Construction of a novel synthetic root-specific promoter and its characterization in transgenic tobacco plants
- 94 Downloads
Synthetic promoter technology offers a framework for designing expression cassettes that could provide precise control of transgene expression. Such artificially designed promoters enable defined transgene regulation, reduce unwanted background expression, and can overcome homology-dependent gene silencing in transgenic plants. In the present study, a synthetic root-specific module was designed using characterized cis-acting elements, fused with minimal promoter (86 bp) from PortUbi882 promoter, and cloned in pCAMBIA1305.1 by replacing CaMV 35S promoter so as to drive GUS expression. Two constructs were made; one had the synthetic module at the 5′ end of the minimal promoter (SynR1), whereas in the other construct, the module was present in both 5′ and 3′ ends (SynR2). Furthermore, the synthetic promoter constructs were transformed in tobacco wherein SynR1 promoter drove constitutive expression, whereas SynR2 conferred root-specific expression though slight leaky expression was present in stem. GUS assay in the roots of transgenic tobacco plants (T1) indicated that SynR2 promoter expressed significantly higher GUS activity than the CaMV 35S promoter. The real-time quantitative PCR (RT-qPCR) analysis of GUS gene further confirmed that SynR2 promoter conferred 2.1-fold higher root-specific expression when compared to CaMV 35S promoter.
KeywordsGUS Root specific Synthetic promoter Tobacco Transgene expression
Ethylene response factor
Domain of function
Basic helix loop helix
5-Bromo-4-chloro-3-indolyl-beta-d-glucuronic acid, cyclohexylammonium salt
The authors are grateful to the Indian Council of Agricultural Research and the Sugarcane Breeding Institute, Coimbatore, for the funding and infrastructure. The first author is also grateful to the São Paulo Research Foundation (FAPESP, Proc. 2015/10855-9) for the postdoctoral research grant.
NS and MC conceived and designed the research. MC and JAN performed the experiments. MC analyzed the results and drafted the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
- Bhullar S, Chakravarthy S, Advani S, Datta S, Pental D, Burma PK (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol 132:988–998CrossRefGoogle Scholar
- Geisler M, Kleczkowski LA, Karpinski S (2006) A universal algorithm for genome-wide in silico identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis. Plant J 45(3):384–398CrossRefGoogle Scholar
- Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907Google Scholar
- Jensen PR, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64:82–87Google Scholar
- Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37(1):49–59CrossRefGoogle Scholar
- Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15(5):690–700Google Scholar
- Sawant S, Kiran K, Mehrotra R, Chaturvedi CP, Ansari SA, Singh P, Lodhi N, Tuli R (2005) A variety of synergistic and antagonistic interactions mediated by cis-acting DNA motifs regulate gene expression in plant cells and modulate stability of the transcription complex formed on a basal promoter. J Exp Bot 56:2345–2353CrossRefGoogle Scholar
- Shokouhifar F, Zamani MR, Motallebi M (2011) Expression pattern of the synthetic pathogen-inducible promoter (SynP-FF) in the transgenic canola in response to Sclerotinia sclerotiorum. Iranian J Biotechnol 9(1):1–10Google Scholar