Sajed Ali1,2, Idrees Ahmad Nasir2, Muhammad Rafiq3, Shahid Javed Butt1, Farooq Ihsan2, Abdul Qayyum Rao2, Tayyab Husnain2. 1. Department of Biotechnology, School of Sciences, University of Management and Technology, Sialkot Campus, Sialkot, Pakistan. 2. Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. 3. Institute of Clinical Psychology, University of Management and Technology, Lahore, Pakistan.
Abstract
Background: Potyvirus-based virus-induced gene silencing (VIGS) is used for knocking down the expression of a target gene in numerous plant species. Sugarcane mosaic virus (SCMV) is a monopartite, positive single strand RNA virus. Objectives: pBINTRA6 vector was modifi ed by inserting a gene segment of SCMV in place of Tobacco rattle virus (TRV) genome part 1 (TRV1 or RNA1) and the two nonstructural proteins of TRV2(RNA2). Materials and Methods: SCMV construct was inoculated into 3-4 weeks Nicotiana benthamiana plant leaves either by using a needleless syringe or applying pricking with a toothpick. Results: The construct (SCMV-RNA2) successfully induced post-transcriptional gene silencing (PTGS) of the target genes GFP and ChlI through agroinoculation proving that SCMV is a substitute of the RNA1, which plays a pivotal role in the systemic gene silencing. 2-3-weeks of post inoculation, target genes' silencing was observed in the newly developed noninoculated leaves. Conclusions: The newly developed construct expresses the knocked down of the endogenous as well as exogenous genes and only four weeks are required for the transient expression of the gene silencing based on SCMV-VIGS system.
Background: Potyvirus-based virus-induced gene silencing (VIGS) is used for knocking down the expression of a target gene in numerous plant species. Sugarcane mosaic virus (SCMV) is a monopartite, positive single strand RNA virus. Objectives: pBINTRA6 vector was modifi ed by inserting a gene segment of SCMV in place of Tobacco rattle virus (TRV) genome part 1 (TRV1 or RNA1) and the two nonstructural proteins of TRV2(RNA2). Materials and Methods:SCMV construct was inoculated into 3-4 weeks Nicotiana benthamiana plant leaves either by using a needleless syringe or applying pricking with a toothpick. Results: The construct (SCMV-RNA2) successfully induced post-transcriptional gene silencing (PTGS) of the target genes GFP and ChlI through agroinoculation proving that SCMV is a substitute of the RNA1, which plays a pivotal role in the systemic gene silencing. 2-3-weeks of post inoculation, target genes' silencing was observed in the newly developed noninoculated leaves. Conclusions: The newly developed construct expresses the knocked down of the endogenous as well as exogenous genes and only four weeks are required for the transient expression of the gene silencing based on SCMV-VIGS system.
Authors: Samuel M D Seaver; Svetlana Gerdes; Océane Frelin; Claudia Lerma-Ortiz; Louis M T Bradbury; Rémi Zallot; Ghulam Hasnain; Thomas D Niehaus; Basma El Yacoubi; Shiran Pasternak; Robert Olson; Gordon Pusch; Ross Overbeek; Rick Stevens; Valérie de Crécy-Lagard; Doreen Ware; Andrew D Hanson; Christopher S Henry Journal: Proc Natl Acad Sci U S A Date: 2014-06-09 Impact factor: 11.205
Authors: José M Alonso; Anna N Stepanova; Thomas J Leisse; Christopher J Kim; Huaming Chen; Paul Shinn; Denise K Stevenson; Justin Zimmerman; Pascual Barajas; Rosa Cheuk; Carmelita Gadrinab; Collen Heller; Albert Jeske; Eric Koesema; Cristina C Meyers; Holly Parker; Lance Prednis; Yasser Ansari; Nathan Choy; Hashim Deen; Michael Geralt; Nisha Hazari; Emily Hom; Meagan Karnes; Celene Mulholland; Ral Ndubaku; Ian Schmidt; Plinio Guzman; Laura Aguilar-Henonin; Markus Schmid; Detlef Weigel; David E Carter; Trudy Marchand; Eddy Risseeuw; Debra Brogden; Albana Zeko; William L Crosby; Charles C Berry; Joseph R Ecker Journal: Science Date: 2003-08-01 Impact factor: 47.728