Molecular cloning and in-silico analysis of Ramy3D promoter and 5′ untranslated region from an Iranian rice (Oryza sativa L.) cultivar “NEMAT”

Document Type: Research Paper


1 Department of biotechnology, Faculty of agriculture and natural resources, Imam Khomeini international university, Qazvin, Iran

2 Dept. of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University (IKIU), Qazvin, 34149-16818, IR. of Iran.


The regulatory sequence of rice alpha amylase 3D gene (Ramy3D) is amongst the most successful expression systems used for recombinant protein expression in plants. In the current study a 995 bp fragment consisting of Ramy3D promoter and its 5′ untranslated region was amplified from the genomic DNA of an Iranian rice cultivar ″Nemat″, using polymerase chain reaction. The amplified fragment was ligated into the pTG19-T vector and the cloned fragment was sequenced. For in silico characterization, the rice specific consensus sequences of TATA-box and YR Rule motifs were scanned against the cloned fragment sequence using FIMO program and the cis acting elements existing in the promoter region were investigated using PlantCare database. A TATA-box motif with the rice specific pattern was identified at upstream position of the transcription start site. The identification of TATA-box in Ramy3D promoter is consistent with its metabolic and tissue specific regulation manner. Several cis regulatory motifs responsible for the metabolic and hormonal regulation of Ramy3D gene were identified including ABRE, G-Box, GC-box, GATA motif and TATCCA T/C motif. In addition, several motifs involved in response to various stimuli such as plant hormones, light and biotic and abiotic stresses were identified which include circadian motif, as-2-box, WUN-motif, TGACG-motif, Skn-1 motif, O2-site, MBS, LAMP-element, I-box, HSE, GCC Box, GATT motif, CGTCA-motif and GAG-motif.


Main Subjects

[1]    Benoist, C., O'hare, K., Breathnach, R., and Chambon, P.J.N.A.R. 1980. The ovalbumin gene-sequence of putative control regions. Nucleic Acids Research, 8 (1): 127-142.

[2]    Chung, C., Niemela, S.L., and Miller, R.H. 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences, 86(7): 2172-2175.

[3]    Civan, P. and Švec, M. 2009. Genome-wide analysis of rice (Oryza sativa L. subsp. japonica) TATA box and Y Patch promoter elements. Genome, 52(3): 294-297.

[4]    4.    de los Reyes, B.G., Mohanty, B., Yun, S.J., Park, M.-R., and Lee, D.-Y. 2015. Upstream regulatory architecture of rice genes: summarizing the baseline towards genus-wide comparative analysis of regulatory networks and allele mining. Rice, 8(1): 8-14.

[5]    5.    Forde, B., Heyworth, A., Pywell, J., and Kreis, M. 1985. Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucleic Acids Research, 13(20): 7327-7339.

[6]    6.    Francisco, J.A., Gawlak, S.L., Miller, M., Bathe, J., Russell, D., Chace, D., Mixan, B., Zhao, L., Fell, H.P., and Siegall, C.B. 1997. Expression and characterization of bryodin 1 and a bryodin 1-based single-chain immunotoxin from tobacco cell culture. Bioconjugate chemistry, 8(5): 708-713.

[7]    7.    Giuliano, G., Pichersky, E., Malik, V., Timko, M., Scolnik, P., and Cashmore, A. 1988. An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene. Proceedings of the National Academy of Sciences, 85(19): 7089-7093.

[8]    8.    Grant, C.E., Bailey, T.L., and Noble, W.S. 2011. FIMO: scanning for occurrences of a given motif. Bioinformatics, 27(7): 1017-1018.

[9]    9.    Grob, U. and Stüber, K. 1987. Discrimination of phytochrome dependent light inducibie from non-light inducibie plant genes. Prediction of a common light-responsive element (LRE) in phytochrome dependent


light inducibie plant genes. Nucleic Acids Research, 15(23): 9957-9973.

[10] 10.  Guo, H. and Moose, S.P. 2003. Conserved noncoding sequences among cultivated cereal genomes identify candidate regulatory sequence elements and patterns of promoter evolution. The Plant Cell, 15(5): 1143-1158.

[11] 11.  Hellwig, S., Drossard, J., Twyman, R.M., and Fischer, R. 2004. Plant cell cultures for the production of recombinant proteins. Nature Biotechnology, 22(11): 1415-1422.

[12] 12.  Hernandez-Garcia, C.M. and Finer, J.J. 2014. Identification and validation of promoters and cis-acting regulatory elements. Plant Science, 217: 109-119.

[13] 13.  Huang, J., Wu, L., Yalda, D., Adkins, Y., Kelleher, S.L., Crane, M., Lonnerdal, B., Rodriguez, R.L., and Huang, N. 2002. Expression of functional recombinant human lysozyme in transgenic rice cell culture. Transgenic Research, 11(3): 229-239.

[14] 14.  Huang, L.-F., Liu, Y.-K., Lu, C.-A., Hsieh, S.-L., and Yu, S.-M. 2005. Production of human serum albumin by sugar starvation induced promoter and rice cell culture. Transgenic Research, 14(5): 569-581.

[15] 15.  Huang, N., Chandler, J., Thomas, B.R., Koizumi, N., and Rodriguez, R.L. 1993. Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.). Plant Molecular Biology, 23(4): 737-747.

[16] 16.  Hwang, Y.-S., Karrer, E., Thomas, B., Chen, L., and Rodriguez, R.L. 1998. Three cis-elements required for rice α-amylase Amy3D expression during sugar starvation. Plant Molecular Biology, 36(3): 331-341.

[17] 17.  Ibraheem, O., Botha, C.E., and Bradley, G. 2010. In silico analysis of cis-acting regulatory elements in 5′ regulatory regions of sucrose transporter gene families in rice (Oryza sativa Japonica) and Arabidopsis thaliana. Computational Biology and Chemistry, 34(5): 268-283.

[18] 18.  Kim, N.-S., Yu, H.-Y., Chung, N.-D., Shin, Y.-J., Kwon, T.-H., and Yang, M.-S. 2011. Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures. Protein Expression and Purification, 76(1): 121-126.

[19] 19.  Kim, T.-G., Baek, M.-Y., Lee, E.-K., Kwon, T.-H., and Yang, M.-S. 2008. Expression of human growth hormone in transgenic rice cell suspension culture. Plant Cell Reports, 27(5): 885-891.

[20] 20.  Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P., and Rombauts, S. 2002. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research, 30(1): 325-327.

[21] 21.  Li, J., Yuan, J., and Li, M. 2014. Characterization of Putative cis-Regulatory Elements in Genes Preferentially Expressed in Arabidopsis Male Meiocytes. BioMed research International, 2014.

[22] 22.  Li, S.F. and Parish, R.W. 1995. Isolation of two novel myb‐like genes from Arabidopsis and studies on the DNA‐binding properties of their products. The Plant Journal, 8(6): 963-972.

[23] 23.  Lu, C.-A., Lim, E.-K., and Yu, S.-M. 1998. Sugar response sequence in the promoter of a rice α-amylase gene serves as a transcriptional enhancer. Journal of Biological Chemistry, 273(17): 10120-10131.

[24] 24.  McDonald, K.A., Hong, L.M., Trombly, D.M., Xie, Q., and Jackman, A.P. 2005. Production of human α‐1‐antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnology Progress, 21(3): 728-734.

[25] 25.  Molina, C. and Grotewold, E. 2005. Genome wide analysis of Arabidopsis core promoters. BMC Genomics, 6(1): 1.

[26] 26.  Park, C.-I., Lee, S.-J., Kang, S.-H., Jung, H.-S., Kim, D.-I., and Lim, S.-M. 2010. Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids. Process Biochemistry, 45(1): 67-74.

[27] 27.  Priest, H.D., Filichkin, S.A., and Mockler, T.C. 2009. Cis-regulatory elements in plant cell signaling. Current Opinion in Plant Biology, 12(5): 643-649.

[28] 28.  Reyes, J.C., Muro-Pastor, M.I., and Florencio, F.J. 2004. The GATA family of transcription factors in Arabidopsis and rice. Plant Physiology, 134(4): 1718-1732.

[29] 29.  Rouster, J., Leah, R., Mundy, J., and Cameron‐Mills, V. 1997. Identification of a methyl jasmonate‐responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. The Plant Journal, 11(3): 513-523.

[30] 30.  Scharf, K.-D., Rose, S., Zott, W., Schöffl, F., Nover, L., and Schöff, F. 1990. Three tomato genes code for heat stress transcription factors with a region of remarkable


 homology to the DNA-binding domain of the yeast HSF. The EMBO Journal, 9(13): 4495.

