Molecular and biochemical characterization of extracellular tannin acyl hydrolase activity from a Mexican isolate of Aspergillus niger

"Microbial tannase, a hydrolysable tannin-degrading enzyme, is extensively used in manufacture of instant tea, beer, wine, and gallic acid. Aspergillus niger strain, obtained from a Mexican tannery wastewaters rich in gallic acid [Quebracho Phenolics-rich Tannery Wastewaters, (QPTW)], displayed a good growth and tannase activity in a minimal medium added with 1% (w/v) QPTW (Kr= 0.451 mm.h-1). Using PCR and RACE 3´ and 5´methodologies, a complete cDNA of a tannase was cloned from this isolate.Nucleotide sequence of complete cDNA was of 4690 bp with a complete ORF of 1833 bp encoding 611 amino acids. Transcriptionalinduction was observed in mineral medium added with carbon sources as tannic acid alone (1 and 10 g/l), as well as mix of glucose(1 and 10 g/l) and tannic acid (1 g/l) in the media. However, neither glucose (1 and 10 g/l) and sucrose (1 and 10 g/l) nor (+)-catechin(1 and 10 g/l) as sole carbon sources displayed gene induction in in vitro assays. A. niger-GTO is a new strain with interesting characteristics for industrial tannase production purposes.

EL GEN CRK33 ES REGULADO POR GIBERELINAS DURANTE EL DESARROLLO DEL FRUTO Arabiopsis thaliana (CRK33 GENE IS REGULATED BY GIBERELINES DURING THE DEVELOPMENT FRUIT IN Arabidopsis thaliana)

ResumenEl fruto representa una parte importante en la dieta humana y animal. En Arabidopsis thaliana, el fruto es un excelente modelo de estudio por la forma de desarrollo y los tejidos que lo conforman. Sin embargo, aún es poca la información que se tiene sobre la regulación de su desarrollo, forma y tamaño. Existen un grupo de quinasas tipo receptor (RLK´s) que participan en diversas etapas del desarrollo de la planta, a pesar de que pocas se han caracterizado funcionalmente se sabe que participan en la transducción de señales. Sin embargo, no se ha reportado su función durante el desarrollo del fruto.En el presente proyecto se aborda el estudio del gen CRK33, perteneciente a la familia de genes que codifican para proteínas quinasas de tipo receptor de RLK en plantas. En estudios recientes se ha reportado la participación e importancia de los reguladores de crecimiento vegetal o fitohormonas en el desarrollo del fruto, entre ellas las giberelinas. Hasta el momento, se ha demostrado que el gen CRK33se expresa en las hojas y frutos cuando son tratados con giberelinas exógenas, sugiriendo que éstas últimas lo regulan, no obstante, se están llevando a cabo análisis que confirmen lo antes mencionado. Por otro lado, se analizan los productos de las cruzas de la línea mutante salk-crk33 con líneas marcadoras del desarrollo del fruto a fin de encontrar cambios en el patrón de expresión. Palabras Clave:Fruto, gen CRK33, giberelinas. AbstractThe fruit represents an important part in the human and animal diet. In Arabidopsis thaliana, the fruit is an excellent study model because of the way of development and the tissues that make it up, however, there is little information about the regulation and induction of it. There is a group of receptor-type kinases (RLK´s) involved in various stages of plant development, although few have been functionally characterized as being involved in signal transduction. However, their function has not been reported during fruit development.The present project addresses the study of the CRK33 gene, belonging to the family of genes coding for protein kinases of receptor type RLK in plants.Recent studies have reported the participation and importance of plant growth regulators or phytohormones in the development of the fruit, including gibberellins.So far, it has been demonstrated that the CRK33 gene is expressed in leaves and fruits when treated with exogenous gibberellins, suggesting that the latter regulates it, however, analyzes are being carried out confirming the aforementioned. On the other hand, the products of the crosses of the salk-crk33 mutant line with fruit development marker lines are analyzed in order to find changes in the expression pattern.Keywords:Fruit, CRK33 gene, gibberellins

MECANISMOS DE SILENCIAMIENTO GÉNICO DURANTE EL DESARROLLO DEL GAMETOFITO FEMENINO EN Arabidopsis thaliana (MECHANISMS OF GENE SILENCING DURING THE DEVELOPMENT OF THE FEMALE GAMETOPHYTE IN Arabidopsis thaliana)

