Volume 9, Issue 4 (2018)                   JMBS 2018, 9(4): 627-633 | Back to browse issues page

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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
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
Abstract:   (3107 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.
Full-Text [PDF 659 kb]   (2812 Downloads)    
Article Type: Research Paper | Subject: Agricultural Biotechnology
Received: 2016/12/13 | Accepted: 2017/06/13 | Published: 2018/12/21

References
1. Tsuji T, Niida Y. Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories. Electrophoresis. 2008;29(7):1473-83. [Link] [DOI:10.1002/elps.200700729]
2. Yeung AT, Hattangadi D, Blakesley L, Nicolas E. Enzymatic mutation detection technologies. Biotechniques. 2005;38(5):749-58. [Link] [DOI:10.2144/05385RV01]
3. Vogiatzakis N, Kekou K, Sophocleous C, Kitsiou S, Mavrou A, Bakoula C, et al. Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol. Mol Biotechnol. 2013;55(1):1-9. [Link] [DOI:10.1007/s12033-007-0062-9]
4. Shi Y, Terry SF, Terry PF, Bercovitch LG, Gerard GF. Development of a rapid, reliable genetic test for pseudoxanthoma elasticum. J Mol Diagn. 2007;9(1):105-12. [Link] [DOI:10.2353/jmoldx.2007.060093]
5. Wang GX, Tan MK, Rakshit S, Saitoh H, Terauchi R, Imaizumi T, et al. Discovery of single-nucleotide mutations in acetolactate synthase genes by Ecotilling. Pestic Biochem Physiol. 2007;88(2):143-8. [Link] [DOI:10.1016/j.pestbp.2006.10.006]
6. Nieto C, Piron F, Dalmais M, Marco CF, Moriones E, Gómez-Guillamón ML, et al. EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility. BMC Plant Biol. 2007;7:34. [Link] [DOI:10.1186/1471-2229-7-34]
7. Brady SM, Provart NJ. Extreme breeding: Leveraging genomics for crop improvement. J Sci Food Agric. 2007;87(6):925-9. [Link] [DOI:10.1002/jsfa.2763]
8. Sokurenko EV, Tchesnokova V, Yeung AT, Oleykowski CA, Trintchina E, Hughes KT, et al. Detection of simple mutations and polymorphisms in large genomic regions. Nucleic Acids Res. 2001;29(22):e111. [Link] [DOI:10.1093/nar/29.22.e111]
9. Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, et al. Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J. 2004;37(5):778-86. [Link] [DOI:10.1111/j.0960-7412.2003.01999.x]
10. Comai L, Henikoff S. TILLING: Practical single‐nucleotide mutation discovery. Plant J. 2006;45(4):684-94. [Link] [DOI:10.1111/j.1365-313X.2006.02670.x]
11. Holkers M, Maggio I, Liu J, Janssen JM, Miselli F, Mussolino C, et al. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 2013;41(5):e63. [Link] [DOI:10.1093/nar/gks1446]
12. Maier DA, Brennan AL, Jiang S, Binder-Scholl GK, Lee G, Plesa G, et al. Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. Hum Gene Ther. 2013;24(3):245-58. [Link] [DOI:10.1089/hum.2012.172]
13. Desai NA, Shankar V. Single-strand-specific nucleases. FEMS Microbiol Rev. 2003;26(5):457-91. [Link] [DOI:10.1111/j.1574-6976.2003.tb00626.x]
14. Mon H, Lee J, Fukushima M, Nagata Y, Fujii M, Xu J, et al. Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system. Appl Microbiol Biotechnol. 2013;97(15):6813-22. [Link] [DOI:10.1007/s00253-012-4583-1]
15. Oleykowski CA, Mullins CRB, Godwin AK, Yeung AT. Mutation detection using a novel plant endonuclease. Nucleic Acids Res. 1998;26(20):4597-602. [Link] [DOI:10.1093/nar/26.20.4597]
16. Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Clepet C, et al. Characterization of Arabidopsis thaliana mismatch specific endonucleases: Application to mutation discovery by TILLING in pea. Plant J. 2007;51(6):1116-25. [Link] [DOI:10.1111/j.1365-313X.2007.03201.x]
17. Zolala J, Bahrami AR, Farsi M, Matin MM, Yassaee VR. Comparison of CEL I gene expression and mismatch-cleavage activity in some Apiaceae plants. Mol Breed. 2009;24(1):17-24. [Link] [DOI:10.1007/s11032-009-9267-x]
18. Mirzaei M, Zolala J, Shalouzad A. Isolation, cloning and molecular characterization of genes encoding single-strand specific endonucleases from Parsley (Petroselinum crispum L.). J Agric Biotechnol. 2015;7(1):201-15. [Persian] [Link]
19. Huang FC, Studart-Witkowski C, Schwab W. Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system. Plant Biotechnol J. 2010;8(7):783-95. [Link] [DOI:10.1111/j.1467-7652.2010.00508.x]
20. Kapila J, De Rycke R, Van Montagu M, Angenon G. An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. 1997;122(1):101-8. [Link] [DOI:10.1016/S0168-9452(96)04541-4]
21. Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: Versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009;7(7):682-93. [Link] [DOI:10.1111/j.1467-7652.2009.00434.x]
22. Wydro M, Kozubek E, Lehmann P. Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana. Acta Biochim Pol. 2006;53(2):289-98. [Link]
23. Yang B, Wen X, Kodali NS, Oleykowski CA, Miller CG, Kulinski J, et al. Purification, cloning, and characterization of the CEL I nuclease. Biochemistry. 2000;39(13):3533-41. [Link] [DOI:10.1021/bi992376z]
24. Kozak M. The scanning model for translation: An update. J Cell Biol. 1989;108(2):229-41. [Link] [DOI:10.1083/jcb.108.2.229]
25. Sambrook J, Green MR. Appendix 1, Reagents and buffers. Molecular cloning: A laboratory manual. [Link]
26. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1-2):248-54. [Link] [DOI:10.1016/0003-2697(76)90527-3]
27. Baron U, Imhoff U, Koch J, Leuer M, Weber J, Olek K, inventors; Biopsytec Analytik Gmbh, assignee. Mutated nucleic acid of a CEL I-endonuclease and method for producing the recombinant, full-length CEL I-protein. WO2004035771. 2003 Oct 9. [Link]
28. Pimkin M, Caretti E, Canutescu A, Yeung JB, Cohn H, Chen Y, et al. Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection. BMC Biotechnol. 2007;7:29. [Link] [DOI:10.1186/1472-6750-7-29]
29. Bendahmane A, Sturbois B, Triques K, Caboche M, inventors; Genoplante-Valor, Institut National de la Recherche Agronomique (INRA), assignees. Method for producing highly sensitive endonucleases, novel preparations of endonucleases and uses thereof. US20120009583A1. 2004 Jul 30. [Link]

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