[31] 31.  Sharma, N., Russell, S.D., Bhalla, P.L., and Singh, M.B. 2011. Putative cis-regulatory elements in genes highly expressed in rice sperm cells. BMC Research Notes, 4(1): 1.

[32] 32.  Sheu, J.J., Jan, S.P., Lee, H.T., and Yu, S.M. 1994. Control of transcription and mRNA turnover as mechanisms of metabolic repression of α‐amylase gene expression. The Plant Journal, 5(5): 655-664.

[33] 33.  Shin, Y.-J., Lee, N.-J., Kim, J., An, X.-H., Yang, M.-S., and Kwon, T.-H. 2010. High-level production of bioactive heterodimeric protein human interleukin-12 in rice. Enzyme and Microbial Technology, 46(5): 347-351.

[34] 34.  Shin, Y.J., Hong, S.Y., Kwon, T.H., Jang, Y.S., and Yang, M.S. 2003. High level of expression of recombinant human granulocyte‐macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnology and Bioengineering, 82(7): 778-783.

[35] 35.  Shinshi, H., Usami, S., and Ohme-Takagi, M. 1995. Identification of an ethylene-responsive region in the promoter of a tobacco class I chitinase gene. Plant Molecular Biology, 27(5): 923-932.

[36] 36.  Simmons, C.R., Huang, N., Cao, Y., and Rodriguez, R.L. 1991. Synthesis and secretion of α‐amylase by rice callus: Evidence for differential gene expression. Biotechnology and Bioengineering, 38(5): 545-551.

[37] 37.  Smale, S.T. and Baltimore, D. 1989. The “initiator” as a transcription control element. Cell, 57(1): 103-113.

[38] 38.  Thomas, B.R., Chandler, J., Simmons, C.R., Huang, N., Karrer, E., Rodriguez, R.L., Ryu, D., and Furusaki, S. 1994. Gene regulation and protein secretion from plant cell cultures: the rice α-amylase system. Advances in Plant Biotechnology.: 37-55.

[39] 39.  Thomas, B.R. and Rodriguez, R.L. 1994. Metabolite signals regulate gene expression and source/sink relations in cereal seedlings. Plant Physiology, 106(4): 1235.

[40] 40.  Toyofuku, K., Loreti, E., Vernieri, P., Alpi, A., Perata, P., and Yamaguchi, J. 2000. Glucose modulates the abscisic acid-inducible Rab16A gene in cereal embryos. Plant Molecular Biology, 42(3): 451-460.

[41] 41.  Toyofuku, K., Umemura, T.-a., and Yamaguchi, J. 1998. Promoter elements required for sugar‐repression of the Ramy3D gene for α‐amylase in rice. FEBS Letters, 428(3): 275-280.

[42] 42.  Trexler, M.M., McDonald, K.A., and Jackman, A.P. 2002. Bioreactor Production of Human α1‐Antitrypsin Using Metabolically Regulated Plant Cell Cultures. Biotechnology progress, 18(3): 501-508.

[43] 43.  Vicente-Carbajosa, J., Moose, S.P., Parsons, R.L., and Schmidt, R.J. 1997. A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. Proceedings of the National Academy of Sciences, 94(14): 7685-7690.

[44] 44.  Washida, H., Wu, C.-Y., Suzuki, A., Yamanouchi, U., Akihama, T., Harada, K., and Takaiwa, F. 1999. Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1. Plant Molecular Biology, 40(1): 1-12.

[45] 45.  Xu, J., Ge, X., and Dolan, M.C. 2011. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. Biotechnology Advances, 29(3): 278-299.

[46] 46.  Yamaguchi-Shinozaki, K. and Shinozaki, K. 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol., 57: 781-803.

[47] 47.  Yamamoto, Y.Y., Ichida, H., Matsui, M., Obokata, J., Sakurai, T., Satou, M., Seki, M., Shinozaki, K., and Abe, T. 2007. Identification of plant promoter constituents by analysis of local distribution of short sequences. BMC Genomics, 8(1): 1.

[48] 48.  Yang, C., Bolotin, E., Jiang, T., Sladek, F.M., and Martinez, E. 2007. Prevalence of the initiator over the TATA box in human and yeast genes and identification of DNA motifs enriched in human TATA-less core promoters. Gene, 389(1): 52-65.

[49] 49.  Yano, A., Maeda, F., and Takekoshi, M. 2004. Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen. Journal of Medical Virology, 73(2): 208-215.