Journal of Experimental Botany, Vol. 54, No. 383, pp. 739-747,
February 1, 2003
© 2003 Oxford University Press
Antisense SNF1-related (SnRK1) protein kinase gene represses transient activity of an 
-amylase (-Amy2) gene promoter in cultured wheat embryos
Received 20 May 2002; Accepted 23 October 2002
1 Crop Performance and Improvement, Long Ashton Research Station, Long Ashton, Bristol BS41 9AF, UK
2 Crop Performance and Improvement, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
3 To whom correspondence should be addressed. Fax: +44 (0)1582 763 010. E-mail nigel.halford{at}bbsrc.ac.uk
A DNA fragment corresponding to part of an SNF1 (sucrose non-fermenting-1)-related protein kinase (SnRK1) transcript was amplified by a polymerase chain reaction (PCR) from a wheat (Triticum aestivum) endosperm cDNA library. It was used to construct a chimaeric gene, pUasSnRKN, comprising a ubiquitin promoter, the SnRK1 PCR product in the antisense orientation and the nopaline synthase (Nos) gene terminator. This construct was used in transient gene expression experiments in cultured wheat embryos together with a series of reporter gene constructs. These included the wheat alpha amylase gene 
-Amy2 promoter with UidA (Gus) coding region (p2GT), rice actin promoter with Gus (pActIDGus), ubiquitin promoter with Gus (pAHC25) and actin promoter with green fluorescent protein (GFP) gene (pAct1Is-GFP1). All of the reporter genes were found to be active when bombarded into scutellae isolated from immature grains at 25 d post-anthesis and incubated on MS medium for 24 h prior to bombardment. However, co-bombardment of p2GT with equimolar amounts of pUasSnRKN resulted in no detectable Gus activity, indicating that the antisense SnRK1 construct repressed the -Amy2 promoter. Co-bombardment with pUasSnRKN had no effect on the activity of the other promoters used in the study. A triple bombardment with p2GT, pAct1Is-GFP-1 and pUasSnRKN resulted in clear green fluorescence, indicating that the bombarded cells were still viable, but no Gus activity. RT-PCR analysis showed clearly that the antisense SnRK1 gene was expressing. Northern and RT-PCR analyses confirmed that SnRK1 and both -amylase genes, -Amy1 and -Amy2, are expressed in cultured wheat embryos harvested from grain 25 d post-anthesis. Expression of -Amy1 and -Amy2 was up-regulated by sugar starvation.
Key words: Carbohydrate metabolism, gibberellin, phosphorylation, seed development, sugar sensing, Triticum aestivum.
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