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Unformatted text preview: Transgenic Research 11: 437445, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands. 437 Cloning of the chrysanthemum UEP 1 promoter and comparative expression in florets and leaves of Dendranthema grandiflora Annadana S. 1 , 2 , Beekwilder M.J. 2 , Kuipers G. 2 , Visser P.B. 2 , Outchkourov N. 2 , Pereira A. 2 , Udayakumar M. 1 , De Jong J. 2 & Jongsma M.A. 2 , 1 Department of Crop Physiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560 065, India 2 Plant Research International B.V., P.O. Box 16, 6700 AA Wageningen, The Netherlands Received 27 March 2001; revised 12 July 2001; accepted 15 October 2001 Key words: chrysanthemum, florets, genetic engineering, GUS, molecular breeding, petal, transgene expression, ubiquitin extension protein Abstract To attain high transgene expression in petal tissue of ray florets of chrysanthemum an endogenous ubiquitin exten- sion protein ( UEP 1) promoter was cloned and tested with the -glucuronidase (GUS) reporter gene. Expression levels were compared with four heterologous promoters: chalcone synthase ( chs-A ) and zinc finger transcription factor ( EPF 2-5) from petunia, eceriferum ( CER 6) from Arabidopsis and multicystatin ( PMC ) from potato. The comparison of the expression levels of the different constructs in ray florets, disc florets, and leaves is presented. The highest mean expression in petal tissue of ray and disc florets was conferred by the UEP 1 promoter, followed by CER 6 and EPF 2-5. The UEP 1 promoter in ray florets confers over 50-fold enhancement in expression as compared to CaMV 35S-based promoters. Introduction Chrysanthemum Dendranthema grandiflora (Ander- son, 1987) is the second largest cut flower produced next to roses ( Rosa hybrida ). Insects cause direct and indirect damage on plants resulting in reduced flower quality and lower marketable value. Two important pests of chrysanthemum are beet armyworm, Spodop- tera exigua (Lepidoptera: noctuidae) (Cuijpers, 1994) and western flower thrips, Frankliniella occidentalis (Thysanoptera)(De Jager et al., 1995). The prospect of achieving insect resistance through genetic engineer- ing has received attention in the recent past. The use of Bt toxins (Dolgov et al., 1995) and proteinase inhib- itors could be a method of developing insect resistant chrysanthemums, but requires high level of expression in leaves and flowers. Studies on transgene expression in chrysanthem- ums have focused on the 35S-CaMV (cauliflower Author for correspondence: (E-mail: firstname.lastname@example.org) mosaic virus) promoter (Ledger et al., 1991; Renou et al., 1993; De Jong et al., 1995). However, with the 35S-CaMV promoter only low levels of expression were observed by us and other groups (Van Wordra- gen et al., 1993; Boase et al., 1998; Sherman et al., 1998). The enhanced d35S-CaMV promoter fused to GUS showed bright blue coloration in histochemical assays, but the quantitative -glucuronidase (GUS) measurements indicated low expression in all tissues...
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