Analysis of MeEf1A6 Gene Promoter Activity with In-vitro and In-vivo using Transient and Stable Expression Techniques in Tobacco Plant (Nicotiana tabacum)
DOI:
https://doi.org/10.5614/3bio.2021.3.1.1Keywords:
Promoter, Transformation, MeEF1A6Abstract
The promoter is a part of the gene that functions in carrying out the gene expression, and its work activity becomes a matter of concern to ensure that expression works effectively. MeEF1A6 (Manihot esculentaElongation Factor 1 Alfa - 6) is a promoter derived from cassava plants (Manihot esculenta). In previous studies, the MeEF1A6 promoter was successfully isolated, introduced, and characterized into the pBI121 plasmid, replacing the CaMV35S promoter. This study aims to analyze the activity of MeEF1A6 promoters in-vivo and in-vitro by using transient and transgenic techniques in tobacco plants. The pBI121 plasmid containing the MeEF1A6 promoter was introduced intoAgrobacterium tumefaciensstrain AGL1 and LBA4404. The promoter's work was then analyzed by the result of introducing it into the tobacco plant using the transient and stable transformation. The whole part of explants was used for transient study and tested in a minimum of two biological replicates. Sixty sheets of explant leaves that have been cut were used for stable transformation. The promoter work analysis was carried out with theGUSgene expression that integrated with the promoter with histochemicalGUSassay. The transient produced a blue color in the roots, stems, and leaves on the whole repetition. The transverse incision in the stem shows the blue color on the epidermis and procambium tissue. Stable transformation using AGL1 as vector produced 43 shoots from 40 calli. A total of 43 shoots were selected with antibiotics and produced 27 plantlets that were successfully grown. Some plantlets are then reacted with x-gluc as histochemical GUS assay substrat and produced a blue color in the explants, indicating that the MeEF1A6 promoter has been successfully introduced. The results indicate that the MeEF1A6 promoter could work on plant tissue in roots, stems, leaves, and tissues that connect meristems such as procambium in tobacco plants. This reinforces the suspicion that the MeEF1A6 promoter performs work constitutionally as a constitutive promoter.
References
Maston, G.A., Evans, S.K., dan Green, M. R: Transcriptional Regulatory Elements in the Human Genome. Genet [Internet]. 2006 Sep [cited 2020 Mar 18]:7:29-59. DOI: 10.1146/annurev.genom.7.080505.115623
Peramuna A, Bae H, Rasmussen E.K, Dueholm B, Waibel T, Critchley J.H, Berzezek K, Roberts M, Simonsen H.T: Evaluation of synthetic promoters in Phycomitrella patens. Biochemical and Biophysical Reserch Communications [Internet]. 2018 Jun [cited 2020 Mar 17] :500: 418-422. DOI: https://doi.org/10.1016/j.bbrc.2018.04.092
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., dan Walter, P.: Molecular Biology of The Cell. 5th edition. Garland science [Internet]. 2008 Jul [cited 2020 Mar 18]. DOI: https://doi.org/10.1002/bmb.20192
Merrick, W. C. dan Nyborg, J: The Protein Biosynthesis Elongation Cycle, Translational control of gene expression, edited Nahum Sonenberg, Jhon W. B. Hersey. Michael B. Mathews, Cold Spring Harbor Laboratory Press [Internet]. 2000 [cited 2020 Mar 17] : 39: 89-125. Available from : https://www.forskningsdatabasen.dk/en/catalog/2440359660
Ejiri S : Moonlighting functions of polypeptide elongation factor 1: from action bundling to zine finger protein R1-associated nuclear localization. Biosci. Biotechnol. Biochem [Internet]. 2014 May [cited 2020 Mar 17]: 66: 1-21. DOI: https://doi.org/10.1271/bbb.66.1
