Expression of Recombinant Alcohol Dehydrogenase in Escherichia coli Strain BL21 (DE3) and In Planta Agrobacterium Transformation of Tomato Seeds
Article Sidebar
Main Article Content
Abstract
Alcohol dehydrogenase is an enzyme that is involved in various roles in plant such as plant development, growth and plant responses to abiotic and biotic stresses. A recombinant alcohol dehydrogenase 1 (Adh1) cDNA (r-msAdh1) from Metroxylon sagu has been previously isolated, and it contained 20 nucleotides derived from Elaeis guineensis at the 5’-end and had a molecular weight of 1.14 kb. The objective of this study is to determine the function of r-msAdh1 via analyses in prokaryotic and eukaryotic hosts. For expression in prokaryotic system, pET-41a(+) with a 8x His tag at the C terminal was used for r-msAdh1 protein purification, and expression was achieved using IPTG for 4-6 h in Escherichia coli strain BL21 (DE3) incubated at low temperature. The induced BL21 strain produced a small amount of soluble r-msAdh1 protein while a large amount was present as insoluble aggregates. Subsequently, the r-msAdh1 cDNA was transformed into tomato seeds (Solanum lycopersicum cv. MT1) via Agrobacterium-mediated in planta transformation. The integration of r-msAdh1 cDNA and the selectable marker were detected in transformed seedlings, T0, using polymerase chain reaction technique. The transformation efficiency was determined to be 33% for r-msAdh1 cDNA and 46% for the selectable marker. For stability analysis of the transgene, eleven T1 generation randomly selected seedlings from the transgenic T0 were analyzed for the presence of the cDNA, and all seedlings were found to contain the full length of r-msAdh1 cDNA. However, out of the eleven T1 transgenic lines produced, only four seedlings were used for expression analysis using the reverse transcriptase PCR (RT-PCR). Two transgenic lines, T19 and T111, were determined to contain r-msAdh1 cDNA and this was verified by nucleotide sequencing. Although only a small number of T1 transgenic seedlings was obtained, this study shows that tomato seeds could be used as a target tissue for Agrobacterium-mediated in planta transformation primarily because the protocol is easy, rapid and cheaper compared to tissue culture-based methods.
Keywords: Alcohol dehydrogenase; Metroxylon sagu; BL21 (DE3); Agrobacterium tumefaciens; tomato seeds
*Corresponding author: Tel.: +60 84 36 75 50, Fax: +60 84 36 73 01
Email: [email protected]
Article Details
Copyright Transfer Statement
The copyright of this article is transferred to Current Applied Science and Technology journal with effect if and when the article is accepted for publication. The copyright transfer covers the exclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, electronic form (offline, online) or any other reproductions of similar nature.
The author warrants that this contribution is original and that he/she has full power to make this grant. The author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors.
Here is the link for download: Copyright transfer form.pdf
References
Roslan, H.A., Sundaraj, Y. and Awang Husaini, A.A.S., 2008. Multiple form of alcohol dehydrogenase (Adh) genes in sago palm: A preliminary study. BANWA, 5(1), 30-41.
Kato-Noguchi, H., 2001. Wounding stress induces alcohol dehydrogenase in maize and lettuce seedlings. Plant Growth Regulation, 35(3), 285-288.
Dolferus, R., Jacobs, M., Peacock, W.J. and Dennis, E.S., 1994. Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiology, 105(4), 1075-1087.
Garabagi, F., Duns, G. and Strommer, J., 2005. Selective recruitment of Adh genes for distinct enzymatic functions in Petunia hybrida. Plant Molecular Biology, 58(2), 283-294.
Yi, S.Y., Ku, S.S., Sim, H.J., Kim, S.K., Park, J.H., Lyu, J.I., So, E.J., Choi, S.Y., Kim, J., Ahn, M.S., Kim, S.W., Park, H., Jeong, W.J., Lim, Y.P., Min, S.R. and Liu, J.R., 2017. An alcohol dehydrogenase gene from Synechocystis sp. confers salt tolerance in transgenic tobacco. Frontier in Plant Science, 8, 1965.
Jeon, Y.J., Fong, J.C., Riyanti, E.I., Neilan, B.A., Roger, P.L. and Svenson, C.J., 2008. Heterologous expression of alcohol dehyrogenase (Adh1) gene from Geobacillus thermoglucosidasius strain M10EXG. Journal of Biotechnology, 135(2), 127-133.
Weckbecker, A. and Hummel, W., 2006. Cloning, expression and characterization of an ®-specific alcohol dehyrogenase from Lactobacillus kefir. Biocatalysis and Biotransformation, 24(5), 380-389.
