Poster: Regulation of gene expression

Abs # 754: Identification of ethylene-responsive elements in the promoter of SmCP, the gene encoding Solanum melongena cysteine proteinase

Presenter:	Rawat, Reetika , rrawat@hkucc.hku.hk
Authors	Rawat, Reetika  (A)   Chye, Mee-Len  (A)
Affiliations:	(A): Department of Botany, The University of Hongkong

The expression of SmCP, the gene that encodes Solanum melongena cysteine proteinase, coincides with developmental events associated with programmed cell death in plant tissues¹. Its expression is ethylene-inducible and is under circadian regulation with peak expression in late light². In order to better understand the regulation of SmCP, we have isolated and analyzed its 1.34-kb 5’-flanking region for cis-regulatory elements. Various lengths of the SmCP 5’-flanking sequence were fused to the GUS reporter gene in binary vector pBI101.3. These SmCP promoter deletion constructs were used in Agrobacterium–mediated tobacco transformation. Histochemical GUS analysis of transgenic tissues revealed that the minimal promoter region -81/+55 containing a TATA box was sufficient to drive GUS expression in roots. For minimal basal expression in stem, leaves and flowers, a -127/+55 region was required. A larger region (-827/+55) produced strong expression in all organs. Analysis of the 5’-flanking region revealed the presence of two 8-bp ethylene-responsive elements (EREs), at -355/-348 and -683/-676, with strong homology to the ERE of the tomato-ripening gene E4. Analysis of transgenic tobacco revealed that the -415/+55 region, with ERE -355/-348, was sufficient to mediate a two-fold ethylene-induction of GUS expression. Five-fold ethylene-induction was seen with the -827/+55 region, in which ERE -683/-676 was also present. Using gel mobility shift assays, we demonstrated that each ERE showed binding to nuclear protein extracts from senescent fruits and aerial parts of 5-week-old seedlings. ¹Xu, FX and Chye, ML (1999) Plant J. 17: 321-327. ²Xu, ZF et al., (2003) Plant Mol. Biol. 51: 9-19. This work was supported by the Research Grants Council of Hong Kong (Project HKU 7181/01M)