Cis-acting motifs in artificially synthesized expression cassette leads to enhanced transgene expression in tomato (Solanum lycopersicum L.)

Efficacy of artificial synthetic expression modules was compared with native CaMV35S and DECaMV35S promoter in transgenic tomato developed by Agrobacterium-mediated transformation. The promoters under trial were CaMV35S-mec (PcamI), CaMV35S (PcamII), DECaMV35S (PcamIII), synthetic minimal expression cassette (Pmec), complete expression cassette (Pcec), double enhancer expression cassette (Pdec) and triple enhancer expression cassette (Ptec) for driving the uidA gene for β-glucuronidase (GUS) activity. The promoter efficiency based on average of GUS expression in T0 and T1 transgenic tomato was in the order Pcec > Pdec > PcamIII > PcamII > PcamI > Ptec > Pmec. The two promoters Pcec and PcamIII were deployed for development of insect-resistant transgenic tomato with optimal expression of modified cry1Ac insecticidal toxin gene from Bacillus thuringiensis (Bt). The transgenic status and copy number of the cry1Ac in T0 transgenic tomato was confirmed through PCR, Southern hybridization, RT-PCR and Western immunoassay, while toxin expression was monitored by DAS-ELISA. The expression level of Cry1Ac toxin driven by Pcec in T0 population ranged from 0.08 to 0.8% of total soluble protein (TSP) that was significantly higher to PcamIII which ranged from 0.02 to 0.13% of TSP. The outcome of insect mortality bioassay with Helicoverpa armigera correlated well with the results of DAS-ELISA. The higher expression of cry1Ac gene driven by Pcec promoter in transgenic tomato did not show any yield penalty and reflected complete protection, while low recovery of promising transgenics expressing Cry1Ac toxin driven by PcamIII was a major limitation for complete protection against the fruit borer insect.