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GrainGenes Reference Report: JEB-54-739

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Reference	JEB-54-739
Title	Antisense SNF1-related (SnRK1) protein kinase gene represses transient activity of an alpha-amylase (alpha-Amy2) gene promoter in cultured wheat embryos
Journal	Journal of Experimental Botany
Year	2003
Volume	54
Pages	739-747
Author	Laurie S McKibbin RS Halford NG
Abstract	Summary: 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 alpha-Amy2 promoter with UidA (Gus) coding region (palpha2GT), 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 postanthesis and incubated on MS medium for 24 h prior to bombardment. However, co-bombardment of palpha2GT with equimolar amounts of pUasSnRKN resulted in no detectable Gus activity, indicating that the antisense SnRK1 construct repressed the alpha-Amy2 promoter. Co-bombardment with pUasSnRKN had no effect on the activity of the other promoters used in the study. A triple bombardment with palpha2GT, 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 alpha-amylase genes, alpha-Amy1 and alpha-Amy2, are expressed in cultured wheat embryos harvested from grain 25 d post-anthesis. Expression of alpha-Amy1 and alpha-Amy2 was up-regulated by sugar starvation
External Databases	Pubmed: 12554717
Keyword	[ Hide all but 1 of 48 ] actin alpha-amylase gene amylase antisense barley bombardment carbohydrate metabolism carbon metabolism cdna cell differential expression dna endosperm fluorescence gene gfp gibberellin grains green fluorescent protein high-level expression kinase media orientation pcr phosphorylation photosynthesis polymerase potato promoter protein protein kinase region reporter rice rt-pcr seed development sequence series sucrose sucrose-inducibility sugar sugar sensing synthase transient triticum ubiquitin promoter viable wheat embryos

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