ResumenSilenciar un gen significa disminuir la expresión del mismo y a partir de los efectos, afectar su función, actuando a diferentes niveles, por ejemplo, silenciamiento transcripcional (TGS), silenciamiento post-transcripcional (PTGS) y traduccional. Los cuales, presentan elementos comunes mediados por ARNs pequeños (RNAp), que proporcionan especificidad y se acomplejan con proteínas efectoras del silenciamiento génico. Recientemente se reportó la participación de ARNs pequeños  en la determinación de la identidad de la célula madre de la megaspora. En este trabajo se plantea una estrategia para estudiar las rutas de silenciamiento que participan en la regulación de la identidad celular durante el desarrollo del gametofito femenino. Para lo anterior se generaron líneas silenciadoras del gen GUS (GUSRNAi). Posteriormente se seleccionaron algunas de estas líneas para ser cruzadas con los marcadores del desarrollo del gemetofito femenino. Se obtuvieron imágenes que reflejaron la actividad de las líneas marcadoras durante el desarrollo reproductivo. En las plantas de la línea GUSRNAi;Marcadora se observó una expresión atenuada o nula del marcador de identidad. Lo anterior demostró la capacidad de la lineas para llevar a cabo silenciamiento génico en células del gametofino femenino.Palabras clave: Gametogénesis, linaje celular, ARNs. AbstracSilencing a gene means decreasing its expression affecting its function, acting at different levels, for example transcriptional silencing (TGS), post-transcriptional silencing (PTGS) and translational silencing. They present common elements mediated by small RNAs (RNAs), which provide specificity and form complexes with gene silencing effector proteins. The participation of small RNAs in the determination of the identity of the megaspore mother cell was recently reported. In this work, a strategy is proposed to study the gene silencing pathways involved in the regulation of cell identity during the development of the female gametophyte. For this, we generated silencing lines of the GUS gene (GUSRNAi). Subsequently, some of these lines were selected to be crossed with the developmental marker of the female gametophyte, ET2209. Images that reflected the activity of the marker lines during reproductive development were obtained. While in plants GUSRNAi;ET2209 we observed an attenuated or null expression of the identity cell marker. The above demonstrated the ability of these lines to carry out gene silencing in female gametophyte cells.Keywords: Gametogenesis, cell lineage, RNAs

Transcriptomic Analysis in Diabetic Nephropathy of Streptozotocin-Induced Diabetic Rats

Diabetic nephropathy (DN) is a major complication of diabetes and is caused by an imbalance in the expression of certain genes that activate or inhibit vital cellular functions of kidney. Despite several recent advances, the pathogenesis of DN remains far from clear, suggesting the need to carry out studies identifying molecular aspects, such as gene expression, that could play a key role in the development of DN. There are several techniques to analyze transcriptome in living organisms. In this study, the suppression subtractive hybridization (SSH) method was used to generate up- and down-regulated subtracted cDNA libraries in the kidney of streptozotocin (STZ)-induced diabetic rats. Northern-blot analysis was used to confirm differential expression ratios from the obtained SSH clones to identify genes related to DN. 400 unique SSH clones were randomly chosen from the two subtraction libraries (200 of each) and verified as differentially expressed. According to blast screening and functional annotation, 20.2% and 20.9% of genes were related to metabolism proteins, 9% and 3.6% to transporters and channels, 16% and 6.3% to transcription factors, 19% and 17.2% to hypothetical proteins, and finally 24.1 and 17.2% to unknown genes, from the down- and up-regulated libraries, respectively. The down- and up-regulated cDNA libraries differentially expressed in the kidney of STZ diabetic rats have been successfully constructed and some identified genes could be highly important in DN

Silencing of a Germin-Like Protein Gene (CchGLP) in Geminivirus-Resistant Pepper (Capsicum chinense Jacq.) BG-3821 Increases Susceptibility to Single and Mixed Infections by Geminiviruses PHYVV and PepGMV

Germin-like proteins (GLPs) are encoded by a family of genes found in all plants, and in terms of function, the GLPs are implicated in the response of plants to biotic and abiotic stresses. CchGLP is a gene encoding a GLP identified in a geminivirus-resistant Capsicum chinense Jacq accession named BG-3821, and it is important in geminivirus resistance when transferred to susceptible tobacco in transgenic experiments. To characterize the role of this GLP in geminivirus resistance in the original accession from which this gene was identified, this work aimed at demonstrating the possible role of CchGLP in resistance to geminiviruses in Capsicum chinense Jacq. BG-3821. Virus-induced gene silencing studies using a geminiviral vector based in PHYVV component A, displaying that silencing of CchGLP in accession BG-3821, increased susceptibility to geminivirus single and mixed infections. These results suggested that CchGLP is an important factor for geminivirus resistance in C. chinense BG-3821 accession

