title: Amplicon-Based RNA Interference Targeting V2 Gene of Cotton Leaf Curl Kokhran Virus-Burewala Strain Can Provide Resistance in Transgenic Cotton Plants.
abstract: The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T0, T1, and T2generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T1generation. Copy number and transgene location were observed using FISH and karyotyping in T2generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed low virus titer compared to wild-type control plants upon challenging them with viruliferous whiteflies in a contained environment. From the results, it was concluded that amplicon V2 RNAi construct was able to limit virus replication and can be used to control CLCuV in the field.
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Aneela
authors.initials: A
authors.lastname: Yasmeen
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Sarfraz
authors.initials: S
authors.lastname: Kiani
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Afshan
authors.initials: A
authors.lastname: Butt
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan. qayyumabdul77@yahoo.com.
authors.forename: Abdul Qayyum
authors.initials: AQ
authors.lastname: Rao
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Faheem
authors.initials: F
authors.lastname: Akram
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Aftab
authors.initials: A
authors.lastname: Ahmad
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Idrees Ahmad
authors.initials: IA
authors.lastname: Nasir
authors.affiliation: Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
authors.forename: Tayyab
authors.initials: T
authors.lastname: Husnain
authors.affiliation: National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
authors.forename: Shahid
authors.initials: S
authors.lastname: Mansoor
authors.affiliation: National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
authors.forename: Imran
authors.initials: I
authors.lastname: Amin
authors.affiliation: National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
authors.forename: Shaheen
authors.initials: S
authors.lastname: Aftab
authors.affiliation: National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Zubair
authors.affiliation: National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
authors.forename: Muhammad Nouman
authors.initials: MN
authors.lastname: Tahir
authors.affiliation: National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
authors.forename: Sohail
authors.initials: S
authors.lastname: Akhtar
authors.affiliation: Jamie Whitten Delta States Research Center, USDA, Stoneville, MS, 38776, USA.
authors.forename: Jodi
authors.initials: J
authors.lastname: Scheffler
authors.affiliation: Jamie Whitten Delta States Research Center, USDA, Stoneville, MS, 38776, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Scheffler
mesh_terms: D053839:Begomovirus / Q000235:genetics / Q000502:physiology*; D036022:Capsid Proteins / Q000235:genetics*; D032461:Chromosomes, Plant / Q000235:genetics; D060467:Disease Resistance; D018390:Gene Targeting; D003368:Gossypium / Q000235:genetics* / Q000254:growth & development / Q000821:virology; D010935:Plant Diseases; D030821:Plants, Genetically Modified / Q000254:growth & development*; D034622:RNA Interference; D019076:Transgenes; D019562:Viral Load; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins
keywords: Begomovirus; CLCuD; Gene transformation; Knockdown; V2 ORF; siRNA