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Construction of a novel synthetic root-specific promoter and its characterization in transgenic tobacco plants
- Mohan, Chakravarthi, Jayanarayanan, Ashwin Narayan, Narayanan, Subramonian
- 3 Biotech 2017 v.7 no.4 pp. 234
- gene expression, gene silencing, promoter regions, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, roots, tobacco, transgenes, transgenic plants
- 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.