Link to home

Development of high expression system of a foreign gene replacing a coat protein region in the cucumber mosaic virus vector through agroinfection

Noriho Fukuzawa: National Institute of Advanced Industrial Science and Technology

<div>To enable further improvement in the expression levels of the foreign gene using viral vector, we have developed the cucumber mosaic virus (CMV)-agroinfection methods using a plant-based platform. CMV is known to have a very broad host range and contains three segmented, genomic RNAs (RNAs 1-3). In this study, we engineered CMV RNA3 to replace the coat protein (CP) gene with a green fluorescence protein (GFP) as a marker protein in a Ti plasmid (pBI-CR3ΔCP-GFP). RNA1 and RNA2 were also designated pBI-CR1 and pBI-CR2. As the platform plant to produce the foreign protein, we prepared transgenic <em>Nicotiana benthamiana</em> expressing either RNA1 or RNA2 (CR1 Tg or CR2 Tg plant). When wild-type plants were infiltrated with <em>Agrobacterium</em> suspension harboring the pBI-CR1, pBI-CR2 and pBI-CR3ΔCP-GFP construct, we observed sporadic GFP fluorescence in the infiltrated leaves. When the pBI-CR1 and pBI-CR3ΔCP-GFP construct were infiltrated in CR2 Tg plants, we found that the GFP fluorescence was dispersed in the infected leaves. However, when CR1 Tg plants were infiltrated with bacteria containing the pBI-CR2 and pBI-CR3ΔCP-GFP construct, the yield of GFP in CR1 Tg plants was calculated to be 750 mg/kg fresh weight within 3 dpi. This study demonstrates that a transgenic plant expressing CMV RNA1 can be used as an excellent plant-based platform for high protein production.</div>