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

BMC Research Notes

Gyanendra Pratap Singh¹  Rahul Singh Jasrotia²  Koushik Biswas²  Harinder Vishwakarma²  Jasdeep Chatrath Padaria² 

[1] Division of Genetics, IARI, Pusa Campus, New Delhi 110012, India;Biotechnology and Climate Change Laboratory, National Research Centre on Plant Biotechnology, Pusa Campu, New Delhi 110012, India

关键词: cv. Raj3765;    Expression;    Homology modeling;    Peroxisomal ascorbate peroxidase;    In-silico;    Cloning;   

Others  :  1127292
DOI  :  10.1186/1756-0500-7-713

 received in 2014-06-03, accepted in 2014-10-02,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Padaria et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Asada K: Ascorbate peroxidase hydrogen peroxide scavenging enzyme in plants. Physiol Plant 1992, 85:235-241.
• [2]Teixeira FK, Menezes-Benavente L, Galvao VC, Margis R, Margis-Pinhiero M: Rice peroxidase gene family encodes functionally diverse isoforms localized in different subcellular compartments. Planta 2006, 224:300-314.
• [3]Lu Z, Liu D, Liu S: Two rice cytosolic ascorbate peroxidase differentially improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep 2008, 26:1909-1917.
• [4]Wang C, Yang CP, Wang YC: Cloning and expression analysis of an APX gene from Betula platyphylla. J NE Forest U 2009, 37:79-88.
• [5]Ma CL, Wang PP, Cao ZY: cDNA cloning and gene expression of APX in Suaeda salsa in response to salt stress. J Plant Physiol 2002, 28:261-266.
• [6]Mittler R, Zilinskas BA: Purification and characterization of pea cytosolic ascorbate peroxidase. Plant Physiol 1991, 97:962-968.
• [7]Yoo TH, Park CJ, Lee GL, Shin RY, Yun JH, Kim KJ, Rhee KH, Paek KH: A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria. Mol Cell 2002, 14(1):75-84.
• [8]Lin L, Wang X, Wang Y: cDNA clone, fusion expression and purification of novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli. Mol Biol Rep 2006, 33(3):197-206.
• [9]Rane J, Pannu RK, Sohu VS, Saini RS, Mishra B, Shoran-Crossa J, Vargas M, Joshi AK: Performance of yield and stability of advanced wheat genotypes under heat stress environments of the Indo-Gangetic plains. Crop Sci 2007, 47:1561-1573.
• [10]Padaria JC, Bhatt D, Biswas K, Singh G, Raipuria R: In-silico prediction of an uncharacterized protein generated from heat responsive SSH library in wheat (Triticum aestivum L.). Plant Omics 2013, 6:150-156.
• [11]DuPont FM, Hurkman WJ, Vensel WH, Tanaka CK, Kothari KM, Chung OK, Altenbach SB: Protein accumulation and composition in wheat grains: effects of mineral nutrients and high temperature. Eur J Agron 2006, 25:96-107.
• [12]Boominathan P, Shukla R, Kumar A, Manna D, Negi D, Verma PK, Chattopadhyay D: Long term transcript accumulation during the development of dehydration adaptation in Cicer arietinum. Plant Physiol 2004, 135:1608-1620.
• [13]Liu L, Zhou Y, Zhou G, Ye R, Zhao L, Li X, Lin Y: Identification of early senescence- associated genes in rice flag leaves. Plant Mol Biol 2008, 67:37-55.
• [14]Sekman AH, Turkan I, Takio S: Differential responses of antioxidative enzymes and lipid peroxidation to salt stress in salt-tolerant Plantago maritima and salt-sensitive Plantago media. Physiol Plant 2007, 131(3):399-411.
• [15]Hernandez JA, Almansa MS: Short term effects of salt stress on antioxidant systems and leaf water relations of pea plants. Physiol Plant 2002, 115:251-257.
• [16]Bas A, Fosberg G, Hammarstrom S, Hammarstrom ML: Utility of the housekeeping genes18S rRNA, β-actin and Glyceraldehyde-3-Phosphate-Dehydrogenase for Normalization in Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction analysis of Gene Expression in Human T Lymphocytes. Scand J Immunol 2004, 59:566-573.
• [17]Bunkelmann JR, Trelease RN: Ascorbate peroxidase- a prominent membrane protein in oilseed glyoxysomes. Plant Physiol 1996, 110:589-598.
• [18]Kubo A, Saji H, Tanaka K, Kondo N: Cloning and sequencing of a cDNA encodingAscorbate peroxidase from Arabidopsis thaliana. Plant Mol Biol 1992, 18(4):691-701.
• [19]Shi WM, Muramoto Y, Ueda A, Takebe T: Cloning of ascobate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana. Gene 2001, 273(1):23-27.
• [20]Zhang H, Wang J, Nickel U, Allen RD, Goodman HM: Cloning and expression of an Arabidopsis gene encoding a putative peroxisomal ascorbate peroxidase. Plant Mol Biol 1997, 34:967-971.
• [21]Katiyar A, Lenka SA, Lakshmi K, Chinnusamy V, Bansal KC: In silico characterization and homology modeling of thylakoid bound ascorbate peroxidase from a drought tolerant wheat cultivar. Genomics Proteomics Bioinformatics 2009, 7:4.
