The application ofNicotiana benthamianaas a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Abstract

AbstractCoding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patternsin vivo. These challenges require the development of alternative CDS cloning technologies.In this study, we found that the genomic intron-containing gene coding sequences (gDNA) fromArabidopsis thaliana, Oryza sativa, Brassica napus, andGlycine maxcan be correctly transcribed and spliced into mRNA inNicotiana benthamiana(N. benthamiana). In contrast, gDNAs fromTriticum aestivumandSorghum bicolordid not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from theN. benthamianaleaves making it conducive to the cloning of CDS of target genes. Our data demonstrate thatN. benthamianacan be used as an effective host for the cloning CDS of plant genes.