Promoter trapping in Arabidopsis thaliana : characterization of T-DNA tagged lines
thesisposted on 15.12.2014, 10:33 by Pedro. Rocha
Two A. thaliana lines transgenic for the promoter-trap vector pgusBin19 were studied. Line AtEN101 lacked a phenotype but reporter gene (gus) expression exhibited temporal and spatial regulation, including in the developing embryo. This pattern was unaltered when AtEN101 was used as a functional marker in two embryo-defective mutant backgrounds tested. In roots of AtEN101 seedlings, gus expression was repressed by ABA and modified by auxins, in a sucrose-modulated manner.;The tagged genomic region in the mutant and wildtype, and cDNAs mapping to it were isolated. The cDNAs derived from two overlapping genes (PKT1 and PKT2) almost identical except for their first exons and introns and encoding putative peroxisomal 3-ketoacyl-CoA thiolases. The T-DNA was inserted in the long intron1 (966 bp) of PKT1, and 0.3 kb upstream from exon1 of PKT2. gus copies were found at both T-NDA junctions. Fusion transcripts including exon1 of PKT1 and T-DNA sequences were identified, and a cryptic 3' splice site identified in the left border region.;The genes were found to be independently expressed, at low levels, in all organs by Northern blot hybridization and RT-PCR. Both putative promoter regions were capable of driving the expression of gus in transient assays. That of PKT1 was also functional in reverse orientation. The thiolase genes are part of a family of at least four members, PKT1 being unique in lacking the characteristic N-terminal peroxisomal targeting signal, PTS2.;A new T-DNA linked semi-dominant mutation causing dwarfism (bashful) was identified in line P26K4, gus-derived sequences were present in its genome but no GUS activity was observed. The mutation affected cell growth and was rescued by epibrassinolide but not gibberellic acid (GA3). Skotomorphogenic development is also affected in the mutant.;The utility of pgusBin19 as a promoter-trap and insertional mutagen in Arabidopsis, and the possible functions of PKT1/PKT2 are discussed.