Yang F, Demma M, Warren V, Dharmawardhane S, Condeelis J : Identification of an actin-binding protein from Dictyostelium as elongation factor 1a. Nature [Internet]. 1990 Oct [cited 2020 Mar 17]:347: 494?496. DOI: doi: 10.1038/347494a0
Sasikumar, A. N., Perez, W. B., dan Kinzy, T. G: The many roles of the eukaryotic elongation factor 1 complex, WIREs RNA [Internet]. 2012 May [cited 2020 Mar 17]:3:543-55. DOI: doi: 10.1002/wrna.1118
Kaur KJ, Ruben L: Protein translation elongation factor-1 alphafrom Trypanosoma brucei binds calmodulin. J Biol Chem [Internet]. 1994 Sept [cited 2020 Mar 17]:269: 23045?23050. Available from : https://www.jbc.org/content/269/37/23045.full.pdf
Duttaroy, A., Bourbeau, D., Wang, X. L.,dan Wang, E : Apoptosis rate can be accelerated or decelerated by overexpression or reduction of the level of elongation factor-1 alpha, Exp. Cell Res [Internet]. 1998 Jan [cited 2020 Mar 17] :238:168?176. DOI: 10.1006/excr.1997.3819
Yamaji Y, Sakurai K, Hamada K, Komatsu K, Ozeki J, et al : Significance of eukaryotic translation elongation factor 1A in tobacco mosaic virus infection. Archives of Virology [Internet]. 2009 Des [cited 2020 Mar 18]:155: 263?268. DOI: 10.1007/s00705-009-0571-x
Toueille M, Saint-Jean B, Castroviejo M, Benedetto JP : The elongation factor 1a: a novel regulator in the DNA replication/repair protein network in wheat cells?. Plant Physiol Biochem [Internet]. 2007 Feb [cited 2020 Mar 17]: 45: 113?118. DOI: 10.1016/j.plaphy.2007.01.006
Suhandono S, Hughes J, Brown K, Hughes M.A : Expression and structure of an elongation factor-1α gene (MeEF1) from cassava (Manihot esculenta Crants). Euphytica [Internet]. 2001 Jun [cited Mar 2020 17]:120: 49-58. DOI: 10.1023/a:1017543318423
Suhandono S, Apriyanto A, Ihsani N : Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters. PloS ONE [Internet]. 2014 Jan [cited 2020 Mar 17] :9(1), e84692. DOI: doi:10.1371/journal.pone.0084692.
Debler J W. : Making electrocompeten agrobacterium tumefaciens [Internet]. 2019 Available from https://dx.doi.org/10.17504/protocols.io.xyqfpvw. [Accessed: 2019-12-01]
Apriyanto, A. Isolation and Characterization of Eukaryotic Elongation Factor 1 Alpha (EF1a) Gene Promoters from Cassava (Manihot esculenta Crantz.) [thesis]. Bandung: Bandung Insititute of technology. 2012.
Fajri Hayatul. Genetic engineering of Nicotiana tabacum cv. SR1 with the Gα gene cDNA [thesis]. Bogor: Bogor Agricultural University. 2015.
Jefferson, R. A., Kavanagh, T.A., dan Bevan, M.W.: GUS fusions: ,B-glucuronidase as a sensitive and versatile gene fusion marker in higher plants, The EMBO journal [Internet].1987 Dec [cited 2020 Mar 17]: 6: 3901-3907. PMCID: PMC553867. PMID: 3327686
Oktaviani Indah. MeEF1A6 promoter activity in Nicotiana tabacum L. Putative transgenic [thesis]. Bandung: Bandung Insititute of technology. 2016
Verdaguer, B., Kochko, A., Beachy, R. N., dan Fauquet, C. : Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter, Plant Molecular Biology [Internet]. 1996 Sept [cited 2020 Mar 17]:31: 1129-1139. DOI: 10.1007/BF00040830
Delporte, A., Holle, S. V, dan Van Damme, E. J. M. : Qualitative and quantitative analysis of the Nictaba promoter activity during development in Nicotiana tabacum, Plant Physiology and Biochemistry [Internet]. 2013 Jun [cited 2020 Mar 18]: 67: 162-168. DOI: 10.1016/j.plaphy.2013.03.005
Prina A.R, Landau A.M, Pacheco M.G. Chimeras and mutant gene transmission. In book Plant Mutation Breeding and Biotechnology, Edition: 2012, Chapter: Chapter 15. FAO/IAEA, Editors: Shu Q.Y., pp.179-187. 2012. Available from : https://www.researchgate.net/publication/262068626_Chimeras_and_Mutant_Gene_Transmission
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