Construction of PVX virus-expression vector to express enterotoxin fusion gene LTB-ST and transformation into tobacco

Abstract

Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST gene was introduced into the PVX-based vector in the site of the ClaI and NotI after digested incompletely by ClaI and NotI. Then the positive clone was picked and was sequenced and the clone with exactly same sequence was named PVX-LTB-ST vector. The recombinant plasmid was transformed into Agrobacterium tumefacience strain MOG101 and LBA4404, respectively. The fusion gene LTB-ST cannot be amplified stably in the MOG101, while it could be amplified stably in LBA4404. Of the two methods which were used to infect tobacco, the plasmid plaiting method could not make the leaves show mosaic symptoms, and plants which were infected using the agroinoculation method showed mosaic symptoms. One step reverse transcriptase (RT)-PCR analysis indicated the LTB-ST gene expressed in the inoculated tobacco plants with mosaic symptoms.

Key words: Enterotoxin fusion gene LTB-ST, PVX virus-expression vector, transient expression, tobacco.