tailieunhanh - Genetic transformation of Citrus paradisi with antisense and untranslatable RNA-dependent RNA polymerase genes of Citrus tristeza closterovirus
Protein and RNA-mediated forms of pathogen-derived resistance (PDR) have been developed against many viruses in different plants. However, no resistance has been reported against Citrus tristeza virus (CTV), a closterovirus, in Citrus species transformed with coat protein genes or other sequences of CTV. | Turk J Agric For 30 (2006) 173-182 © TÜB‹TAK Genetic Transformation of Citrus paradisi with Antisense and Untranslatable RNA-dependent RNA Polymerase Genes of Citrus tristeza closterovirus Bayram ÇEV‹K1,*, Richard F. LEE2, Charles L. NIBLETT3 1 Süleyman Demirel University, Faculty of Agriculture, Department of Plant Protection, 32260 Isparta - TURKEY 2 USD-ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, 92521, USA 3 University of Florida, Department of Plant Pathology, Gainesville, FL 326113, USA Received: Abstract: Protein and RNA-mediated forms of pathogen-derived resistance (PDR) have been developed against many viruses in different plants. However, no resistance has been reported against Citrus tristeza virus (CTV), a closterovirus, in Citrus species transformed with coat protein genes or other sequences of CTV. The successful use of replication-associated genes in RNA-mediated resistance in other crops prompted the use of the RNA-dependent RNA polymerase (RdRp) gene of CTV for the development of RNA-mediated PDR in Citrus. The RdRP gene was amplified from CTV isolate DPI3800 from Florida and used to generate antisense (RdRp-AS) and untranslatable (RdRp-UT) constructs with point mutation consecutive stop codons in the 5’ end of the RdRp gene for use in plant transformation. A total of 3120 etiolated epicotyl segments of Duncan grapefruit (Citrus paradisi Macf. cv. Duncan) were transformed with these constructs using Agrobacterium tumefaciens-mediated transformation. From these segments 1040 kanamycin-resistant shoots were regenerated, and a total of 131 putative transgenic shoots were identified by fluorescent microscopy and histochemical β-glucuronidase (GUS) assays. One hundred GUS positive plants were rooted and 66 plants survived and were established on soil. A total of 41 plants were tested by polymerase chain reaction (PCR) for the presence of the GUS gene and for the transgenes. Eighteen GUS-positive and .
đang nạp các trang xem trước
