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JXB Advance Access published online on December 10, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern273
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.)

Tao Yin1 *, Hanying Wu2 *, Shanglong Zhang1,{dagger}, Hongyu Lu3, Lingxiao Zhang4, Yong Xu2, Daming Chen1,5 and Jingmei Liu2,{dagger}

1College of Agriculture and Biotechnology, Zhejiang University, PR China
2National Engineering Research Center for Vegetable, PR China
3Department of Breeding and Genetics, China Pharmaceutical University, PR China
4Delta Research and Extension Center, Mississippi State University, Stoneville, Mississippi, USA
5Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

{dagger} To whom correspondence should be addressed. E-mail: shlzhang{at}zju.edu.cn, jingmeiliu{at}mail.tsinghua.edu.cn.

A 1.8 kb 5'-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from –986 to –959 and from –472 to –424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative β-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were ~10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves.

Key words: ADP-glucose pyrophosphorylase, cis-element, fruit-specific promoter, transcriptional factors, transgenic tomato, watermelon

* These authors contributed equally to this work.

Received 16 June 2008; Revised 29 July 2008 Accepted 9 October 2008


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