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Transformation of Brassica napus cv. Westar with the beet western yellows virus coat protein gene

University of British Columbia

Double stranded (ds) complementary DNA (cDNA) copies of the beet western yellows virus (BWYV) coat protein (CP) gene were synthesized from genomic BVVYV RNA using reverse transcriptase (RT) followed by the polymerase chain reaction (PCR). The ds cDNA copy of the BWYV CP was then cloned into plasmid pRT103, a plant expression vector which contains the cauliflower mosaic virus (CaMV) 35S promoter and polyadenylation signal. The CP cDNA was inserted in the plus-sense orientation (clone BW102D) and in the anti-sense orientation (clone BW137D) relative to the CaMV 35S promoter. The CP coding regions of both pRT103 constructs were sequenced and found to correspond closely to those of previously published BWYVCP sequences. In vitro translation analysis in wheat germ extracts of synthetic transcripts of the cloned BWYV CP DNA produced only one major protein of ca. 22.5kDa, the expected size for the BWYV coat protein. The CaMV 35S promoter-BWYVCP-polyadenylation cassette from clones BW102D and BW137D were then cloned separately into the Hind III restriction enzyme site between the left and right borders of the T-DNA region and next to the npt II cassette of the two binary plasmids, pCGN1548 and pCGN1557 (Calgene). After mobilization of the four resulting binary plasmid constructs (pCGN154802, pCGN154837, pCGN155702 and pCGN155737) into Agrobacterium tumefaciens EHA101, the A. tumefaciens were used in the plant transformation procedure described by Moloney et al. (1989) utilizing cotyledonary explants of Brassica napus cv. Westar. Thirty eight regenerated plants were recovered under the selection of the antibiotic, kanamycin. Southern blot analysis of Hind digested plant DNA indicated that the BWYV CP gene was present in the genome of three transformed plants (154802-1, 154802-3 and 1557-7). PCR analysis of the same plant genomic DNA followed by Southern blot analysis of the PCR products indicated that several plants contained the BWYV CP gene integrated into the plant genome (plants 155702-1 to -8, -10 to -14, 1557-7 and 154802-1 to -5). RNA transcripts of either the BWYV CP gene or the co-transformed npt II gene could not be detected using Northern blot analysis of the RNA extracted from regenerated plants. However, germination of the R1 seeds from the regenerated plants on kanamycin indicated that the npt II gene product which confers kanamycin resistance was functional in many of the progeny of the regenerated plants. BWYV CP subunits could not be detected byenzyme-linked immunosorbent assay (ELISA) using a polyclonal antiserum which reacts with BWYV CP subunits. Preliminary evaluation of the progeny of two promising lines (154802-3 and 1557-7), demonstrated to have the BWYV CP gene integrated into the plant genome, did not reveal significant levels of resistance when challenged with the homologous virus using the aphid vector, Myzus persicae (Sulz.)

Thesis/Dissertation

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2008-09-02

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http://hdl.handle.net/2429/1603

Plant Science

University of British Columbia

UBCV

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