Aislamiento de la región promotora del gen FaPAL2 de Fragaria x ananassa Cv. “Camino Real” y evaluación de su funcionalidad en respuesta a la irradiación UV-C

La proteína fenilalanina amonio-liasa o PAL es una enzima clave en la ruta de síntesis de los flavonoides; en fresa se han reportado 6 genes que la codifican, entre ellos el FaPAL2. Los flavonoides son metabolitos secundarios que participan en la protección contra luz UV de las plantas, además, son de gran interés farmacéutico debido a las propiedades antioxidantes, antibacterianas, antiinflamatorias, antimutagénicas y anticancerígenas que poseen. Se ha correlacionado el aumento de flavonoides en fresas irradiadas con luz UV-C con altos niveles de expresión del gen FaPAL. Para poder estudiar y controlar la expresión de genes de interés es indispensable conocer la funcionalidad de los promotores, por lo que la presente investigación se planteó por objetivo identificar y aislar el promotor del gen FaPAL2 mediante la técnica TAIL PCR, para posteriormente evaluar su actividad ante respuesta a la luz UV-C en frutos de Fragaria x ananassa cv. “Camino Real” vía Agrobacterium tumefaciens utilizando el gen reportero GUS. Se consiguió aislar y secuenciar el promotor del gen FaPAL2, para después generar un constructo genético y evaluar su expresión genética transitoria en frutos agroinfiltrados de fresa. Se identificó una tinción histológica positiva de los frutos agroinfiltrados, tanto irradiados como no irradiados, lo que indica que el promotor del gen FaPAL2 actúa positivamente en respuesta a luz UV-C, pero no de manera exclusiva.

Overexpression of HOP2 induces developmental defects and compromises growth in Arabidopsis

HOMOLOGOUS PAIRING 2 (HOP2) is a predominantly meiotic protein that plays a pivotal role in homologous chromosome pairing in organisms as diverse as yeast and mammals. While generating HOP2::GFP reporter lines, we identified two Arabidopsis T-DNA insertion mutants, stunted1(std1) and stunted2 (std2) that exhibit pleiotropic phenotypes, including fasciated stems, altered phyllotaxy, floral organ defects, reduced fecundity, and an overall reduction in growth properties. TAIL-PCR followed by sequencing revealed several insertions near genes, but genotyping showed that none of the insertions are causal. Analysis the std mutants by qRT-PCR, and analysis of dexamethasone inducible HOP2 transgenic plants demonstrated that the std phenotypes are associated with ectopic/overexpression of HOP2. Based on the postulated mechanisms of HOP2 action, we speculate on how overexpression leads to these developmental/growth defects.

Agrobacterium Tumefaciens-mediated T-DNA Insertional Mutagenesis of Fusarium Oxysporum

BackgroundFusarium species are important pathogenic organisms, which can cause many diseases in plants and humans. Characterizing the mechanism underlying their pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated genetic transformation has been widely used for the molecular analysis of many species. ResultsIn this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and efficient procedure for the transformation of resistant Fusarium oxysporum mediated by A. tumefaciens. The transformation efficiency was as high as 250 mutants per 104 conidia. A total of 1,450 stably transformed mutants were generated, resulting in a small-scale library of F. oxysporum mutants containing T-DNA tags. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. Additionally, the sequences flanking the inserted T-DNA were obtained by touchdown-TAIL PCR, the insertion sites and genes associated with the phenotypic changes could be determined.ConclusionsThe developed method may enable to analyze gene functions and study biological characteristics, which will lay the foundation for future analyses of the mechanism underlying F. oxysporum pathogenicity and resistance. Furthermore, it may be applicable to investigations of other important pathogenic fungi.

A Metalloaminopeptidase from Flavobacterium breve: Characterization and Molecular Cloning

Aminopeptidase from Flavobacterium breve, was purified by a three step FPLC column chromatography to homogeneity from the culture filtrate. The aminopeptidase gene was cloned by using TAIL-PCR technique. The gene encodes for a polypeptide composed of 497 amino acids with a theoretical molecular weight of 58 kDa. SDS-PAGE detection revealed that the protein is of 52 kDa. The native enzyme showed high affinity to Leu-pNA (km 0.0515 mM), and kcat /km of 88.8 s-1mM-1 . The enzyme had an optimum pH 7.5 and was stable from pH 6 to 9. The purified aminopeptidase was stable up to 60 oC and the optimum temperature for the maximum activity was at 70 oC. The amino acid sequence showed 47% identity to aminopeptidase of Aeromonas caviae (family M14), a Zn2+ dependent metallozyme.

Interlaboratory comparison of fig (Ficus carica L.) microsatellite genotyping data and determination of reference alleles

<p>Microsatellites have been identified as the marker of choice in plant genotyping projects. However, due to length discrepancies obtained between different laboratories for the same allele, interlaboratory comparison of fingerprinting results is often a difficult task. The objectives of this study were to compare genotyping results of two laboratories, to evaluate genetic parameters of microsatellite markers and to determine reference allele sizes for fig cultivars from the Istrian peninsula.</p><p>Genotyping results of ninety fig (<em>Ficus carica</em> L.) accessions were comparable between the laboratories despite differences observed when comparing electropherograms of different capillary electrophoresis systems. Differences in lengths of the same alleles were detected due to different PCR methods and laboratory equipment, but the distances between alleles of the same locus were preserved. However, locus FSYC01 exhibited one allele dropout which led to misidentification of 28 heterozygotes as homozygote individuals suggesting this locus as unreliable. Allele dropout was assigned to the tail PCR technology or to a touchdown PCR protocol.</p><p>Genotypes of twenty-four reference cultivars from the Istrian peninsula were confirmed by both laboratories. These results will contribute to the usage of markers with greater reliability, discrimination power and consequently, to more reliable standardization with other fig genotyping projects.</p>