RESUMO
Microspores, with a haploid number of chromosomes, are destined to produce the male gametophyte, which hosts the male gametes that fertilize the female egg cell. During microsporogenesis, a particular stage of development is amenable to be switched to undergo embryogenesis and developed into a haploid plant. By doubling the chromosomes, a doubled haploid plant, homozygous for all the gene loci, is produced. These plants are useful to study the expression of recessive genes and in plant breeding as a rapid pathway to achieve homozygosity.
Assuntos
Datura metel/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Células Germinativas Vegetais/crescimento & desenvolvimento , Técnicas de Embriogênese Somática de Plantas/métodos , Datura metel/genética , Flores/genética , Haploidia , Melhoramento Vegetal/métodosRESUMO
To identify the original plant of Daturae Flos from its adulterants by DNA barcoding, the sequences of ITS2, psbA-trnH, matK, rbcL of four species including Datura metel, Darura innoxia, Darura stramonium and Brugmansia arborea were compared and analyzed. The PCR and sequencing success rate of the four regions (ITS2, psbA-trnH, matK, rbcL) was 100%, 90%, 100% and 85%, respectively. Sequences were assembled with CodonCode Aligner. K2P distances were calculated and NJ tree was performed by MEGA 4.1. Thirty SNPs were found among ITS2 sequences, and 33 insert/deletes were found among psbA-trnH intergenic regions. The interspecific K2P distance of ITS2 and psbA-trnH was obviously higher than that of the intraspecific one. As to matK and rbcL, there was no "Barcoding Gap" existing between inter- and intra-specific distances. The NJ trees of the four regions/combinations were built separately. Samples of Brugmansia arborea were clustered into one clade, and the other species of Datura L. formed another clade. The results showed that either ITS2 or psbA-trnH was useful to identify Daturae Flos from its adulterants.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Datura metel/genética , Datura/genética , Contaminação de Medicamentos , Plantas Medicinais/genética , Sequência de Bases , DNA Intergênico/genética , DNA de Plantas/genética , Datura/classificação , Datura stramonium/genética , Flores/genética , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Solanaceae/genética , Especificidade da EspécieRESUMO
Hyoscyamine 6ß-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6ß-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6ß-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K(m) values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50µM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14M(-1) and 0.56M(-1), respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.
