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      Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of Brugmansia candida (Solanaceae)

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          Abstract

          Brugmansia candida (Solanaceae) is a native tree distributed across South-American and produces the pharmacologically- important group of tropane alkaloids including scopolamine. This biocompound is synthesised from hyoscyamine by action of Hyoscyamine 6-β hydroxylase (H6H, EC 1.14.11.11) at the end of the tropane alkaloid pathway. Here are reported the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the cDNA obtained from B. candida anthers and hairy root transformed cultures. Bioinformatic analysis of the nucleotide sequence revealed an uninterrupted ORF of 1038 bp and the predicted aminoacid sequence could be 344 aminoacid long. A database search showed that this sequence has high homology (97% identity) to Hyoscyamus niger H6H protein (Genbank accession number AAA33387.1).

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          Validating internal controls for quantitative plant gene expression studies

          Amy M. Brunner, Igor Yakovlev, Steven Strauss (2004)
          Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.
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            Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger.

            J Matsuda, S Okabe, T. Hashimoto … (1991)
            Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger.
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              New genes in alkaloid metabolism and transport.

              Takashi Hashimoto, Yasuyuki YAMADA (2003)
              The biosynthetic pathway of plant alkaloids is composed of several distinct enzymes of varying substrate specificities. Homology-based cloning of candidate genes and their subsequent functional testing in heterologous expression systems are accelerating the pace at which the gene catalogues of alkaloid biosynthesis are expanding. Availability of diverse genes involved in the biosynthesis, catabolism, transport, and regulation of pharmaceutically important alkaloids should steadily advance our molecular understanding of alkaloid biology and will enable us to devise more rational strategies for metabolic engineering.
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                Author and article information

                Journal
                Journal ID (publisher-id): ejb
                Title: Electronic Journal of Biotechnology
                Abbreviated Title: Electron. J. Biotechnol.
                Publisher: Pontificia Universidad Católica de Valparaíso (Valparaíso, , Chile )
                ISSN (Electronic): 0717-3458
                Publication date (Print and electronic): June 2006
                Volume: 9
                Issue: 3
                Affiliations
                [02] Buenos Aires orgnameFundación Pablo Cassará orgdiv1Centro de Ciencia y Tecnología Dr. Cesar Milstein Argentina pmarconi@ 123456fundacioncassara.org.ar
                [01] Buenos Aires orgnameUniversidad de Buenos Aires orgdiv1Facultad de Farmacia y Bioquímica orgdiv2Departamento Microbiología, Inmunología y Biotecnología Argentina alejandra_cardillo@ 123456yahoo.com
                Article
                Publisher ID: S0717-34582006000300004 Publisher ID: S0717-3458(06)00900304
                DOI: 10.4067/S0717-34582006000300004
                SO-VID: bbfd166c-9d95-49b0-8078-1b879787d835
                License:

                This work is licensed under a Creative Commons Attribution 4.0 International License.

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                Figures: 0, Tables: 0, Equations: 0, References: 7, Pages: 0
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                SciELO Chile

                Categories
                Subject: RESEARCH ARTICLES

                Keywords: transformation,sequence analysis,scopolamine,hyoscyamine,hairy roots,H6H
                Data availability:
                Keywords: transformation, sequence analysis, scopolamine, hyoscyamine, hairy roots, H6H

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