Evaluación del potencial biotecnológico de un promotor derivado del virus de la distorsión de la hoja de maracuyá (PLDV), un begomovirus que infecta maracuyá / Evaluation of the biotechnological potential of a promoter derived from passion fruit leaf distortion virus (PLDV), a begomovirus that infects passionfruit

RESUMEN Los avances biotecnológicos en plantas requieren la bioprospección de nuevos promotores para la expresión de genes de interés agronómico, en particular, es necesario caracterizar nuevos promotores con expresión tejido específica. El objetivo de esta investigación fue evaluar la actividad de expresión del promotor del gen AV1 que codifica para la proteína de la cápside (CP) del virus de la distorsión de la hoja de maracuyá (Passion fruit leaf distortion virus, PLDV) mediante ensayos transitorios de biobalística de baja presión. Se realizó un análisis de la región promotora del gen AV1 empleando herramientas bioinformáticas. Se construyó una fusión traduccional (CP-PLDV-GUS), que porta la región promotora del gen AV1 de PLDV fusionada al gen reportero uidA (GUS). CP-PLDV-GUS fue bombardeado sobre hojas de plántulas de tabaco cultivadas in vitro empleando una pistola de genes. Como control positivo se utilizó el plásmido pBI121 que porta el gen GUS bajo el control del promotor 35S de CaMV. Se llevaron a cabo 11 repeticiones, donde la unidad experimental fue la hoja y la variable de respuesta, la expresión transitoria del gen GUS representado por el número de puntos azules observados en las hojas bombardeadas. Como resultado, el análisis estadístico no paramétrico demostró que existe evidencia muestral suficiente para confirmar que, tanto el promotor AV1 del PLDV y 35S de CaMV presentan una actividad de expresión semejante. Finalmente, el promotor del gen AV1 de PLDV mostró una fuerte actividad de expresión del gen reportero en las células del mesófilo de las hojas, el cual podría ser usado para conferir expresión tejido específica en plantas transgénicas.

ABSTRACT Biotechnological advances in plants require the bioprospecting of new promoters for the gene´s expression of agronomic interest, in particular, it is necessary to characterize new promoters with tissue-specific expression The objective of this research was to evaluate the expression activity of the AV1 gene promoter that codes for the capsid protein (CP) of the Passion fruit leaf distortion virus (PLDV) by means of transient tests of low pressure biobalistics. An analysis of the promoter region was carried out using bioinformatics tools. A CP-PLDV-GUS translational fusion was constructed, which carries the promoter region of the AV1 gene of PLDV fused to the uidA reporter gene (GUS). CP-PLDV-GUS was bombarded on leaves of tobacco seedlings grown in vitro using a gene gun. As a positive control pBI121 carrying the GUS gene under the control of the 35S promoter of CaMV was used. It was carried out 11 repetitions where the experimental unit was the leaf and the response variable the transient expression of the GUS gene represented by number of blue dots observed in the bombarded leaves. As a result, the non-parametric statistical analysis showed that there is sufficient sample evidence to confirm that both the AV1 promoter of PLDV and 35S of CaMV exhibit similar expression activity. Finally, the promoter of the AV1 gene of PLDV showed a strong activity of expression of the reporter gene in the leaf mesophyll cells, which could be used to confer tissue-specific expression in transgenic plants.

Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Background: Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. Results: A −1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis onset and development were fused to the uidA (encoding a ß-glucuronidase, GUS) reporter gene to evaluate its expression in transgenic leaf explants. DNA segments of −1,620 and −1048 bp in length directed uidA expression with patterns in leaf explants similar to those occurring during somatic embryogenesis. When a −792-bp fragment was used, uidA expression disappeared only in leaf explants and pro-embryogenic mass but persisted in developing embryos. No uidA expression was detected in any embryogenic stage when a −618-bp fragment was used. Conclusion: DNA deletions showed that a −1048-bp sequence located upstream of the CcSERK1 gene is sufficient to direct gene expression during the onset and the development of C. canephora somatic embryogenesis. The DNA segment located between −1048 and −792 bp (containing BBM and WUS REs) is needed for gene expression before embryogenesis onset but not during embryo development. The promoter segment between −792 and −618 bp (including GATA, ARR1AT, and ANT REs) regulates gene expression in developing embryos.

Optimization of coffee (Coffea arabica) transformation parameters using uidA and hpt genes: effect of osmotic pre-treatment, helium pressure and target distance / Optimización de los parámetros de transformación del café (Coffea arabica) utilizando los genes uidA y hpt: efecto del pretratamiento osmótico, presión de helio y distancia objetivo

The aim of this work was to optimize the biolistic delivery parameters that affect the DNA delivery and stable expression of marker genes into coffee tissues (Coffea arabica. L. cvs. Caturra and Catuaí). The effect of osmotic preculture length, osmotic concentration of medium, Helium pressure and target distance on transient expression of the uidA gene in coffee leaves and somatic embryos were tested. The highest transient uidA expression was obtained when Caturra (18.3±2.8) and Catuaí (6.8±2.0) leaves and Catuaí embryos (80.0±7.4) were cultured for 5h on Yasuda medium complemented with 0.5M Mannitol +0.5M Sorbitol. The combination of 1100psi and a target distance of 9cm resulted in the highest number of blue spots per Caturra leaf segment (23.6±3.9), whereas for the Catuaí variety the combination of 1100psi and a target distance of six (10.2±1.9) and nine (8.2±1.9) cm gave the highest number of blue spots per leaf segment. The optimized protocol was tested with pCAMBIA 1 301 (uidA gene and the hpt gene), pCAMBIA 1 305.2 (uidA version GUSPlus ™ and the hpt gene) and pCAMBIA 1 301-BAR (uidA gene and the bar gene). The highest number of blue spots was obtained when Caturra (54.6±5.7) and Catuaí (28.9±4.3) leaves were bombarded with pCAMBIA 1 305.2. Selection of bombarded coffee tissues with 100mg/l hygromicyn caused the oxidation of tissues. Rev. Biol. Trop. 57 (Suppl. 1): 151-160. Epub 2009 November 30.

La presente investigación tuvo como objetivo optimizar los parámetros que afectan la incorporación y expresión de genes marcadores mediante biobalística en segmentos de hoja y embriones somáticos de café (Coffea arabica. L. cvs. Caturra y Catuaí). La mayor expresión transitoria del gen uidA en segmentos de hoja de Caturra (18.3±2.8) y Catuaí (6.8±2.0) y embriones somáticos de Catuaí (80.0±7.4) se obtuvo al cultivar los explantes por cinco horas previo al bombardeo en el medio Yasuda complementado con 0.5M mannitol+0.5M sorbitol. Asimismo, se obtuvo una mayor expresión transitoria del gen uidA al bombardear los segmentos de hoja de Caturra y Catuaí y embriones somáticos de Catuaí con una presión de helio de 1 100psi y una distancia de bombardeo de 6 o 9 cm.