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Unformatted text preview: ORIGINAL ARTICLE The promoter–terminator of chrysanthemum rbcS1 directs very high expression levels in plants Received: 23 July 2002/ Accepted: 11 November 2002/Published online: 10 January 2003 ȑ Springer-Verlag 2003 Abstract Transgenic plants are increasingly used as production platforms for various proteins, yet protein expression levels in the range of the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase have not yet been achieved by nuclear transformation. Suitable gene regulatory 5 ¢ and 3 ¢ elements are crucial to obtain adequate expression. In this study an abundantly tran- scribed member ( rbcS1 ) of the ribulose-1,5-bisphosphate carboxylase small-subunit gene family of chrysanthe- mum ( Chrysanthemum morifolium Ramat.) was cloned. The promoter of rbcS1 was found to be homologous to promoters of highly expressed rbcS gene members of the plant families Asteraceae, Fabaceae and Solanaceae. The regulatory 5 ¢ and 3 ¢ non-translated regions of rbcS1 were engineered to drive heterologous expression of various genes. In chrysanthemum, the homologous rbcS1 cassette resulted in a b-glucuronidase (gusA) ac- cumulation of, at maximum, 0.88% of total soluble protein (population mean 0.17%). In tobacco ( Nicotiana tabacum L.), the gusA expression reached 10% of total soluble protein. The population mean of 2.7% was found to be 7- to 8-fold higher than for the commonly used cauliﬂower mosaic virus (CaMV) 35S promoter (population mean 0.34%). RbcS1-driven expression of sea anemone equistatin in potato ( Solanum tuberosum L.), and potato cystatin in tomato ( Lycopersicon esculentum Mill.) yielded maximum levels of 3–7% of total soluble protein. The results demonstrate, that the compact 2-kb rbcS1 expression cassette provides a novel nuclear transformation vector that generates plants with expression levels of up to 10% of total protein. Keywords Chrysanthemum Æ Gene expression (high level) Æ b-Glucuronidase Æ Promoter Æ Ribulose-1,5-bisphosphate carboxylase (rbcS) Abbreviations CaMV: cauliﬂower mosaic virus Æ GUS, gusA : b-glucuronidase Introduction In genetically modified plants, high-level, tissue-specific transgene expression is required for a variety of traits, and numerous promoters to ensure this have been de- scribed in the literature. Known strong constitutive promoters in plants include the cauliﬂower mosaic virus (CaMV) 35S promoter (Ow et al. 1987), opine synthase promoters (Harpster et al. 1988), actin promoters (McElroy et al. 1991) and ubiquitin promoters (Kawalleck et al. 1993). Other non-constitutive strong promoters include the light-regulated chlorophyll a / b- binding protein promoter (Nap et al. 1993) or the promoters of the small subunit of ribulose bisphosphate carboxylase ( rbcS ) (Khoudi et al. 1997; Gittins et al....
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This note was uploaded on 08/01/2009 for the course HORT hor-11-12 taught by Professor Park during the Spring '09 term at A.T. Still University.
- Spring '09