Abstract
Bamboo mosaic virus has a single-strand RNA genome which consists of five open reading frames (ORFs). ORF1 encodes a ~155 kDa replicase that contains a capping enzyme domain, a helicase-like domain, and a RNA-dependent RNA polymerase domain (RdRp). In previous study, a putative Nicotiana benthamiana methyltransferase (NbMTs1) was found to interact with RdRp in a yeast two-hybrid system screening against a leaf cDNA library of N. benthamiana. The NbMTs1 can inhibit the replication of virus, and it may be the part of defense mechanism in plants. However, the effects of the NbMTs1 overexpression or NbMTs1 gene silence on other plant virus replication are not clear yet. In this study, we generated N. benthamiana lines overexpressing NbMTs1 and gene silencing transgenic plants, respectively. We constructed pEpyon-MTs by inserting NbMTs1 fragment into pEpyon plasmid to overexpress NbMTs1, and constructed pEpyon-MTs-rev by inserting antisense NbMTs1 fragment into pEpyon plasmid to silence the NbMTs1 expression in the transgenic plants. The recombinant plasmids were transformed into N. benthamiana via Agrobacterium LBA4404-mediated transformation. The transgenic plant lines were first selected by Kanamycin resistance phenotype, and further confirmed by genomic PCR analysis. The quantity of NbMTs1 mRNA in transgenic plants was detected by RT-PCR. We had obtained 8 pEpyon-MTs and 6 pEpyon-MTs-rev transgenic plant lines. We will inoculate transgenic N. benthamiana with several different plant virus and analyze the effect of NbMTs1 expression on plant virus replication in transgenic plants.
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