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Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

Padaria JC, Vishwakarma H, Biswas K, Jasrotia RS, Singh GP - BMC Res Notes (2014)

Bottom Line: A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling.TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting.TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology and Climate Change Laboratory, National Research Centre on Plant Biotechnology, Pusa Campu, New Delhi 110012, India. jasdeep_kaur64@yahoo.co.in.

ABSTRACT

Background: Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India.

Results: qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting.

Conclusions: TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

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Related in: MedlinePlus

Active sites and interaction of receptor-ligand. Model of generated TapAPX protein showing ten active sites by Q-SiteFinder tool (in different colours) containing different amino acid residues. First five active sites shown in space fill (A). Molecular interaction studies between TapAPX model and substrate H2O2 by Autodock vina software. Green dots represent the Hydrogen bonding between ASN55 and SER57 (B).
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Fig6: Active sites and interaction of receptor-ligand. Model of generated TapAPX protein showing ten active sites by Q-SiteFinder tool (in different colours) containing different amino acid residues. First five active sites shown in space fill (A). Molecular interaction studies between TapAPX model and substrate H2O2 by Autodock vina software. Green dots represent the Hydrogen bonding between ASN55 and SER57 (B).

Mentions: Ten different active sites were identified (Figure 6A) in the generated TapAPX 3D protein model by Q-SiteFinder (Table 2). H2O2 ligand molecule was retrieved from pubchem database [PubChem:CID_784] for docking studies of the generated protein structure. Nine different confirmations of docking between the receptor (TapAPX) and the ligand molecule (H2O2) were obtained using Autodock vina. The best docked interaction model of TapAPX with H2O2 (hydrogen peroxide) was analyzed by Autodock tool. Each ligand represents a specific binding energy where the ligand containing lowest binding energy conformation was considered the most acceptable docking structure. The docked confirmation with lowest binding energy score i.e. -3.3 was selected for further analysis. Docking analysis clearly indicates that the ligand molecule was involved in the interaction with TapAPX model and that H2O2 formed H bond between 2 amino acid residues i.e. ASN55 and SER57 (Figure 6B).Figure 6


Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

Padaria JC, Vishwakarma H, Biswas K, Jasrotia RS, Singh GP - BMC Res Notes (2014)

Active sites and interaction of receptor-ligand. Model of generated TapAPX protein showing ten active sites by Q-SiteFinder tool (in different colours) containing different amino acid residues. First five active sites shown in space fill (A). Molecular interaction studies between TapAPX model and substrate H2O2 by Autodock vina software. Green dots represent the Hydrogen bonding between ASN55 and SER57 (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4209082&req=5

Fig6: Active sites and interaction of receptor-ligand. Model of generated TapAPX protein showing ten active sites by Q-SiteFinder tool (in different colours) containing different amino acid residues. First five active sites shown in space fill (A). Molecular interaction studies between TapAPX model and substrate H2O2 by Autodock vina software. Green dots represent the Hydrogen bonding between ASN55 and SER57 (B).
Mentions: Ten different active sites were identified (Figure 6A) in the generated TapAPX 3D protein model by Q-SiteFinder (Table 2). H2O2 ligand molecule was retrieved from pubchem database [PubChem:CID_784] for docking studies of the generated protein structure. Nine different confirmations of docking between the receptor (TapAPX) and the ligand molecule (H2O2) were obtained using Autodock vina. The best docked interaction model of TapAPX with H2O2 (hydrogen peroxide) was analyzed by Autodock tool. Each ligand represents a specific binding energy where the ligand containing lowest binding energy conformation was considered the most acceptable docking structure. The docked confirmation with lowest binding energy score i.e. -3.3 was selected for further analysis. Docking analysis clearly indicates that the ligand molecule was involved in the interaction with TapAPX model and that H2O2 formed H bond between 2 amino acid residues i.e. ASN55 and SER57 (Figure 6B).Figure 6

Bottom Line: A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling.TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting.TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology and Climate Change Laboratory, National Research Centre on Plant Biotechnology, Pusa Campu, New Delhi 110012, India. jasdeep_kaur64@yahoo.co.in.

ABSTRACT

Background: Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India.

Results: qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting.

Conclusions: TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

Show MeSH
Related in: MedlinePlus