Fine?￠????tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system

摘要

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA?￠????2, termed CPMV?￠???? HT, in which the sequence to be expressed is positioned between a modified 5?￠???2 UTR and the 3?￠???2 UTR has been successfully used for the plant?￠????based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5?￠???2 UTR can dramatically influence expression levels, the role of the 3?￠???2 UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3?￠???2UTR of CPMV RNA?￠????2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress?￠????HT?￠????GFP. The results showed that the presence of a 3?￠???2 UTR in the CPMV?￠???? HT system is important for achieving maximal expression levels. Removal of the entire 3?￠???2 UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y?￠????shaped secondary structure formed by nucleotides 125?￠????165 of the 3?￠???2 UTR plays a key role in its function; mutations that disrupt this Y?￠????shaped structure have an effect equivalent to the deletion of the entire 3?￠???2 UTR. Our results suggest that the Y?￠????shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5?￠???2 and 3?￠???2 UTRs in CPMV?￠???? HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.