Plant viral vectors have shown significant promse for studies of gene function, through either up-regu-lation or down-regulation of gene expression. However, there have remained issues of efficiency of generating constructs, and of subcellular localization of expression; both issues are addressed here. Alternanthera mosaic virus (AltMV; genus Potexvirus) is distinguished from the type member of the genus, Potato virus Xby features of viral movement and variation within triple gene block protein 1 (TGB1). AltMV TGB1 variants TGB1L88 and TGB1P88 confer strong and weak silencing suppression, respectively, depending on the presence of L or P at residue 88. Because AltMV replication is associated with chloroplasts, we compared the relative efficiency of RNA interference (RNAi) vectors derived from AltMV and Tobacco rattle virus (TRV) to silence a chloroplast-encoded gene. An AltMV RNAi vector expressing a fragment of the chloroplast β ATPase gene reduced β-ATPase expression 1.5 times more than the TRV RNAi vector expressing the same fragment. In addition, we used AltMV (TGB1P88) to create a whitefly (Bemisia tabaci) RNAi vector. For this purpose, we first introduced the Gateway cloning cassette into the AltMV multiple cloning site, into which polymerase chain reaction (PCR) products from a whitefly cDNA library could be easily cloned. Second, a mixture of five different PCR fragments of about 250 bp were used to test cloning efficiency of the newly-created AltMV-P-att vector. Third, random 250 bp fragments of Gateway cDNA libraries from B. tabaci and Nicotiana benthamiana were efficiently cloned into the Gateway-modified AltMV-att vector, demonstrating for the first time a high throughput RNAi system based on AltMV. This strategy could be applied to other RNAi systems.
|ジャーナル||Journal of the Faculty of Agriculture, Kyushu University|
|出版ステータス||出版済み - 2 1 2015|
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