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  • 1
    Digitale Medien
    Digitale Medien
    Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds (1994)
    Springer
    Plant molecular biology 26 (1994), S. 189-202 
    Details
    ISSN: 1573-5028
    Schlagwort(e): fatty acid synthesis ; malonyl CoA-ACP transacylase ; seed development ; transgenic rape ; transgenic tobacco
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: 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.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1007/BF00039531
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  • 2
    Digitale Medien
    Digitale Medien
    Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes (1989)
    Springer
    Plant molecular biology 13 (1989), S. 491-502 
    Details
    ISSN: 1573-5028
    Schlagwort(e): Petunia hybrida ; Antirrhinum majus ; flavonoid synthesis ; dihydroflavonol-4-reductase ; regulatory genes
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays. The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively). Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1007/BF00027309
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  • 3
    Digitale Medien
    Digitale Medien
    Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression (1990)
    Springer
    Plant molecular biology 15 (1990), S. 95-109 
    Details
    ISSN: 1573-5028
    Schlagwort(e): chalcone synthase gene (A) ; flower-specific transient expression ; Petunia hybrida ; promoter analysis ; TACPyAT sequence
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract In order to scan the 5′ flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5′ end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from −67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from −142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (−59 and −52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1007/BF00017727
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  • 4
    Digitale Medien
    Digitale Medien
    Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect (1990)
    Springer
    Plant molecular biology 14 (1990), S. 457-466 
    Details
    ISSN: 1573-5028
    Schlagwort(e): antisense RNA ; chalcone synthase (CHS) gene ; Petunia hybrida
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene. Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3′ half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5′ half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes. Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1007/BF00027492
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  • 5
    Digitale Medien
    Digitale Medien
    cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli (1991)
    Springer
    Plant molecular biology 17 (1991), S. 895-909 
    Details
    ISSN: 1573-5028
    Schlagwort(e): acyl carrier protein ; Brassica napus ; enoyl-ACP reductase ; fatty acid synthesis ; seed development ; nuclear-encoded chloroplast proteins
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific λgt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1007/BF00037070
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