• [22]Kaya C, Aydemir S, Sonmez O, Ashraf M, Dikilitas M: Regulation of growth and some key physiological processes in salt-stressed maize (Zea mays L.) plants by exogenous application of asparagine and glycerol. Acta Bot Croat 2013, 72:157-168.
• [23]Morimoto RI: Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Gene Dev 1998, 12:3788-3796.
• [24]Kumar RR, Sharma SK, Gadpayle KA, Singh K, Sivaranjani R, Goswami S, Rai RD: Mechanism of action of hydrogen peroxide in wheat thermotolerant-interaction between antioxidants isoenzymes, proline and cell membrane. Afr J Biotechnol 2012, 11(78):14368-14379.
• [25]Heath RL, Packer L: Photoperoxidation in isolated chloroplasts I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 1968, 125:189.
• [26]Bhatt D, Saxena SC, Jain S, Dobriyal AK, Majee M, Arora S: Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana. Mol Biol Rep 2013, 40(2):1155-1165.
• [27]Sambrook J, Russell DW: Molecular Cloning. In A Laboratory Manual. 3rd edition. New York: Cold Spring Harbor, USA; 2001.
• [28]Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M: In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 2006, 1:2856-2860.
• [29]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.
• [30]Slevers F, Wilm A, Dineen D, Gibson TJ, Karpus K, Li W, Lopez R, Macwiliam H, Remmert M, Sodling J, Thompson JD, Higgins DJ: Fast, scalable generation of high quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011, 7:539.
• [31]Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24:1596-1599.
• [32]Sigrist CJA, de Castro E, Cerutti L, Cuche BA, Hulo N, Bridge A, Bougueleret L, Xenarios I: New and continuing developments at PROSITE. Nucleic Acids Res 2012, 41:344-347.
• [33]Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD: The Pfam protein families database. Nucleic Acids Res 2012, 40:290-301.
• [34]Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Lu S, Marchler GH, Song JS, Thanki N, Yamashita RA, Zhang D, Bryant SH: CDD: conserved domains and protein three- dimensional structure. Nucleic Acids Res 2013, 41:348-352.
• [35]Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A: Protein Identification and Analysis Tools on the ExPASy Server. In The Proteomics Protocols Handbook. Edited by Walker JM. New Jersey: Humana; 2005:571-607.
• [36]Garnier J, Gibrat JF, Robson B: GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 1996, 266:540-553.
• [37]Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
• [38]Kelley LA, Sternberg MJE: Protein structure prediction on the web: a case study using the Phyre server. Nat Protoc 2009, 4:363-371.
• [39]Lambert C, Leonard N, De-Bolle X, Depiereux E: ESyPred3D: Prediction of proteins 3D structures. Bioinformatics 2002, 18:1250-1256.
• [40]Chen CC, Hwang JK, Yang JM: (PS)2: protein structure prediction server. Nucleic Acid Res 2006, 34:152-157.
• [41]Arnold K, Bordoli L, Kopp J, Schwede T: The SWISS-MODEL Workspace: A web- based environment for protein structure homology modelling. Bioinformatics 2006, 22:195-201.
• [42]Bates PA, Kelley LA, MacCallum RM, Sternberg MJE: Enhancement of protein modelling by human intervention in applying the automatic programs 3D-JIGSAW and 3D-PSSM. Proteins 2001, 45:39-46.
• [43]Zhang Y: I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 2008, 9:40. BioMed Central Full Text
• [44]Laskoswki RA, MacArthur MW, Moss DS, Thorton JM: PROCHECK: a program to check the stereo chemical quality of protein structures. J Appl Crystallogr 1993, 26:283-291.
• [45]Luthy R, Bowie JU, Eisenberg D: Assessment of protein models with three-dimensional profiles. Nature 1992, 356:83-85.
• [46]Benkert P, Kunzli M, Schwede T: QMEAN server for protein model quality estimation. Nucleic Acids Res 2009, 37:510-514.
• [47]DeLano WL: The PyMOL Molecular Graphics System. DeLano Scientific. California: San Carlos; 2002.
• [48]Gelly JC, Joseph AP, Srinivasan N, de Brevern AG: iPBA: a tool for protein structure comparison using sequence alignment strategies. Nucleic Acids Res 2011, 39:18-23.
• [49]Laurie AT, Jackson RM: SiteFinder: an energy-based method for the prediction of protein-ligand binding sites. Bioinformatics 2005, 1909:1916.
• [50]Trott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem 2010, 31:455-461.
• [51]Bolton E, Wang Y, Thiessen PA, Bryant SH: PubChem: Integrated Platform of Small Molecules and Biological Activities. In Annual Reports in Computational Chemistry, Volume 4. Washington, DC: American Chemical Society; 2008.
• [52]O’Boyle NM, Banck M, James CA, Morley C, Vandermeersch T, Hutchison GR: Open Babel: An open chemical toolbox. J Cheminformatics 2011, 3:33. BioMed Central Full Text
• [53]Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ: Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 2009, 16:2785-2791.