RESUMO
The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.
Assuntos
Oryza/genética , Plantas Geneticamente Modificadas/genética , Agrobacterium/fisiologia , Vetores Genéticos/genética , Oryza/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Transformação Genética/genéticaRESUMO
A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Ácido Glutâmico/metabolismo , Manihot/crescimento & desenvolvimento , Manihot/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Agricultura , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Daucus carota/genética , Perfilação da Expressão Gênica , Glucuronidase/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transformação GenéticaRESUMO
Este estudio fue realizado en los hospitales Docente Maternoinfantil "10 de Octubre" e Infantil "Angel A. Aballi", ambos en Ciudad de La Habana. Se registraron todos los recien nacidos con asfixia severa al nacer en un periodo de 7 anos (80-87) y se siguieron durante los primeros anos de vida. Nuestra investigacion quedo constituida por 461 recien nacidos con asfixia severa al nacer, que fue designado como el grupo A (casos) y 329 que fueron recien nacidos aparentemente sanos y fue designado grupo B (control). Las principales conclusiones a que se llegaron en nuestra investigacion fueron las siguientes : aquellos que presentaron trastornos del lenguaje, en la minoria de ellos, estuvo asociado al cociente de desarrollo bajo y en estos mas de la mitad a un retardo del lenguaje de tipo severo. La gran mayoria de estos ninos que presentaron trastornos del lenguaje ligero y moderado se recuperaron con tratamiento logopedico, en cambio los severos mejoraron, pero no se recuperaron
