Transient expression of GFP in plant tissue culture in vitro using viral-based module system
Aim. Agrobacterium-mediated transient expression using viral-based vectors is one of the most effective method for recombinant protein production in plant. Transient expression of GFP using viral-based module system was studied in plant tissue culture in vitro. Methods. Regenerated shoots and callus clones of Nicotiana benthamiana were agroinfiltrated with viral-based module system. Protein extracts from GFP-positive tissues were resolved by non-reducing polyacrylamide gel electrophoresis. GFP from gel was eluted and GFP fluorescence was measured by fluorescence spectrometry with excitation filter at 395 nm and emission filter at 510 nm. Results. Regenerated shoots and callus clones lines accumulated GFP at 242.0±0.7 and 221.6±4.1 μg per g fresh tissue, respectively. The obtained level of transient expression is comparable with other plant production systems in early stage development. Conclusions. The developed technique shows promise for production of therapeutic proteins and antigens in the short term (14–16 days) by transient expression system in plant tissue culture in vitro.
Keywords: viral-based module system, transient expression, recombinant proteins, plant tissue culture, GFP.
Bhaskar P.B., Venkateshwaran V., Wu L., Ane´ J.-M., Jiang J. Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS ONE. 2009. V. 4(6). e5812. doi: 10.1371/journal.pone.0005812.
Sawers R.J.H., Farmer P.R., Moffett P., Brutnell T.P. In planta transient expression as a system for genetic and biochemical analysis of chlorophyll biosynthesis. Plant Methods. 2006. V. 2: 15. doi: 10.1186/1746–4811–2–15.
An S.H., Choi H.W., Hong J.K., Hwang B.K. Regulation and function of the pepper pectin methylesterase inhibitor (CaPMEI1) gene promoter in defence and ethylene and methyl jasmonate signalling in plants. Planta. 2009. V. 230. P. 1223–1237. doi: 10.1007/s00425-009-1021-4.
Kishchenko E.M., Kuchuk N.V. Vliianie ekzogennykh uglevodov na effektivnost' geneticheskoy transformatsii tabaka i sakharnoy svekly s pomoshch'iu Agrobacterium tumefaciens. Fiziologiia i biokhimiia kul't. rasteniy. 2005. V. 37(2). P. 160–166. [in Russian]
He Y., Jones H.D., Chen S., Chen X.M., Wang D.W., Li K.X., Wang D.S., Xia L.Q. Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv. Stewart) with improved efficiency. J.Exp. Bot. 2010. V. 61(6). P. 1567–1581. doi: https://doi.org/10.1093/jxb/erq035.
Marillonnet S., Giritch A., Gils M., Kandzia R., Klimyuk V., Gleba Y. In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. USA. 2004. V. 101. P. 6852–6857. doi: 10.1073/pnas.0400149101.
Marillonnet S., Thoeringer C., Kandzia R., Klimyuk V., Gleba Y. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat. Biotechnol. 2005. V. 23. P. 718–723. doi: 10.1038/nbt1094.
Shamloul M., Trusa J., Mett, V., Yusibov V. Optimization and utilization of Agrobacterium–mediated transient protein production in Nicotiana. J. Vis. Exp. 2014. V. 86. e51204. doi: 10.3791/51204.
Lindbo J.A. High-efficiency protein expression in plants from agroinfection compatible Tobacco mosaic virus expression vectors. BMC Biotech. –2007. V. 7:52. doi: 10.1186/1472-6750-7-52.
Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962. V. 15. P. 473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x.
Maniatis T., Frich E., Sembruk Dzh. Metody geneticheskoy inzhenerii. Molekuliarnoe klonirovanie: Per. s angl. M.: Mir, 1984. 545 p. [in Russian]
Biokhimiia. Metodychni vkazivky do vykonannia laboratornykh robot dlia studentiv spetsial'nosti 227 – Fizychna reabilitatsiia / Ukl.: O.M. Savchenko, V.M. Cheliabiieva, O.I. Syza. Chernihiv: ChNTU, 2016. 87 p. [in Ukrainian]
Skarjinskaia M., Karl J., Araujo A., Ruby K., Rabindran S., Streatfield S.J., Yusibov V. Production of recombinant proteins in clonal root cultures using episomal expression vectors. Biotechnol. Bioeng. 2008. V. 100(4). P. 814–819. doi: 10.1002/bit.21802.