Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean.
Plant virus-based vectors provide valuable tools for expression of foreign proteins in plants and for gene function studies. None of the presently available virus vectors is suitable for use in soybean. In the present study, we produced Bean pod mottle virus (BPMV)-based vectors that are appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The genes of interest were inserted into the RNA2-encoded polyprotein open reading frame between the movement protein (MP) and the large coat protein (L-CP) coding regions. Additional proteinase cleavage sites were created to flank the foreign protein by duplicating the MP/L-CP cleavage site. To minimize the chances of homologous recombination and thus insert instability, we took advantage of the genetic code degeneracy and altered the nucleotide sequence of the duplicated regions without affecting amino acid sequences. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. These included the reporter proteins GFP and DsRed, phosphinothricin acetyltransferase (encoded by the herbicide resistance bar gene), and the RNA silencing suppressors encoded by Tomato bushy stunt virus, Turnip crinkle virus, Tobacco etch virus, and Soybean mosaic virus. The possible use of BPMV as a VIGS vector to study gene function in soybean was also demonstrated with the phytoene desaturase gene. Our results suggest that the BPMV-based vectors are suitable for expression of foreign proteins in soybean and for functional genomics applications.
Zhang
Chunquan
C
Department of Plant Pathology, 201F Plant Science Building, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0312, USA.
Ghabrial
Said A
SA
D000595:Amino Acid Sequence; D001483:Base Sequence; D004351:Drug Resistance; D015870:Gene Expression; D020868:Gene Silencing; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D006540:Herbicides / Q000494:pharmacology; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010942:Plant Viruses / Q000235:genetics*; D030821:Plants, Genetically Modified; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012680:Sensitivity and Specificity; D013025:Glycine max / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D006540:Herbicides; D012367:RNA, Viral; D011994:Recombinant Proteins; D010088:Oxidoreductases; C022398:phytoene dehydrogenase