Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method

Dina Sri Yulita, Aqwin Polosoro, Atmitri Sisharmini, Aniversari Apriana, Febi Nurilmala, Kurniawan Rudi Trijatmiko

Abstract


Cold shock protein (Csp) is an essential bacterial protein for increasing abiotic stress tolerance, especially cold stress. Several studies discovered that overexpression of the gene successfully improves the tolerances of several types of plant not only under cold stress, but also other abiotic stresses, e.g. hot and drought conditions. The objectives of this study were to construct a binary vector containing the LcCsp gene modified from Lactobacillus casei and transform the gene into Nipponbare rice genome. The native LcCsp gene sequence, however, has low GC content (46.7%) while rice as transformation target plant has 52% GC content. The native LcCsp gene sequence, therefore, was optimized to the level close to 52.7% similar to GC content of the rice genome. This LcCsp gene was synthesized by using DNA printing technology (gBlocks® Gene Fragments Entry, IDT). The synthetic LcCsp gene was successfully inserted into the pCAMBIA1300-int binary vector driven by Ubiquitin1 promoter and NOS terminator. The T-DNA cassette was successfully transformed into Nipponbare rice genome by Agrobacterium tumefaciens strain LBA4404 using immature embryo transformation protocol. A total of 51 T0 Nipponbare lines survived from hygromycin selections and 21 lines were successfully acclimatized. Molecular analysis of the candidate lines showed that all Nipponbare transgenic putative lines contain the LcCsp gene demonstrating high transformation efficiency of 11.8%. The rice lines resulted from this study should be further analyzed and might be useful for developing rice transgenic lines tolerance to heat, drought, or saline stress condition.


Keywords


Rice; LcCsp gene; abiotic stresses; Lactobacillus casei; transformation

Full Text:

PDF

References


Apriana, A., Sisharmini, A., Enggarini, W. & Khumaida, N. (2011) Introduksi konstruk over-ekspresi kandidat gen OsWRKY76 melalui Agrobacterium tumefaciens pada tanaman padi Nipponbare. Jurnal AgroBiogen, 7 (1), 19–27.

Arumuganathan, K. & Earle, E.D. (1991) Nuclear DNA content of some important plant species. Plant Molecular Biology Reporter. [Online] 9 (3), 208–218. Available from: https://doi.org/10.1007/BF02672069 [Accessed 3 July 2017].

Barahimipour, R., Strenkert, D., Neupert, J., Schroda, M., Merchant, S.S. & Bock, R. (2015) Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii. The Plant Journal. [Online] 84 (4), 704–717. Available from: https://doi.org/10.1111/tpj.13033 [Accessed 1 January 2018].

Barnes, W.M. (1992) The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. Gene. [Online] 112 (1), 29–35. Available from: https://doi.org/https://doi.org/10.1016/0378-1119(92)90299-5 [Accessed 5 August 2017].

Bihani, P., Char, B. & Bhargava, S. (2011) Transgenic expression of sorghum DREB2 in rice improves tolerance and yield under water limitation. Journal of Agricultural Science. [Online] 149 (1), 95–101. Available from: https://doi.org/10.1017/S0021859610000742 [Accessed 3 June 2016].

Broadbent, J.R., Neeno-Eckwall, E.C., Stahl, B., Tandee, K., Cai, H., Morovic, W., Horvath, P., Heidenreich, J., Perna, N.T., Barrangou, R. & Steele, J.L. (2012) Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation. BMC Genomics. [Online] 13, 533. Available from: https://doi.org/10.1186/1471-2164-13-533 [Accessed 13 June 2014].

Castiglioni, P., Warner, D., Bensen, R.J., Anstrom, D.C., Harrison, J., Stoecker, M., Abad, M., Kumar, G., Salvador, S., D'Ordine, R., Navarro, S., Back, S., Fernandes, M., Targolli, J., Dasgupta, S., Bonin, C., Luethy, M.H. & Heard, J.E. (2008) Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions. Plant Physiology. [Online] 147 (2), 446–455. Available from: https://doi.org/10.1104/pp.108.118828 [Accessed 16 June 2016].

