Antisense SNF1-related (SnRK1) protein kinase gene represses transient activity of an alpha-amylase (alpha-Amy2) gene promoter in cultured wheat embryos.
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 post-anthesis 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.
Laurie
Sophie
S
Crop Performance and Improvement, Long Ashton Research Station, Long Ashton, Bristol BS41 9AF, UK.
McKibbin
Rowan S
RS
Halford
Nigel G
NG
D000594:Amino Acid Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D016375:Antisense Elements (Genetics) / Q000235:genetics; D002241:Carbohydrates / Q000494:pharmacology; D003001:Cloning, Molecular; D046508:Culture Techniques; D004794:Enzyme Repression / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D016375:Antisense Elements (Genetics); D002241:Carbohydrates; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D000516:alpha-Amylases; D005966:Glucuronidase