Effect of Sub-inhibitory Amounts of Nisin and Mineral Salts on Nisin Production by Lactococcus lactis UQ2 in Skim Milk

The effect of nisin regulatory system of the quorum-sensing mechanism and mineral salts on the production of nisin A by the native strain Lactococcus lactis UQ2 growing in skim milk was evaluated using a static culture. A 6 × 3 full factorial design with two replicates was conducted, aiming to study nisin production during growth of L. lactis UQ2 in skim milk as model food. At appropriate time intervals, the produced nisin, microbial population, and medium pH were measured. Sub-inhibitory amounts of commercial nisin (IN; 0, 0.05, 0.65, 1.25, 1.87, and 2.5 μg/L) were added as inducer to skim milk. A mixture of Mg/Mn (MS; 0, 0.5/0.1, and 0.2/0.04 g/L) was also added. These two factors (IN, MS) and their interactions were highly significant for nisin production by L. lactis UQ2. The highest nisin production (75 ± 7 IU/mL) was achieved at 10 h of incubation, for treatment containing 1.87 μg/L of IN and MS 0.5/0.1 g/L, while only 3.5 ± 0.5 IU/mL were produced by control cultures at 6 h. In contrast with other reports, nisin production started at mid-log phase, and the maximum activity was observed well beyond the beginning of the stationary phase (6 h). This was attributed to the effect of IN. Semi-quantification of thhttps://digital.csic.es/listadoMetadatos.jsp?ID=autores&vocabulary=autores&plataforma=pasarelae structural nisin gene nisA by reverse transcriptase-polymerase chain reaction indicated that it was expressed 2.2 times more than the control treatment. L. lactis UQ2 is different from most strains of this genus, because of its poor lactose consumption and lactic acid production when growing in skim milk. Given the capacity of nisin production and the well-known antimicrobial properties of this bacteriocin, this strain may be useful to enhance the safety of low acidity dairy products such as Mexican-style fresh cheese. © 2009 Springer Science + Business Media, LLC.Peer Reviewe

<b>Molecular and biochemical</b><b> characterization of extracellular tannin acyl hydrolase activity from a Mexican isolate of <i style="">Aspergillus niger</i></b>

942-947Microbial tannase, a hydrolysable tannin-degrading enzyme, is extensively used in manufacture of instant tea, beer, wine, and gallic acid. Aspergillus niger strain, obtained from a Mexican tannery wastewaters rich in gallic acid [Quebracho Phenolics-rich Tannery Wastewaters, (QPTW)], displayed a good growth and tannase activity in a minimal medium added with 1% (w/v) QPTW (Kr= 0.451 mm.h-1). Using PCR and RACE 3´ and 5´methodologies, a complete cDNA of a tannase was cloned from this isolate.Nucleotide sequence of complete cDNA was of 4690 bp with a complete ORF of 1833 bp encoding 611 amino acids. Transcriptionalinduction was observed in mineral medium added with carbon sources as tannic acid alone (1 and 10 g/l), as well as mix of glucose(1 and 10 g/l) and tannic acid (1 g/l) in the media. However, neither glucose (1 and 10 g/l) and sucrose (1 and 10 g/l) nor (+)-catechin(1 and 10 g/l) as sole carbon sources displayed gene induction in in vitro assays. A. niger-GTO is a new strain with interesting characteristics for industrial tannase production purposes

Diversidad genética de aislados de "Rhizoctonia solani" (Kuhn) de chile en México

