期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 9, 页码 -出版社
MDPI
DOI: 10.3390/ijms23094938
关键词
CMV; capsid protein; Nicotiana benthamiana; gene silencing; shRNA
资金
- Ministerio de Ciencia, Tecnologia e Innovacion de Colombia-MINCIENCIAS [265-2017]
- Universidad de Caldas [0178917]
This study successfully synthesized virus-derived artificial small hairpin RNAs (shRNAs) using a dsDNA template and triggered the RNAi mechanism in Nicotiana benthamiana plants against CMV. The gene silencing was confirmed using PCR, immunological-based assays, and qPCR.
Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV.
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