RESUMEN
Sanguinarine is the main active component of the Papaver plants, and protopine-6-hydroxylase(P6 H), involved in the sanguinarine biosynthetic pathway, can oxidize protopine to 6-hydroxyprotopine. The investigation on the diversity of P6 H genes in the medicinal Papaver plants contributes to the acquirement of P6 H with high activity to increase the biosynthesis of sanguinarine. Five P6 H genes in P. somniferum, P. orientale, and P. rhoeas were discovered based on the re-sequencing data of the Papaver species, followed by bioinformatics analysis. With the elongation factor 1α(EF-1α), which exhibits stable expression in the root and stem, as the internal reference gene, the transcription levels of P6H genes in roots and stems of the Papaver plants were detected by real-time fluorescent quantitative PCR. As indicated by the re-sequencing results, there were two genotypes of P6H in P. somniferum and P. orientale, respectively, and only one in P. rhoeas. The bioinformatics analysis showed that the P6 H proteins of the three Papaver plants contained the conserved domain cl12078, which is the characteristic of p450 supergene family, and transmembrane regions. The existence of signal peptide remained verification. Real-time fluorescent quantitative PCR results revealed that the transcription level of P6 H in roots of P. somniferum was about 1.44 times of that in stems(α=0.05). The present study confirmed genetic diversity of P6 H in the three medicinal Papaver plants, which lays a basis for the research on the biosynthesis pathway and mechanism of sanguinarine in Papaver species.
Asunto(s)
Benzofenantridinas , Alcaloides de Berberina , Sistema Enzimático del Citocromo P-450/genética , Variación Genética , Papaver/genéticaRESUMEN
Codeine-O-demethylase (CODM) is a key enzyme in the biosynthesis of codeine and morphine. In this study, CODM gene sequences were cloned from Papaver somniferum and Papaver rhoeas, and were compared with each other by sequence alignment and bioinformatics analysis. The results showed that there were three genotypes of CODM in Papaver somniferum and five genotypes of CODM in Papaver rhoeas. Bioinformatics analysis showed that all CODM proteins had no signal peptide sequence, and these proteins were predicted to be non-secretory proteins, belonging to the Pcbc supergene family. Although the amino acid sequences of CODM in poppies are the same, the expression levels of CODM in different poppy resources are significantly different. It is speculated that the variation of transcription level of CODM may be related to its non-coding region sequence, which lays a foundation for further research on the synthesis and regulation mechanism of alkaloids in poppies.
RESUMEN
Objective To disclose the inter-species differences among Papaver somniferum L, Papaver rhoeas L and Cannabis sativa L using AFLP analysis. MethodsThe root, stem, foliage, flower, seed of Papaver somniferum L, foliage of Papaver rhoeas L and Cannabis sativa L were collected respectively. DNA was isolated with AxyPrep Kit, and double-digested by EcoR I and Mse I, then oligonucleotide adapters were ligated. After Pre-amplification, selective amplification was performed using 6 pairs of fluorescent primer. The DNA fragments were analyzed using a CEQ 8000 DNA Fragment Analyzer. Results27 to 46, 5 to 20 and 4 to 31 pieces of amplified products were detected from Papaver somniferum L, Cannabis sativa L and Papaver rhoeas L respectively, and significant difference was observed among these three groups of products. The identical species-specific peaks were identified from root, stem, leaf, flower and seed DNA from the same Papaver somniferum L. ConclusionThe diacritical results from Papaver somniferum L, Papaver rhoeas L and Cannabis sativa L, as well as the identical results of different parts from the same plant demonstrated that AFLP analysis could be used possibly to determine the species origin of unknown plants samples.