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| Western blot detection of PMI protein in transgenic rice |
| RONG Rui-juan1*, WU Peng-cheng2*, LAN Jin-ping1, WEI Han-fu2, WEI Jian1, CHEN Hao2, SHI Jia-nan1, HAO Yu-jie2, LIU Li-juan1, DOU Shi-juan1, LI Li-yun1, WU Lin3, LIU Si-qi3, YIN Chang-cheng2, LIU Guo-zhen1 |
1 College of Life Sciences, Hebei Agricultural University, Baoding 071001, P.R.China
2 Beijing Protein Innovation Co. Ltd., Beijing 101318, P.R.China
3 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, P.R.China |
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摘要 Phosphomannose isomerase (PMI) encoding gene manA is a desirable selective marker in transgenic research. Understanding of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain (about 0.08 mg). PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.
Abstract Phosphomannose isomerase (PMI) encoding gene manA is a desirable selective marker in transgenic research. Understanding of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain (about 0.08 mg). PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.
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Received: 21 March 2015
Accepted:
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| Fund: This work was supported in part by the Natural Science Foundation of Beijing, China (5121001) and the Cultivate New Varieties of Genetically Modified Organisms Technology Major Projects, the Ministry of Science and Technology of China (2009ZX08012-006B). |
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Corresponding Authors:
LIU Guo-zhen, Tel: +86-312-7528787, Fax: +86-312-7528250, E-mail: gzhliu@genomics.org.cn;
YIN Chang-cheng, Tel: +86-10-80493132, Fax: +86-10-80485325, E-mail: yincc@genomics.cn
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Cite this article:
RONG Rui-juan, WU Peng-cheng, LAN Jin-ping, WEI Han-fu, WEI Jian, CHEN Hao, SHI Jia-nan, HAO Yu-jie, LIU Li-juan, DOU Shi-juan, LI Li-yun, WU Lin, LIU Si-qi, YIN Chang-cheng, LIU Guo-zhen.
2016.
Western blot detection of PMI protein in transgenic rice. Journal of Integrative Agriculture, 15(4): 726-734.
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| Bakhsh A, Rao A Q, Shahid A A, Husnain T, Riazuddin S. 2010. CaMV 35S is a developmental promoter being temporal and spatial in expression pattern of insecticidal genes (cry1ac & cry2a) in cotton. Australian Journal of Basic and Applied Science, 4, 37–44.Boscariol R, Almeida W, Derbyshire M, Mourao Filho F, Mendes B. 2003. The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Reports, 22, 122–128.Boyko A, Molinier J, Chatter W, Laroche A, Kovalchuk I. 2010. Acute but not chronic exposure to abiotic stress results in transient reduction of expression levels of the transgene driven by the 35S promoter. New Biotechnology, 27, 70–77.Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.B?íza J, R??i?ková N, Niedermeierová H, Dusbábková J, Vlasák J. 2010. Phosphomannose isomerase gene for selection in lettuce (Lactuca sativa L.) transformation. Acta Biochimica Polonica, 57, 63–68.Cao Y, Sun J, Zhu J, Li L, Liu G. 2010. Primer CE: Designing primers for cloning and gene expression. Molecular Biotechnology, 46, 113–117.Dahl R, Bravo Y, Sharma V, Ichikawa M, Dhanya R P, Hedrick M, Brown B, Rascon J, Vicchiarelli M, Mangravita-Novo A, Yang L, Stonich D, Su Y, Smith L H, Sergienko E, Freeze H H, Cosford N D. 2011. Potent, selective, and orally available benzoisothiazolone phosphomannose isomerase inhibitors as probes for congenital disorder of glycosylation Ia. Journal of Medicinal Chemistry, 54, 3661–3668.Duan Y, Zhai C, Li H, Li J, Mei W, Gui H, Ni D, Song F, Li L, Zhang W. 2012. An efficient and high-throughput protocol for Agrobacterium-mediated transformation based on phosphomannose isomerase positive selection in japonica rice (Oryza sativa L.). Plant Cell Reports, 31, 1611–1624.Gui H, Li X, Liu Y, Han K, Li X. 2014. The relationship between PMI (manA) gene expression and optimal selection pressure in Indica rice transformation. Plant Cell Reports, 33, 1081–1090.Hansen G, Wright M S. 1999. Recent advances in the transformation of plants. Trends in Plant Science, 4, 226–231.Hou M, Xu W, Bai H, Liu Y, Li L, Liu L, Liu B, Liu G. 2012. Characteristic expression of rice pathogenesis-related proteins in rice leaves during interactions with Xanthomonas oryzae pv. oryzae. Plant Cell Reports, 31, 895–904.Joersbo M, Donaldson I, Kreiberg J, Petersen S G, Brunstedt J, Okkels F T. 1998. Analysis of mannose selection used for transformation of sugar beet. Molecular Breeding, 4, 111–117.Lamblin F, Aimé A, Hano C, Roussy I, Domon J, Van Droogenbroeck B, Lainé E. 2007. The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum). Plant Cell Reports, 26, 765–772.Li J, Xu R F, Qin R Y, Ma H, Li H, Zhang Y P, Li L, Wei P C, Yang J B. 2014. Isolation and functional characterization of a novel rice constitutive promoter. Plant Cell Reports, 33, 1651–1660.Li X, Bai H, Wang X, Li L, Cao Y, Wei J, Liu Y, Liu L, Gong X, Wu L, Liu S, Liu G. 2011. Identification and validation of rice reference proteins for Western blotting. Journal of Experimental Botany, 62, 4763–4772.Ma H, Shieh K. 2006. Western blotting method. The Journal of American Science, 2, 23–27.Prado J R, Segers G, Voelker T, Carson D, Dobert R, Phillips J, Cook K, Cornejo C, Monken J, Grapes L. 2014. Genetically engineered crops: From idea to product. Annual Review of Plant Biology, 65, 769–790.Priya R, Khan M, Rao M K, Parida M. 2014. Cloning, expression and evaluation of diagnostic potential of recombinant capsid protein based IgM ELISA for chikungunya virus. Journal of Virological Methods, 203, 15–22.Schnurr J, Guerra D. 2000. The CaMV-35S promoter is sensitive to shortened photoperiod in transgenic tobacco. Plant Cell Reports, 19, 279–282.Svensson J, Andersson C, Reseland J E, Lyngstadaas P, Bulow L. 2006. Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli. Protein Expression and Purification, 48, 134–141.Terada R, Shimamoto K. 1990. Expression of CaMV35S-GUS gene in transgenic rice plants. Molecular Genetics and Genomics, 220, 389–392.Thiruvengadam M, Hsu W, Yang C. 2011. Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (Oncidium Gower Ramsey). Plant Cell Tissue and Organ Culture, 104, 239–246.Wang H, Petri C, Burgos L, Alburquerque N. 2013. Phosphomannose-isomerase as a selectable marker for transgenic plum (Prunus domestica L.). Plant Cell Tissue and Organ Culture, 113, 189–197.Williamson J D, Hirsch-Wyncott M E, Larkins B A, Gelvin S B. 1989. Differential accumulation of a transcript driven by the CaMV 35S promoter in transgenic tobacco. Plant Physiology and Biochemistry, 90, 1570–1576.Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim-Moore L. 2001. Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Reports, 20, 429–436.Zahradnik C, Kolm C, Martzy R, Mach R L, Krska R, Farnleitner A H, Brunner K. 2014. Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: A practical approach. Analytical and Bioanalytical Chemistry, 27, 6835–6842. |
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