Dellaporta, S.L., Wood, J. & Hicks, J.B. (1983) A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter. [Online] 1 (4), 19–21. Available from: https://doi.org/10.1007/BF02712670 [Accessed 20 June 2013].

Enggarini, W., Polosoro, A., Sustiprijatno & Trijatmiko, K.R. (2017) Introduksi konstruk gen CsNitr1-L dengan promotor Ubiquitin melalui Agrobacterium tumefaciens dan deteksi molekulernya pada padi kultivar Nipponbare. Jurnal Biologi Indonesia. [Online] 13 (2), 261–270. Tersedia pada: https://doi.org/10.14203/jbi.v13i2.3400 [Diakses 22 Desember 2017].

Fuglsang, A. (2003) Codon optimizer: A freeware tool for codon optimization. Protein Expression and Purification. [Online] 31 (2), 247–249. Available from: https://doi.org/10.1016/S1046-5928(03)00213-4 [Accessed 24 August 2013].

Fukunaga, N., Sahara, T. & Takada, Y. (1999) Bacterial adaptation to low temperature: Implications for cold-inducible genes. Journal of Plant Research. [Online] 112 (2), 263–272. Available from: https://doi.org/10.1007/PL00013883 [Accessed 24 August 2013].

Guo, X., Bao, J. & Fan, L. (2007) Evidence of selectively driven codon usage in rice: Implications for GC content evolution of Gramineae genes. FEBS Letters. [Online] 581 (5), 1015–1021. Available from: https://doi.org/10.1016/j.febslet.2007.01.088 [Accessed 22 July 2013].

Jackson, M.A., Sternes, P.R., Mudge, S.R., Graham, M.W. & Birch, R.G. (2014) Design rules for efficient transgene expression in plants. Plant Biotechnology Journal. [Online] 12 (7), 925–933. Available from: https://doi.org/10.1111/pbi.12197 [Accessed 20 January 2016].

Jeong, Y.S., Ku, H.K., Kim, J.K., You, M.K., Lim, S.H., Kim, J.K. & Ha, S.H. (2017) Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm. Plant Biotechnology Reports. [Online] 11 (3), 171–179. Available from: https://doi.org/10.1007/s11816-017-0440-0 [Accessed 23 March 2018].

Kato, Y., Sakala, R.M., Hayashidani, H., Kiuchi, A., Kaneuchi, C. & Ogawa, M. (2000) Lactobacillus algidus sp., Nov., a psychrophilic lactic acid bacterium isolated from vacuum-packaged refrigerated beef. International Journal of Systematic and Evolutionary Microbiology. [Online] 50 Pt. 3, 1143–1149. Available from: https://doi.org/10.1099/00207713-50-3-1143 [Accessed 21 June 2014].

Levy, A., Conway, J.M., Dangl, J.L. & Woyke, T. (2018) Elucidating bacterial gene functions in the plant microbiome. Cell Host & Microbe. [Online] 24 (4), 475–485. Available from: https://doi.org/10.1016/j.chom.2018.09.005 [Accessed 23 May 2019].

Matsumoto, T., Wu, J., Itoh, T., Numa, H., Antonio, B. & Sasaki, T. (2016) The Nipponbare genome and the next-generation of rice genomics research in Japan. Rice. [Online] 9 (1), 33. Available from: https://doi.org/10.1186/s12284-016-0107-4 [Accessed 18 May 2018].

Opabode, J.T. (2006) Agrobacterium-mediated transformation of plants: Emerging factors that influence efficiency. Biotechnology and Molecular Biology Review. [Online] 1 (1), 12–20. Available from: https://academicjournals.org/journal/BMBR/article-abstract/0F759E540214 [Accessed 12 June 2016].

Peng, Z., Lu, T., Li, L., Liu, X., Gao, Z., Hu, T., Yang, X., Feng, Q., Guan, J., Weng, Q., Fan, D., Zhu, C., Lu, Y., Han, B. & Jiang, Z. (2010) Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences. BMC Plant Biology. [Online] 10, 116. Available from: https://doi.org/10.1186/1471-2229-10-116 [Accessed 10 July 2019].

