Creating a novel engineered tobacco chassis with downregulated expression of β-1,2-xylosyltransferase

In genetic engineering research, the use of plants as bioreactors for the production of therapeutic recombinant proteins has become a prominent research subject in the past decade. At present, the primary issue with animal derived therapeutic proteins produced by plants is the potential absence of mammalian glycoproteins. Specifically, β-1,2-xylosyltransferase (CXT1P) and α-1,3-fucosyltransferase (FUT4) can trigger immune or allergic reactions. CXT1P is the first glycosyltransferase identified in Staphylococcus aureus and has a clear role in capsule membrane biosynthesis. It is also the only strain known to induce structural modifications in the capsule polysaccharide GalXM. In this study, we prepared an RNA interference (RNAi) construct by downregulating CXT1P expression. A specific fragment of 387 bp was selected from the first exon of CXT1P by BLAST analysis, and transformed into tobacco plants by Agrobacterium-mediated transformation. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that CXT1P gene expression was reduced in transgenic tobacco lines. Moreover, western-blotting and ELISA analyses showed that protein expression in transgenic tobacco lines was also reduced. The generated RNAi lines were stable and viable, and did not show any obvious phenotypes, thus providing a powerful tool to produce CXT1P therapeutic proteins in tobacco plants. Taken together, this study provides important information for the future development of plant-based expression systems for the production of anticancer therapeutic proteins.