Volume 9, Issue 4 (2018)
JMBS 2018, 9(4): 627-633 |
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Farsi M, Mirzaei M, Zolala J. Transient Expression of Recombinant PARS II Endonuclease Enzyme Using Agroinfiltration in Tobacco. JMBS 2018; 9 (4) :627-633
URL: http://biot.modares.ac.ir/article-22-14011-en.html
URL: http://biot.modares.ac.ir/article-22-14011-en.html
1- Crop Biotechnology & Breeding Department, Agriculture Faculty, Ferdowsi University of Mashhad, Mashhad, Iran, Ferdowsi University of Mashhad, Azadi Saqure, Mashhad, Iran. Postal Code: 9177948974 , farsi@um.ac.ir
2- Crop Biotechnology & Breeding Department, Agriculture Faculty, Ferdowsi University of Mashhad, Mashhad, Iran
3- Agricultural Biotechnology Department, Agriculture Faculty, Shahid Bahonar University of Kerman, Kerman, Iran
2- Crop Biotechnology & Breeding Department, Agriculture Faculty, Ferdowsi University of Mashhad, Mashhad, Iran
3- Agricultural Biotechnology Department, Agriculture Faculty, Shahid Bahonar University of Kerman, Kerman, Iran
Abstract: (4186 Views)
Aims: The production of recombinant proteins in transgenic plants (molecular farming) is considered a functional aspect of genetic engineering. Unlike animal and bacterial cell-based production systems, proteins produced by plants are very safe and have low production costs due to the absence of common pathogens in humans and animals. The aim of this study was the transient expression of recombinant PARS II endonuclease enzyme using agroinfiltration in tobacco.
Materials and Methods: In this experimental study, the possibility of producing a recombinant form of PARS II endonuclease was investigated, using transient expression system via Agrobacterium. The pBI-Pars expression construct (based on the binary vector pBI121) containing the full sequence of the PARS II encoder, upstream kozak, and a downstream 8x-His tag sequence, was infiltrated into Nicotiana tabacum leaves with Agroinfiltration method. After 72 hours, the expression of PARS II gene in agroinfiltrated leave samples was confirmed through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and protein Dot-blot, using Anti-His antibody at the levels of mRNA and protein, respectively.
Findings: The accuracy of the constructed expression construct was confirmed, and the results of Dot-blot by Anti-His antibody confirmed the expression of the recombinant PARS II protein, while no recombinant protein expression was observed in agroinfiltrated control plants with pBI121 construct. Significant amounts of recombinant PARS II nucleases were produced in tobacco leaves.
Conclusion: Agroinfiltration is an effective and short-term method for mass production of pure recombinant PARS II nucleases in tobacco.
Materials and Methods: In this experimental study, the possibility of producing a recombinant form of PARS II endonuclease was investigated, using transient expression system via Agrobacterium. The pBI-Pars expression construct (based on the binary vector pBI121) containing the full sequence of the PARS II encoder, upstream kozak, and a downstream 8x-His tag sequence, was infiltrated into Nicotiana tabacum leaves with Agroinfiltration method. After 72 hours, the expression of PARS II gene in agroinfiltrated leave samples was confirmed through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and protein Dot-blot, using Anti-His antibody at the levels of mRNA and protein, respectively.
Findings: The accuracy of the constructed expression construct was confirmed, and the results of Dot-blot by Anti-His antibody confirmed the expression of the recombinant PARS II protein, while no recombinant protein expression was observed in agroinfiltrated control plants with pBI121 construct. Significant amounts of recombinant PARS II nucleases were produced in tobacco leaves.
Conclusion: Agroinfiltration is an effective and short-term method for mass production of pure recombinant PARS II nucleases in tobacco.
Article Type: Research Paper |
Subject:
Agricultural Biotechnology
Received: 2016/12/13 | Accepted: 2017/06/13 | Published: 2018/12/21
Received: 2016/12/13 | Accepted: 2017/06/13 | Published: 2018/12/21
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