The 2012 ASHS Annual Conference

Since its discovery, RNA silencing and specifically strategies for dsRNA production has become an attractive molecular tool to confer specific plant resistance against Potyvirus, one of the largest and agriculturally most important genus of plant viruses. With this in mind, we report here the construction of an RNA silencing-trigger (dsRNA producer) vector commonly known as intron-hairpin RNAi (ihRNAi) using 217 bp of coat protein (cp) cistron amplified from PRSV-P infected papaya plants and cloned in pCR^® 2.1-TOPO^®. Our strategy consisted of construction (cloning) of sense and antisense versions of this cp fragment into restriction sites Xho I/Kpn I (for sense arm) and Xba I/Cla I (for antisense arm) included in pHANNIBAL plasmid. Correct ligation was confirmed by means of restriction analysis (with both aforementioned pair of enzymes), real-time PCR (using a LightCycler^®1.5 thermal cycler and a Taqman probe labeled with 6-FAM and TAMRA as reporter and quencher dyes, respectively) and DNA sequencing (with BigDye^® Terminator method and an ABI PRISM^® 310 Genetic Analyzer, Sequencing Service at Institute of Neurobiology-UNAM, Juriquilla, Qro., México). Results indicated that this construction is available for subcloning into binary vectors for plant transformation experiments. Fund FRABA-Colima University, agreement 771/11 and SEP