A 28 nt long synthetic 5'UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

Abstract

Background: 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, 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.

Results: The 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.

Conclusions: synJ, 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.

Figure 1

Schematic representation of binary vectors (within T-DNA borders) developed for the transformation of tobacco and cotton. The left and right borders of the T-DNA are designated as LB and RB, respectively. The selection marker gene (nptII) is driven by the nos promoter (Pnos) which also has a polyA signal of octopine synthase gene (ocspA). Different promoters (35S and nos) and reporter genes (gus and gfp) were used for creating various expression cassettes with a polyA signal of 35S (35SpA). A given combination of promoter and 5^′UTR constituted an upstream regulatory module (URM).

Figure 2

GUS activity observed in cotton callus transformed with (A) 35S(RT), (B) 35S(pBI) and (C) 35S(synJ). GUS activity was studied after 40–45 days of transformation. GUS activity recorded in callus transformed with 35S(synJ) was many fold higher than 35S(RT) and 35S(pBI) (see scales of Y-axis). Each circle represents the absolute GUS activity (pmolMU/min/μg protein) measured in one independent callus. GUS activity in this as well as in all the following figures is a mean of two independent reactions carried out for two time points. N in this figure and in later figures denotes the number of callus analyzed in each case.

Figure 3

GUS activity observed in cotton callus transformed with the constructs 35S(synJ), 35S(Ω) and 35S(AMV). Each bar represents the mean GUS activity of the two experimental replicates measured with one independent callus. N denotes the number of callus analyzed in each case. The line represents the GUS activity of 1500 pmolMU/min/μg protein.

Figure 4

Comparative analysis of nos(nos), nos(synJ) and nos(RT) constructs. GUS activity in transformed cotton callus containing the expression constructs nos(nos), nos(synJ) and nos(RT) as observed in two independent experiments. Each circle represents GUS activity measured in one independent callus.

Figure 5

Comparison of GUS activity driven by 35S(synJ) and 35S(nos). GUS activity as observed in cotton callus when transformed with the constructs 35 S(synJ) and 35 S(nos) in three independent transformation experiments. Each circle represents GUS activity measured in one independent callus.

Figure 6

Comparative analysis of 35S(RT), 35S(pBI) and 35S(synJ) constructs in the leaves of tobacco transgenic plants. GUS activity has been represented as measured in the leaves of tobacco transgenic plants transformed with the constructs (A) 35S(RT), (B) 35S(pBI) and (C) 35S(synJ). The level of GUS activity with 35S(synJ) was much higher than the other two constructs. The scale of the Y-axis (i.e. GUS activity pmolMU/min/μg protein) is different for each graph.

Figure 7

Comparison of 35S(RT), 35S(pBI) and 35S(synJ) in the leaf, stem and root tissues of tobacco transgenic plants as a Box and Whisker plot. The horizontal lines in the Box and Whisker plot represent 10, 25, 50, 75, and 90 percentiles. Extreme values are depicted as circles at the top and bottom of the plot.

Figure 8

Comparison of GUS activity driven by 35S(synJ), 35S(Ω) and 35S(AMV). GUS activity measured in the (A) leaf (B) stem and (C) root tissues of tobacco transgenic lines transformed with the three constructs 35S(synJ), 35S(Ω) and 35S(AMV) presented as a Box and Whisker plot.

Figure 9

Comparison of transcript levels driven by 35S(RT) and 35S(synJ). Normalized levels of gus transcripts as observed in individual transgenic lines of tobacco developed with constructs 35S(RT) and 35S(synJ) carrying different 5^′UTRs: RT and synJ. There was no major difference in the range of transcript levels observed between transgenics developed with 35S(RT) and 35S(synJ).