One of the major constraints for the production of pepper are pathogenic fungi causing diseases known as "pepper blight" or "damping off". This disease can be devastating when weather conditions are favorable for the pathogen. Although different means of control (chemical and cultural) have benn used but none has been successful. An alternative to control is to produce resistant germplasm, however in order to establish an effective breeding program is necessary to know the distribution and genetic diversity of the pathogens involved, particularly Rhizoctonia solani, which by its ubiquity represents a potential danger in all producing areas. Thus, the objective was to characterize R. solani in North Central area from Mexico and determine its genetic diversity. To achieve with this goal are considered the states of Chihuahua, Durango, Zacatecas, San Luis Potosi, Colima, Queretaro and Guanajuato where in 2009 were collected adult plants of pepper with pepper blight symptoms, the fungus was isolated and found an incidence of 33%, finding it in both stem and root. Mycelial cells were multinucleated, a characteristic from pathogenic strains. The anastomosis testing showed that in Mexico are present the groups GA4, GA-2. 1, GA-IIB, GA-2IV, GA7, GA11, GA12 and GA13. The genetic diversity of this fungus was very high, so that the relationships demonstrated by the construction of dendrogram show no homogeneous trends so as the main groups formed contain elements of all statesUna de las principales limitantes para la producción de chile son los hongos patógenos causantes de la enfermedad conocida como "marchitez del chile" o "secadera". Esta enfermedad puede ser devastadora cuando las condiciones climáticas son favorables para el patógeno. A pesar de que se han intentado diferentes medios de control (químicos y culturales) ninguno ha tenido éxito. Una alternativa para su control es producir germoplasma resistente, sin embargo para poder establecer un programa de mejoramiento efectivo es necesario conocer la distribución y diversidad genética de los patógenos involucrados, particularmente de Rhizoctonia solani, que por su ubicuidad representa un peligro potencial en todas las zonas productoras. Por ello el objetivo fue caracterizar a R. solani en las zona Centro Norte de México y determinar su diversidad genética. Para cumplir con este objetivo se consideraron los estados de Chihuahua, Durango, Zacatecas, San Luis Potosí, Colima, Querétaro y Guanajuato donde en 2009 se colectaron plantas adultas de Chile con síntomas de marchitez, se aisló al hongo y se encontró una incidencia del 33%, encontrándose tanto en tallo como en raíz. Las células miceliales fueron multinucleadas, características de las cepas patogénicas. Las pruebas de anastomosis demostaron la presencia en México de los grupos GA4, GA-2.1, GA-IIB, GA-2IV, GA7, GA11, GA12 y GA13. La diversidad genética de este hongo fue muy alta, de tal manera que las relaciones demostradas por la construcción de dendrogramas no muestran tendencias homogéneas pues los principales grupos formados contienen elementos de todos los estado

Selección de genotipos de chile resistentes al complejo patogénico de la marchitez

In Mexico the most important root disease of the chili pepper crop is the wilt disease, it is primarily controlled with fumigants and fungicides that help to select resistant isolates and cause environment and health damage.A safe environmental option could be the resistant varieties cultivation, but there are few disease resistance varieties. This study's aim was to isolate the pathogens associated with chili pepper wilt disease in central and north of Mexico and to identify chili pepper resistant genotypes. During 2006 and 2007, chili pepper plants with wilt disease symptoms were collected in 118 lots from Chihuahua, Colima, Durango, Guanajuato, Querétaro, San Luis Potosí and Zacatecas, from which pathogens were isolated and pure cultures were obtained. They were individually or in mixtures inoculated to select resistant germplasm, in 44 chili pepper accessions of INIFAP's germplasm bank and 141 collections from Durango, Guanajuato, Michoacán, San Luis Potosí and Zacatecas. Fusarium spp., was isolated with a 42.6% frequency, Rhizoctonia solani 37% and Phytophthora capsici 3.9%. 26 collections were identified with at least one of them resistant to Fusarium spp., and six to R. solani. Only BG102 and BG107 accessions from the gene bank wereresistant to P. capsici and to the group of three pathogens. These are potential materials to be used in chili pepper genetic improvement.En México la enfermedad de raíz más importante del cultivo de chile es la marchitez, esta se controla principalmente con fumigantes y fungicidas que contribuyen a seleccionar aislados resistentes, y provocan daños al ambiente y a la salud. Una opción inocua con el ambiente podría ser el cultivo de variedades resistentes; sin embargo, hay pocas variedades con resistencia a esta enfermedad. El objetivo del presente trabajo fue aislar los patógenos asociados a la marchitez del chile, en la región centro y norte de México e identificar genotipos de chile resistentes. Durante 2006 y 2007, se colectaron plantas de chile con síntomas de marchitez en 118 lotes de los estados de Chihuahua, Colima, Durango, Guanajuato, Querétaro, San Luis Potosí y Zacatecas, a partir de las cuales se aislaron los patógenos y se obtuvieron cultivos puros. Estos se inocularon individualmente o en mezcla para seleccionar germoplasma resistente en 44 accesiones de chile del banco de germoplasma del INIFAP y 141 colectas procedentes de Durango, Guanajuato, Michoacán, San Luis Potosí y Zacatecas. Fusarium spp. fue aislado con una frecuencia de 42.6%, Rhizoctonia solani 37%, y Phytophthora capsici 3.9%. Se identificaron 26 colectas con al menos un individuo resistente a Fusarium spp., y seis a R. solani. Sólo las accesiones BG102 y BG107 del banco de germoplasma fueron resistentes a P. capsici y a la mezcla de los tres patógenos. Estos materiales tienen potencial para usarse en programas de mejoramiento genético del chile