Financial support: BID 1201 OC-AR/PICT 12178 and CONICET.
Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of Brugmansia candida (Solanaceae)
Alejandra B. Cardillo
Departamento Microbiología, Inmunología y Biotecnología
Facultad de Farmacia y Bioquímica
Universidad de Buenos Aires
Junin 956 (1113)
Ana M. Giulietti
Patricia L. Marconi*
Keywords: H6H, hairy roots, hyoscyamine, scopolamine, sequence analysis, transformation.
(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 18.104.22.168)
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
is a South-American native tree that belongs to the Solanaceae
family. Previous reports found that this plant is a high tropane
alkaloid producer. Hyoscyamine and scopolamine are the most relevant
tropane alkaloids widely used due to their effects on parasimpathic
nervous system. These alkaloids can not be substituted by any other
class of compounds and therefore their demand continues. Hyoscyamine
6-β hydroxylase (H6H, EC 22.214.171.124) catalyses hydroxylation of hyoscyamine
leading to 6,7-β-epoxide of hyoscyamine (scopolamine) at the end of
the tropane alkaloid pathway. This enzyme has been isolated and the
corresponding gene cloned from Hyoscyamus
The present work reports the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the messenger obtained from B. candida anthers and hairy root cultures.
organs: root tips; apical stem and leaves and seeds were harvested
from plants that grew at the “Jardín Botánico” of
were surface sterilized by immersion in NaClO (4%) for 30 min, and
rinsed three times with sterile distilled water. Thereafter, seeds
were placed on B5/2 medium (hormone-free, half-stregth Gamborg B5
medium) supplemented with sucrose 15 g/l and agar 8 g/l. Incubation
was carried out at
Hairy root (HR) cultures of B. candida were obtained from 3-4 weeks old seedlings after infection with Agrobacterium rhizogenes strain LBA 9402. The HR growth at the infection sites were excised and cultured individually on B5/2 liquid medium supplemented with sucrose 15 g/l, ampicillin 2 g/l and agar 8 g/l. The resulting HR were incubated on a gyratory shaker at 100 rpm in the same conditions described above. The HR were routinely sub-cultured every 2 weeks reducing 1:10 the concentration of antibiotics until the elimination of Agrobacterium. Transformation event was confirmed according to methods described previously (Pitta Alvarez et al. 2003).
Total RNA was isolated from organs and HR mentioned above with Trizol-Reagent and compared to the extraction with RNeasy Plant Kit (Qiagen). Integrity and size distribution of purified total RNA were checked by gel electrophoresis on denaturing conditions. The cDNA synthesis was performed using Superscript II reverse Transcriptase (Life Technologies).
primers were designed based on the sequence of the h6h gene from Hyoscyamus
Agarose gel electrophoresis was performed as described by Sambrook et al. (1989). The amplified fragments of the expected size were isolated from agarose gel and purified using the GFX columns (Amersham). They were cloned in the TOPO vector according to the manufacturer instructions (Invitrogen).
Escherichia coli strain DH5α was transformed with the construction obtained by chemical transformation. Positive clones obtained from screening were analyzed by restriction mapping and confirmed by sequencing.
The samples were sequenced by the DNA ABI 373A automated sequencer, based on the Sanger method. The results were analysed by bioinformatics tools.
The induction of HR from B. candida plants using de A. rhizogenes LBA9402 was successfully obtained with a frequency of transformation around 80%. This is according to previous experiments done with this plant material (Pitta Alvarez et al. 2003). The transformation process was checked by PCR reaction (data not shown).
The root tips were isolated from roots growing into de soil. Shoot, leaves, anther were obtained from the same plants.
Different preparations of total RNA were obtained from 3 weeks-old HR cultures and from different tissues and organs of flowering plants.
Concerning to RNA isolation, the RNeasy Plant Mini Kit allowed us to obtain higher total RNA levels comparing to Trizol procedure. The results are shown with anthers due to the stronger specific signal obtained with this organ. Also, the integrity and size distribution of total RNA were checked by denaturing agarose gel electrophoresis as described in Materials and Methods. The ribosomal RNA appeared as sharp bands when Total RNA was extracted using the RNeasy Kit (Figure 1). Also, the 28S ribosomal RNA band presented an intensity of approximately twice that of the 18S RNA band (Figure 1).
In contrast, the ribosomal bands in Trizol methodology were not sharp and appeared smaller sized RNA bands. In this case, the RNA sample suffered major degradation during preparation (Figure 1).
The relative abundance of h6hmRNA was determined by RT-PCR in root tips, stem, leaves, anthers and HR cultures. No signal was detected in apical shoot and leave samples from flowering plants. HR and root tips samples showed a weakly signal. However, anthers showed the strongest signal.
transcription was carried out using an oligo-dT primer which allowed
detection of multiple species of cDNAs from the total RNA. The RT
reaction was amplified by PCR and appropriate specific primers to
evaluate the h6hmRNA presence. The primers were designed using the
known sequence of the Hyoscyamus
Amplified PCR product was cloned using the TOPOVector Technology. EcoRI restriction mapping performed on positive clones showed the release of 1 Kb fragment as expected (data not shown).
The 1Kb PCR product was sequenced (Figure 3). Furthermore, using the sequence information, internal primers were designed in order to confirm H6H sequence.
bioinformatic analysis revealed an uninterrupted ORF of 1038 bp. The
predicted aminoacid sequence is 344 aminoacids long. A database search
showed that this sequence has high homology (97% identity) to H.
Using RT-PCR methodology combined with specific primer design has led to obtain a rapid cloning method. The expression of h6h gene was investigated by RT-PCR in different organs and tissues from flowering plants and HR cultures. Total RNA extracted was analyzed but the different degrees of variation observed in the expression of H6H among samples could not be explained (Brunner et al. 2004). It should also be remembered that B. candida is a self-fertile natural hybrid which will presumably increase the potential range for variation among the population (Giulietti et al. 1993). The highest concentration of h6hmRNA was identified in anther samples but these aspects need to be investigated in further experiments.
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