Liang, J.J., Zhang, M.L., Ding, M., Mai, Z.M., Wu, S.X., Du, Y. and Feng, J.X., 2014. Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization. BMC Biotechnology, 14(45), https://doi: 10.1186/1472-6750-14-45.
Utekal, P., Toth, C., Illesova, A., Kois, P., Bocanova, L., Turna, J., Drahovska, H. and Stuchlik, S., 2014. Expression of soluble Saccharomyces cerevisiae alcohol dehydrogenase in Escherichia coli applicable to oxido-reduction bioconversions. Biologia, 69(6), 722-726.
Wang, N., Shi, H., Yao, Q., Zhou, Y., Kang, L., Chen, H. and Chen, K., 2011. Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori. Genetic and Molecular Biology, 34(2), 240-243.
Yin, J., Li, G., Ren, X. and Herrler, G., 2007. Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. Journal of Biotechnology, 127(3), 335-347.
Rosano, G.L. and Ceccarelli, E.A., 2014. Recombinant protein expression in Escherichia coli: advances and challenges. Frontier in Microbiology, 5, 172.
Joly, J.C., Leung, W.S. and Swartz, J.R., 1998. Overexpression of Escherichia coli oxidoreductases increases recombinant insulin-like growth factor-I accumulation. Proceedings of the National Academy of Science, 95(6), 2773-2777.
Cario, M.M., Cubarsi, R. and Villaverde, A., 2000. Fine architecture of bacterial inclusion bodies. FEBS Letters, 471(1), 7-11.
Slouka, C., Kopp, J., Spadiut, O. and Herwig, C., 2018. Perspective of inclusion bodies for bio-based products: curse or blessing?. Applied Mirobiology and Biotechnology, 103(3), 1143-1153.
Lakshmi, G.J., Kotra, S.R., Peravali, J.B., Kumar, P.P.B.S. and Rao, K.R.S.S., 2014. Molecular cloning, high level expression and activity analysis of constructed human interleukin-25 using industrially important IPTG inducible Escherichia coli BL21 (DE3). International Journal of Bio-Science and Bio-Technology, 6(3), 19-30.
Soltanmohammadi, B., Jalali-Javaran, M., Rajabi-Memari, H. and Mohebodini, M., 2014. Cloning, transformation and expression of proinsulin gene in tomato (Lycopersicum esculentum Mill.). Jundishapur Journal of Natural Pharmaceutical Products, 9(1), 9-15.
Di Matteo, A., Rigano, M.M., Sacco, A., Frusciante, L. and Barone, A., 2011. Genetic transformation in tomato: novel tools to improve fruit quality and pharmaceutical production. In: M. Alvarez, ed. Genetic Transformation. Rijeka: InTech., pp.55.
Barone, A., Chiusano, M.L., Ercolano, M.R., Giuliano, G., Grandillo, S. and Frusciante, L., 2008. Structural and functional genomics of tomato. International Journal of Plant Genomics, 2008, 820274.
Fartyal, D., Agarwal, A., James, D., Borphukan, B., Ram, B., Sheri, V., Agrawal, P.K., Achary, V.M.M. and Reddy, M.K., 2018. Developing dual herbicide tolerant transgenic rice plants for sustainable weed management. Scientific Reports, 8, 11598, https://doi.org/10.1038/s41598-018-29554-9
Alphey, N. and Bonsall, M.B., 2018. Genetics-based method for agricultural insect pest management. Agricultural and Forest Entomology, 20(2), 131-140.
Dong, O.X. and Ronald, P.C., 2019. Genetic engineering for disease resistance in plants: Recent progress and future perspectives. Plant Physiology, 180(1), 26-38.
Mehta, R.A., Cassol, T., Li, N., Ali, N., Handa, A.K. and Mattoo, A.K., 2002. Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality and vine life. Nature Biotechnology, 20(6), 613-618.
Klee, H.J. and Giovannoni, J.J., 2011. Genetics and control of tomato fruit ripening and quality attributes. Annual Review of Genetics, 45, 41-59.
Baghour, M., Gálvez, F.J., Sánchez, M.E., Aranda, M.N., Venema, K. and Rodríguez-Rosales, P., 2019. Overexpression of LeNHX2 and S1SOS2 increases salt tolerance and fruit production in double transgenic tomato plants. Plant Physiology and Biochemistry, 135, 77-86.
Ruf, S., Hermann, M., Berger, I.J., Carrer, H. and Bock, R., 2001. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nature Biotechnology, 19(9), 870-875.
Sedeek, K.E.M., Mahas, A. and Mahfouz, M., 2019. Plant genome engineering for targeted improvement of crop traits. Frontier in Plant Science, 10, 114, https://doi.org/10.3389/fpls.2019.00114
Yasmeen, A., Mirza, B., Inayatullah, S., Safdar, N., Jamil, M. and Ali, S., 2009. In Planta transformation of tomato. Plant Molecular Biology Reporter, 27(1), 20-28.
