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  • 1
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; enoyl-ACP reductase ; fatty acid synthesis ; GUS ; 5′-flanking region ; transgenic tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The NADH-specific enoyl-acyl carrier protein (ACP) reductase, which catalyses the last reducing step during the fatty acid biosynthesis cycle, is encoded in Arabidopsis thaliana encoded by a single housekeeping gene (ENR-A) which is differentially expressed during plant development. To identify elements involved in its tissue-specific transcriptional control, a fragment comprising the 1470 bp region directly upstream of the ATG start codon of the ENR-A gene was fused to the uidA (GUS) reporter gene and analysed in transgenic Nicotiana tabacum plants. GUS activity found during development of the transgenic plants was similar to endogenous ENR protein levels found in both tobacco and Arabidopsis plants, except for developing flowers. In floral tissue the promoter fragment showed very little activity in contrast to the relatively high level of endogenous ENR expression. Successive deletions from the 5′ and 3′ regions of the promoter fragment revealed the presence of at least three elements which control GUS expression in different stages of development in the transgenic tobacco plants. First, expression in young developing leaves required both the presence of sequences between −329 to −201 relative to the transcription start and part of the untranslated leader comprising the first intron. Second, root-specific GUS expression was still observed after deletion of the 5′-upstream sequences up to 19 bp of the transcription initiation site. Further, the additional removal of the intron from the untranslated leader increased root-specific expression by ca. 4- to 5-fold. Third, high expression in seeds was still observed with the minimal upstream promoter segment of 19 bp. This seed expression level was found to be independent of the presence or absence of the intron in the untranslated leader. Finally, 3′ deletion of the leader sequence up to 17 bp of the transcription start greatly impaired GUS activity during all stages of plant development, suggesting that the deleted sequence of the leader either functions as an enhancer for transcription initiation or stabilizes the mRNA.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: fatty acid synthesis ; malonyl CoA-ACP transacylase ; seed development ; transgenic rape ; transgenic tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.
    Type of Medium: Electronic Resource
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