Production of human papillomavirus type 16 E7 oncoprotein fused with β-glucuronidase in transgenic tomato and potato plants

Abstract

The human papillomavirus type 16 (HPV 16) oncogene E7 fused with the gene for β-glucuronidase (gus) was used in plant transformation experiments. The E7 gene modified for lower cancerogenicity and fused with the 5′ end of the gus in cassettes with cauliflower mosaic virus 35S promoter and transcription terminator produced high contents of fusion proteins in potato protoplasts. Expression vectors harbouring E7 fusion cassettes were used for Agrobacterium tumefaciens LBA4404 mediated transformation of either potato (Solanum tuberosum L. cv. Bintje) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker). A fusion gene was found in all rooted regenerants using polymerase chain reaction with primers providing amplified fragments from E7 and gus genes. GUS activity was revealed in all regenerants obtained. Nevertheless, the level of GUS expression in different constructs varied much more than in transient expression experiments with potato protoplasts. Especially, expression level in plants carrying vectors with the whole E7 gene fused with gus was lowered by 2–3 orders of magnitude comparing with fusion of the first 41 codons of E7 and gus. Southern hybridisation of 18 tomato and 23 potato regenerants revealed mostly multiple tandem integration of T-DNA into the plant genome and Western blot proved the presence of the fusion protein in 9 tomato and 11 potato plants out of 41 tested individuals.