Safdar, N. and Mirza, B., 2014. An experimental comparison of different transformation procedures assessed in tomato cv Rio Grande with yeast HAL 1 gene. Turkish Journal of Biochemistry, 39(3), 245-252.
Shah, S.H., Ali, S., Jan, S.A. and Ali, U.G., 2015. Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.). Plant Cell, Tissue and Organ Culture, 120(3), 1139-1157.
Miduthuri, S. S., Kumari, A., Pandey, A.K., Sharma, S., Sharma, P., Sreelakshmi, Y. and Sharma, R., 2017. Generation of genetically stable transformants by Agrobacterium using tomato floral buds. Plant Cell, Tissue and Organ Culture, 129(2), 299-312.
Wee. C.C. and Roslan, H.A., 2012. Isolation of Alcohol Dehydrogenase cDNA and basal regulatory region from Metroxylon sagu. ISRN Molecular Biology, 2012, 1-10.
Bergmeyer. H.U., 1986. Methods of Enzymatic Analysis. 3rd ed. Weinheim: Verlag Chemie.
Dyson, M.R., Shadbolt, S.P., Vincent, K.J., Perera, R.L. and McCafferty, J., 2004. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnology, 4(32), https://doi.org/10.1186/1472-6750-4-32
Sørensen, H.P. and Mortensen, K.K., 2005. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factories, 4, 1, https://doi.org/10.1186/1475-2859-4-1
Jong, W.S.P., Vikström, D., Houben, D., van den Berg van Saparoea, H.B., de Gier, J.W. and Luirink, J., 2017. Application of an E. coli signal sequence as a versatile inclusion body tag. Microbial Cell Factories, 16(1), 50, https://doi.org/10.1186/s12934-017-0662-4
Bornhorst, J.A. and Falke, J.J., 2000. Purification of proteins using polyhistidine affinity tags. Methods in Enzymology, 326, 245-254.
Jeong, H., Barbe, V., Lee, C.H., Vallenet, D., Yu, D.S., Choi, S.H., Couloux, A., Lee, S.W., Yoon, S.H., Cattolico, L., Hur, C.G., Park, H.S., Ségurens, B., Kim, S.C., Oh, T.K., Lenski, R.E., Studier, F.W., Daegelen, P. and Kim, J.F., 2009. Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3). Journal of Molecular Biology, 394(4), 644-652.
Desfeux, C., Clough, S.J. and Bent, A.F., 2000. Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiology, 123(3), 895- 904.
Vicker, J.E., Graham, G.C. and Henry, R.J., 1996. A protocol for the efficient screening of putatively transformed plants forbar, the selectable marker gene, using the polymerase chain reaction. Plant Molecular Biology Reporter, 14(4), 363-368.
Mammedov, T.G., Pienaar, E., Whitney, S.E., TerMaat, J.R., Carvill, G., Goliath, R., Subramanian, A. and Viljoen, H.J., 2008. A fundamental study of the PCR amplification of GC-rich DNA templates. Computational Biology and Chemistry, 32(6), 452-457.
Malabadi, R.B. and Nataraja, K., 2007. A biolistic approach for the production of transgenic plants using embryogenic tissue in Pinus kesiya Royle Ex. Gord (Khasi pine). Biotechnology, 6(1), 86-92.
Afolabi, A.S., Worland, B., Snape, J.W. and Vain, P., 2004. A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configuration. Theory and Applied Genetics, 109(4), 815-826.
Sallaud, C., Meynard, D., van Boxtel, J., Gay, C., Bès, M., Brizard, J.P., Larmande, P., Ortega, D., Raynal, M., Portefaix, M., Ouwerkerk, P.B.F, Rueb, S., Delseny, M. and Guiderdoni, E., 2003. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theory and Applied Genetics, 106(8), 1396-1408.
Kohli, A., Miro, B. and Twyman, R.M., 2010. Transgene integration, expression and stability in plants: strategies for improvements. In C., Kole, C. H., Michler, A. G. Abbot, and T. C. Hall, eds. Transgenic Crop Plants. Berlin and Heidelberg: Springer Berlin Heidelberg, pp. 201-237.
Hasan, M., Khan, A.J., Khan, S., Shah, A.H., Khan, A.R. and Mirza, B., 2010. Transformation of tomato (Lycopersicon esculentum Mill.) with Arabidopsis early flowering gene Apetalai (API) through Agrobacterium infiltration of ripened fruits. Pakistan Journal of Biotechnology, 40(1), 161-173.
Brooks, E.M., Sheflin, L.G. and Spaulding, S.W., 1995. Secondary structure in the 30-UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR. BioTechniques, 19(5), 806-812, 814-815.