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Abstract
BackgroundA high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5′UTR (synJ), which enhances gene expression in tobacco and cotton.ResultsThe influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene gus and gfp, with and without synJ as its 5′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of gus gene, encoding the synJ as 5′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-hpt and pBGEN02-ALSdm each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants.ConclusionssynJ, a synthetic 5′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in dicotyledonous plants. synJ can be incorporated as the 5′UTR of transgenes, especially in cases where high levels of expression is required. A set of vectors has also been designed to facilitate this process.
Highlights
A high level of transgene expression is required, in several applications of transgenic technology
We demonstrate the potential of a small (28 nt long) synthetic 50 untranslated region (50UTR), hitherto called as synJ, to enhance transgene expression levels, which is at par or even higher than what could be achieved with the Ω and Alfalfa mosaic virus (AMV) leader sequences in transgenic tobacco lines and in callus cultures of cotton. synJ was designed as a multiple cloning site containing EcoRI, SnaI and NcoI restriction enzyme recognition sites for developing expression cassettes with the 35S promoter [7]
The reporter gene encodes for a 50UTR of the mRNA which is the main variable in different expression cassettes
Summary
A high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 50UTRs have been shown to enhance transgene expression. We present a 28 nt long synthetic 50UTR (synJ), which enhances gene expression in tobacco and cotton. Achieving high levels of transgene expression is one of the challenges for applications of transgenic technology. Gene expression can be regulated by modulating transcriptional parameters and by using post-transcriptional parameters like using optimal codons or by including 50 and 30 leader sequences. A lot of research has been focused on the transcriptional control mechanisms like testing various promoters [4,5,6], developing synthetic promoters [7,8] and using enhancer domains [9,10]. The importance of 50UTRs on gene expression has not been explored to its fullest potential
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