plant Proteins; D009195:Peroxidase
keywords: Amphipathic α helices; Boiling soluble proteins; Dehydrins; K- segment; String; wBsSRP