Limits...
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.

Show MeSH

Related in: MedlinePlus

Heat stress study of recombinantE. coliBL21 (pET28a-TapAPX)cells. OD reading of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells grown at different temperatures after IPTG induction (A). SDS-PAGE analysis of total protein (10 μg) of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells subjected to heat stress, M-Marker (B). *indicates significant difference as determined by simple pair wise t-test comparison (α = 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Heat stress study of recombinantE. coliBL21 (pET28a-TapAPX)cells. OD reading of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells grown at different temperatures after IPTG induction (A). SDS-PAGE analysis of total protein (10 μg) of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells subjected to heat stress, M-Marker (B). *indicates significant difference as determined by simple pair wise t-test comparison (α = 0.05).

Mentions: E. coli cultures harbouring the recombinant plasmid pET-28a-TapAPX grown at temperature viz 37°C, 39°C, 41°C and 43°C higher than optimum temperature for E. coli growth showed continuous increased growth in comparison to E. coli cells having pET-28a vector only, as evident by O.D. (Optical Density) at A600 of E. coli cultures at different temperatures (Figure 3A, Additional file 2: Table S2). Total protein from bacterial cells of E. coli transformed with pET-28a-TapAPX showed over expression of pAPX gene as evident on SDS-PAGE where no expression of TapAPX gene was observed in case of E. coli transformed with pET-28a vector (Figure 3B).Figure 3


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)

Heat stress study of recombinantE. coliBL21 (pET28a-TapAPX)cells. OD reading of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells grown at different temperatures after IPTG induction (A). SDS-PAGE analysis of total protein (10 μg) of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells subjected to heat stress, M-Marker (B). *indicates significant difference as determined by simple pair wise t-test comparison (α = 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Heat stress study of recombinantE. coliBL21 (pET28a-TapAPX)cells. OD reading of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells grown at different temperatures after IPTG induction (A). SDS-PAGE analysis of total protein (10 μg) of E. coli BL21 (pET28) cells and E. coli BL21 (pET28-TapAPX) cells subjected to heat stress, M-Marker (B). *indicates significant difference as determined by simple pair wise t-test comparison (α = 0.05).
Mentions: E. coli cultures harbouring the recombinant plasmid pET-28a-TapAPX grown at temperature viz 37°C, 39°C, 41°C and 43°C higher than optimum temperature for E. coli growth showed continuous increased growth in comparison to E. coli cells having pET-28a vector only, as evident by O.D. (Optical Density) at A600 of E. coli cultures at different temperatures (Figure 3A, Additional file 2: Table S2). Total protein from bacterial cells of E. coli transformed with pET-28a-TapAPX showed over expression of pAPX gene as evident on SDS-PAGE where no expression of TapAPX gene was observed in case of E. coli transformed with pET-28a vector (Figure 3B).Figure 3

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