congresos y reuniones científicas
Analysis and sequencing of h6hmRNA, last enzyme in
Buenos Aires
Congreso; BairesBiotec 2005; 2005
Institución organizadora:
Introduction Brugmansia candida is a South-American native tree that belongs to the Solanaceae family. Previous reports found that this plant is a high tropane alkaloid producer. Scopolamine and hyoscyamine are the most relevant tropane alkaloids widely used due to their effects on parasimpathic nervous system. These alkaloids can not be substituted by other class of compounds and therefore the demand for them will continue. Hyoscyamine 6-b hydroxylase (H6H, EC catalyses hydroxylation of hyoscyamine leading to 6,7-b-epoxide of hyoscyamine (scopolamine) in the end of the tropane alkaloid pathway. The present work reports the tissue and organ-specific expression of h6hmRNA by RT-PCR analyzes and the isolation, cloning and sequencing of the messenger obtained from B. candida flowering plants and hairy roots cultures.   Methodology Plant material: Different organs (tips of roots, stem, leaves and mother cells of microspores) and seeds of mature plants were harvested from flowering stage plants in the “Jardín Botánico” of Buenos Aires (Argentina). Hairy root cultures: seeds were surface sterilized by immersion in NaClO (4%) for 30 min, rinsed three times with sterile distilled water. Seeds were placed on B5/2 medium (Gamborg B5 medium diluted to half that the normal concentration of salts) supplemented with sucrose 15 g/l and agar 8 g/l. Incubation was carried out at 24ºC ±  2ºC with a 16 h photoperiod. HR of B. candida were obtained from 3 – 4 weeks old seedlings after infection with Agrobacterium rizogenes strain LBA 9402. The HR growth at the infections sites were excised and cultured individually on B5/2 liquid medium supplemented with sucrose 15 g/l, Ampicilline 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 subcultured every 2 weeks reducing gradually the concentration of antibiotics until were obtained axenic cultures. Transformation event was confirmed according to methods described previously (Pitta Alvarez, 1995). RT-PCR and PCR analysis: Total RNA was isolated from organs and HR mentioned above with Trizol-Reagent as previously described (Marconi et al, 2001) 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). Primer design: the resulting primers were: 5’ATGGCTACTTTTGTGTCG3’ and 5’CACTCTAGACATATGAGT 3’ PCR cycle parameters: Melting step: 94ºC for 4 min, annealing step: 40ºC for 1 min, elongation step: 72ºC for 3 min. At termination of 30 cycles, there was a further, final elongation step of 72º C for 7 min. Cloning in the pCR2.1-TOPO vector: 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 DH5a was transformed with the construction obtained by chemical transformation. Positive clones obtained from screening were analyzed by restriction mapping and confirmed by sequencing. Automated Sequencing: The samples were sequenced by the DNA ABI 373A automated sequencer, based on the Sanger method. The results were analysed by bioinformatics tools.   Results and Discusion Different preparations of total RNA were obtained from 3 weeks-old HR cultures and from root tips, shoot, leaves, anther or microspore mother cells of flowering plants. The RNeasy Plant Mini Kit allowed us to obtain higher total RNA levels comparing to Trizol procedure. Anthers were used to show the results due to the stronger specific signal obtained. Also, the integrity and size distribution of TotalRNA were checked by denaturing agarose gel electrophoresis as described in M&M. The ribosomal bands obtained appeared as sharp bands in the Total RNA obtained from RNeasy Plant Mini Kit methodology. Also, the 28S ribosomal RNA band presented an intensity of approximately twice that of the 18S RNA band. In contrast, the ribosomal bands in Trizol methodology suffered major degradation during preparation. Concerning to shoots and leaves samples from flowering plants no signal was detected. However, samples of HR and root tips showed a weakly signal. The relative abundance of h6hmRNA was determined by RT-PCR in tips of roots, stem, leaves and anthers from flowering plants and HR cultures. Reverse 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 a PCR and appropriate specific primers to evaluate the h6hmRNA presence. The primers were designed using the known sequence of the Hyoscyamus niger h6h gene due to the homology founded among the members of the Solanaceae family (Genbank, M62719). Amplified PCR products were inserted in a TOPO vector (Invitrogen). The restriction analysis showed the released of the expected 1 kb fragment. The 1Kb PCR product was sequenced. The bioinformatics analysis revealed an uninterrupted ORF of 1038 bp. The predicted aminoacid sequence was 344 aminoacid long. A database search showed that this sequence had high homology (97% identity) to H. niger H6H protein (Genbank accession number AAA33387.1). These results are coincident to previous reports about the H6H from other related species (Matsuda et al., 1991). Also, the 2OG-Fe(II) oxygenase superfamily domain is also conserved in the H6H gene from B. candida. In addition, the analysis shows that the H6H sequence has similarity to other hydroxylaces including those involved in the formation of ethylene and anthocyanins.   Conclusion Using RT-PCR methodology combined with specific primers design has led to obtain a rapid cloning method. The expression of H6H gene was investigated by RT-PCR in different organ 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 explain (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 amongst the population (Giulietti et al, 1993). The highest concentration of H6H mRNA was identified in anthers but this aspects needs to be investigated in further experiments.   Refferences Brunner, A, Yakovlev, I and Strauss, S. (2004). BMC Plant Biol. 4: 14-21. Giulietti, A, Pan, Rhodes, (1993). Planta Medica 59: 391-484. Matsuda, J., Okabe, S., et al. (1991). J. Biol. Chem. 266: 9460-9464. Pitta-Alvarez, S, A. Giulietti. (1995). In vitro Cell. Dev. Biol. Plant, 31: 215-220. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Laboratory Manual, 2nd ed. Cold Spring Harbor NY.