, Volume 102, Issue 4, pp 635-644

Designing of an artificial expression cassette for the high-level expression of transgenes in plants

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Abstract 

A dataset of highly expressed plant genes was developed from the nucleic acids sequence database. The characteristic features of the nucleotide sequences in TATA-box, transcription initiation, untranslated leader and translation initiation regions in the highly expressible genes in plants and the conserved sequences present 500 bp upstream of transcription initiation site were identified. These features were employed to theoretically design a ’minimal expression cassette’ and a promoter-upstream ’activation module.’ The ’minimal expression cassette’ was sufficient to express the gusA reporter gene in transient transformation of tobacco leaf. The context on the 3′ side of the initiator codon, conserved in a majority of the highly expressible genes, gave approximately a ninefold increase in the expression of β-glucuro- nidase. The artificially designed, upstream ’activation module’ enhanced gusA expression further by about 30-fold in transiently transformed tobacco leaves. A 450-bp-long complete expression cassette, containing both the ’minimal expression cassette’and the ’activation module’ expressed gusA at a high level in cotton leaves, potato tubers and cabbage stem also. In stably transformed tobacco plants, the ’complete expression cassette’ expressed gusA at levels higher than the native CaMV 35S promoter. Histological studies established that the ’complete expression cassette’ was expressed at a high level in different cell types in the roots, leaves, vascular tissues and flower parts of the transgenic tobacco plants. The results substantiate the functional validity of the features identified by us and demonstrate the potential of computational biology in designing artificial expression cassettes for applications in biotechnology.