Abstract
In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L-1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
More From: The Plant journal : for cell and molecular biology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.