Strictosidine synthase from Rauvolfia serpentina: analysis of a gene involved in indole alkaloid biosynthesis.
The gene for strictosidine synthase (str1), the enzyme which catalyzes the stereospecific condensation of tryptamine and secologanin to form the key indole alkaloid 3 alpha(S)-strictosidine has been isolated from genomic libraries prepared from Rauvolfia serpentina (India) and from Rauvolfia mannii (West Africa). The gene, str1, contained no introns and showed 100% nucleotide sequence homology over 1180 bp, encompassing the entire reading frame, between the two species. Transcription of the R. serpentina gene was found to start 81 nucleotides upstream from the AUG (26 nucleotides downstream from the TATA box). Transient expression assays in Nicotiana plumbaginifolia protoplasts of the R. serpentina str1 5'-noncoding region fused to the beta-glucuronidase reporter gene revealed promoter activity equivalent to 4 +/- 2% of that of 35 S CaMV promoter control. A series of truncated segments of the str1 promoter region indicated the presence of three areas of slight, but reproducible, negative control. Gel retardation assays demonstrated that several regions of the 5'-flanking sequences specifically bound nuclear protein from R. serpentina and that at least one region does not bind R. mannii nuclear protein. A survey of the expression of str1 in the R. serpentina plant suggested that strictosidine synthase poly(A)+ RNA was present predominantly, but not exclusively, in the root. This result correlated well with the distribution of both enzyme activity and indole alkaloids which were also predominant in the root, but, in general, distributed throughout the shrub.
Bracher
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Lehrstuhl für Pharmazeutische Biologie, Universität München, Germany.
Kutchan
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TM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D019759:Carbon-Nitrogen Lyases; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D017343:Genes, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011926:Rauwolfia / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D046948:Secologanin Tryptamine Alkaloids / Q000096:biosynthesis*; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D046948:Secologanin Tryptamine Alkaloids; D004247:DNA; D014166:Transferases; D005966:Glucuronidase; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase