Cloning and heterologous expression of Os-AP2/ERF-N22 drought inducible rice transcription factor in E. coli
DOI:
https://doi.org/10.56093/ijas.v88i10.84209Keywords:
Abiotic stress, AP2/ERF, Drought, Heterologous expression, Rice, Transcription factorAbstract
AP2/ERF a plant specific transcription factor plays a crucial role in the expression and regulation of abiotic stress related genes. In this study, we have characterized a rice transcription factor named Os-AP2/ERF-N22. It contains single AP2 domain which spans from 5th to 70th amino acid residue in the protein which is 243 amino acid residue long. The partial sequence of the gene encoding Os-AP2/ERF transcription factor was amplified and cloned into pET29a bacterial expression vector. The histidine-tagged truncated Os-AP2/ERF-N22 protein was expressed in BL21(DE3) strain of E. coli after induction with 0.5mM IPTG. A time course induction study was used to optimize the protein expression and the recombinant Os-AP2/ERF-N22 was purified using Ni-NTA agarose column chromatography. The 26.74 kDa recombinant Os-AP2/ERF-N22 was visualized using SDS-PAGE analysis and its expression was further confirmed by western blot analysis using anti-his primary antibody and alkaline phosphatase conjugated secondary antibody. Antibody against the purified truncated AP2/ERF-N22 was custom synthesized and it was used to quantitate the expression of Os-AP2/ERF-N22 transcription factor in rice at the protein level.
Downloads
References
Ahmadikhah A and Marufinia A. 2016. Effect of reduced plant height on drought tolerance in rice. 3Biotech 6(2): 221, 1–9. DOI: https://doi.org/10.1007/s13205-016-0542-3
Cha R S and Thilly W G. 1993. Specificity, efficiency and fidelity of PCR. Genome Research 3: S18–S29. DOI: https://doi.org/10.1101/gr.3.3.S18
Guo M, Liu J H, Ma X, Luo D X, Gong Z H and Lu M H. 2016. The plant heat stress transcription factors (HSFs): structure, regulation and function in response to abiotic stresses. Frontiers in Plant Science 7 (114): 1–13. DOI: https://doi.org/10.3389/fpls.2016.00114
Jofuku K D, Boer B, Montagu M V and Okamuro J K. 1994. Control of Arabidopsis flower and seed development by the homeotic gene APETELA2. Plant Cell 6: 1211–25. DOI: https://doi.org/10.1105/tpc.6.9.1211
Kim A R, Lim H, Cho J, Kim C, Ji S U, Park S, and Lee G. 2015. Overexpression of BrTSR53 gene improves tolerance of rice plant to salt stress. Plant Breeding and Biotechnology 3(4): 376–83. DOI: https://doi.org/10.9787/PBB.2015.3.4.376
Lata C, Mishra A K, Muthamilarasan M, Bonthala V S, Khan Y and Prasad M. 2014. Genome-wide investigation and expression profiling of AP2/ERF transcription factor superfamily in foxtail millet (Setaria italica L.). PLoS ONE 9 (11): e113092. DOI: https://doi.org/10.1371/journal.pone.0113092
Lin D G and Wang C S. 2014. Extraction of total protein from rice plant. Bio-protocol 4(21): 1–8. DOI: https://doi.org/10.21769/BioProtoc.1277
Mawlong I, Ali K, Kurup D, Yadav S and Tyagi A. 2014. Isolation and characterization of an AP2/ERF-type drought stress inducible transcription factor encoding gene from rice. Journal of Plant Biochemistry and Biotechnology 23(1): 42–51. DOI: https://doi.org/10.1007/s13562-012-0185-3
Mawlong I, Ali K, Srinivasan R, Rai R D and Tyagi A. 2015. Functional validation of a drought-responsive AP2/ERF family transcription factor-encoding gene from rice in Arabidopsis. Molecular Breeding 35: 163–74. DOI: https://doi.org/10.1007/s11032-015-0290-9
Pantuwan G, Fukai S, Cooper M, Rajatasereekul S and O’Toole JC. 2000. Field screening for drought resistance. (In) Increased lowland rice production in the Mekong region. Proceedings of International Workshop (Vientiane, Laos): 69–77.
Rashid M, Guangyuan H, Guangxiao Y, Hussain J and Xu Y. 2012. AP2/ERF transcription factor in rice: Genome-wide canva and syntenic relationships between monocots and Eudicots. Evolutionary Bioinformatics 8: 321–55. DOI: https://doi.org/10.4137/EBO.S9369
Sakuma Y, Liu Q, Dubouzet J G, Abe H, Shinozaki K and Shinozaki K Y. 2002. DNA-binding specificity of the ERF/ AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communication 290: 998–1009. DOI: https://doi.org/10.1006/bbrc.2001.6299
Saleh A and Pages M. 2003. Plants AP2/ERF transcription factors. Genetica 35(1): 37–50. DOI: https://doi.org/10.2298/GENSR0301037S
Sambrook J and Russel D W. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour.
Song X, Li Y and Hou X. 2013. Genome-wide analysis of the AP2/ERF transcription factor superfamily in Chinese cabbage (Brassica rapassp. pekinensis). BMC Genomics 14: (573): 1–15. DOI: https://doi.org/10.1186/1471-2164-14-573
Srivastav A, Mehta S, Lindlof A and Bhargava S. 2010. Over-represented promoter motifs in abiotic stress-induced DREB genes of rice and sorghum and their probable role in regulation of gene expression. Plant Signal Behaviour 5(7): 775–84. DOI: https://doi.org/10.4161/psb.5.7.11769
Wu H, Lv H, Li L, Liu J, Mu S and Li X. 2015. Genome-wide analysis of the AP2/ERF transcription factors family and the expression patterns of DREB genes in moso bamboo (Phyllostachys edulis). PLoS ONE 10(5): e0126657. DOI: https://doi.org/10.1371/journal.pone.0126657
Yamaguchi-Shinozaki K and Shinozaki K. 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology 57: 781–803. DOI: https://doi.org/10.1146/annurev.arplant.57.032905.105444
Zhuang J, Cai B, Peng R H, Zhu B,Jin X F and Xue Y. 2008. Genomewide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochemical and Biophysical Research Communication 371: 468–74. DOI: https://doi.org/10.1016/j.bbrc.2008.04.087
Downloads
Published
Issue
Section
License
Copyright (c) 2018 The Indian Journal of Agricultural Sciences
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
The copyright of the articles published in The Indian Journal of Agricultural Sciences is vested with the Indian Council of Agricultural Research, which reserves the right to enter into any agreement with any organization in India or abroad, for reprography, photocopying, storage and dissemination of information. The Council has no objection to using the material, provided the information is not being utilized for commercial purposes and wherever the information is being used, proper credit is given to ICAR.
How to Cite
