RESUMEN
Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000- containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 µg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes.
Asunto(s)
Asteraceae/química , Asteraceae/genética , Botánica/métodos , ADN de Plantas/aislamiento & purificación , Técnicas Genéticas , Cloroformo/química , ADN de Plantas/análisis , ADN de Plantas/metabolismo , Fluorometría , Técnicas Genéticas/economía , Pentanoles/química , Hojas de la Planta/química , Hojas de la Planta/genética , Polietilenglicoles/química , Reacción en Cadena de la Polimerasa , ARN Ribosómico 5S/genética , Ribulosa-Bifosfato Carboxilasa/genética , EspectrofotometríaRESUMEN
The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.
Asunto(s)
Proteínas Recombinantes/aislamiento & purificación , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Serina Proteinasa/metabolismo , Solanum tuberosum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/genética , Transformación GenéticaRESUMEN
New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.
Asunto(s)
Inhibidores Enzimáticos , Proteínas Fúngicas/antagonistas & inhibidores , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Poligalacturonasa/antagonistas & inhibidores , Solanum/genética , Secuencia de Aminoácidos , Clonación Molecular , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Análisis de Secuencia de Proteína , Solanum/microbiologíaRESUMEN
Eighteen clones representing copies of four Kunitz-type proteinase inhibitor group B genes (PKPI-B) obtained by polymerase chain reaction cloning of potato (Solanum tuberosum L. cv. Istrinskii) genomic DNA were sequenced and analyzed. Three new genes were found and named PKPI-B1, PKPI-B2, and PKPI-B10: these were represented by five, two, and seven clones, respectively. The remaining four clones corresponded to the formerly characterized PKPI-B9 gene. These data show that at least four PKPI-B encoding genes are harbored in the genome of potato cv. Istrinskii. Their analysis suggests that variability of PKPI-B encoding genes in potato is limited and could be explained by cross-hybridization events in the ancestor forms rather than by random mutagenesis.