Peng, X.J., Ma, X.Y., Fan, W.H., Su, M., Cheng, L.Q., Alam, I., Lee, B.H., Qi, D.M., Shen, S.H. & Liu, G.S. (2011) Improved drought and salt tolerance of Arabidopsis thaliana by transgenic expression of a novel DREB gene from Leymus chinensis. Plant Cell Reports. [Online] 30 (8), 1493–1502. Available from: https://doi.org/10.1007/s00299-011-1058-2 [Accessed 23 June 2019].

Sachs, R., Max, K.E.A., Heinemann, U.D.O. & Balbach, J. (2012) RNA single strands bind to a conserved surface of the major cold shock protein in crystals and solution. RNA. [Online] 18 (1), 65–76. Available from: https://doi.org/10.1261/rna.02809212 [Accessed 21 February 2014].

Sakata, K., Antonio, B.A., Mukai, Y., Nagasaki, H., Sakai, Y., Makino, K. & Sasaki, T. (2000) INE: A rice genome database with an integrated map view. Nucleic Acids Research. [Online] 28 (1), 97–101. Available from: https://doi.org/10.1093/nar/28.1.97 [Accessed 23 June 2018].

Sambrook, J., Fritsch, E. & Maniatis, T. (1989) Molecular cloning: A laboratory manual. 2nd edition. New York, Cold Spring Harbor Laboratory Press.

Slamet-Loedin, I.H., Chadha-Mohanty, P. & Torrizo, L. (2013) Agrobacterium-mediated transformation: Rice transformation. Cereal Genomics Methods and Protocols. [Online] 1099, 261–271. Available from: https://link.springer.com/protocol/10.1007/978-1-62703-715-0_21 [Accessed 17 August 2019].

Somantri, I.H., Ambarwati, A.D., Dewi, I.S., Apriana, A. & Santoso, T.J. (2001) Transformasi padi dengan bombardmen mikroprojektil. [Online] Tersedia pada: http://biogen.litbang.pertanian.go.id/terbitan/pdf/prosiding2002_102-110_idahanarida.pdf [Diakses 11 Mei 2016].

Tyagi, A.K. & Mohanty, A. (2000) Rice transformation for crop improvement and functional genomics. Plant Science. [Online] 158 (1), 1–18. Available from: https://doi.org/https://doi.org/10.1016/S0168-9452(00)00325-3 [Accessed 9 August 2016].

Windiastri, V.E., Pantouw, C.F., Astuti, D., Widyajayantie, D., Estiati, A. & Nugroho, S. (2018) Transformasi genetik faktor transkripsi OsMYB6 dan OsMYB7 pada kultivar padi Nipponbare untuk manipulasi kadar lignin. [Online] Tersedia pada: https://smujo.id/psnmbi/article/view/2909 [Diakses 22 Juni 2019].

Yu, T.F., Xu, Z.S., Guo, J.K., Wang, Y.X., Abernathy, B., Fu, J.D., Chen, X., Zhou, Y.B., Chen, M., Ye, X.G. & Ma, Y.Z. (2017) Improved drought tolerance in wheat plants overexpressing a synthetic bacterial cold shock protein gene SeCspA. Scientific Reports. [Online] 7, 44050. Available from: https://doi.org/10.1038/srep44050 [Accessed 30 June 2019].

Yuliawan, T. & Handoko, I. (2016) The effect of temperature rise to rice crop yield in Indonesia uses Shierary rice model with geographical information system (GIS) feature. Procedia Environmental Sciences. [Online] 33, 214–220. Available from: https://doi.org/10.1016/j.proenv.2016.03.072 [Accessed 5 May 2018].




DOI: http://dx.doi.org/10.21082/jbio.v16n1.2020.p25-34

Refbacks

  • There are currently no refbacks.




Copyright (c) 2020 Jurnal AgroBiogen

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

 Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.


 

         

P-ISSN : 1907-1094
E-ISSN : 2549-1547


Jurnal AgroBiogen

Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian

Jl. Tentara Pelajar 3A, Bogor 16111
Jawa Barat, Indonesia
Telp.: (0251) 8339793, 8337975
Call Center: 08211181677
Faks.: (0251) 8338820
E-mail: jurnal.agrobiogen@gmail.com
Situs: http://biogen.litbang.pertanian.go.id



View My Stats