RESUMO
The biotechnological approach to improve performance or yield of crops or for engineering metabolic pathways requires the expression of a number of transgenes, each with a specific promoter to avoid induction of silencing mechanisms. In maize (Zea mays), used as a model for cereals, an efficient Agrobacterium tumefaciens-mediated transformation system has been established that is applied for translational research. In the current transformation vectors, the promoters of the 35S gene of the cauliflower mosaic virus and of the ubiquitin gene of maize are often used to drive the bialaphos-selectable marker and the transgene, respectively. To expand the number of promoters, genes with either constitutive or seed-specific expression were selected in Brachypodium distachyon, a model grass distantly related to maize. After the corresponding Brachypodium promoters had been fused to the ß-glucuronidase reporter gene, their activity was followed throughout maize development and quantified in a fluorimetric assay with the 4-methylumbelliferyl ß-D-glucuronide substrate. The promoters pBdEF1α and pBdUBI10 were constitutively and highly active in maize, whereas pBdGLU1 was clearly endosperm-specific, hence, expanding the toolbox for transgene analysis in maize. The data indicate that Brachypodium is an excellent resource for promoters for transgenic research in heterologous cereal species.
Assuntos
Brachypodium/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Ubiquitina/genética , Zea mays/genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ubiquitina/metabolismo , Zea mays/metabolismoRESUMO
The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/enzimologia , Pirofosfatases/metabolismo , Sementes/enzimologia , Zea mays/enzimologia , Adenosina Difosfato Ribose/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Secas , NAD/metabolismo , Estresse Oxidativo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Pirofosfatases/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Estresse Fisiológico , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
In higher plants, genetic transformation, which is part of the toolbox for the study of living organisms, had been reported only 30 years ago, boosting basic plant biology research, generating superior crops, and leading to the new discipline of plant biotechnology. Here, we review its principles and the corresponding molecular tools. In vitro regeneration, through somatic embryogenesis or organogenesis, is discussed because they are prerequisites for the subsequent Agrobacterium tumefaciens-mediated transferred (T)-DNA or direct DNA transfer methods to produce transgenic plants. Important molecular components of the T-DNA are examined, such as selectable marker genes that allow the selection of transformed cells in tissue cultures and are used to follow the gene of interest in the next generations, and reporter genes that have been developed to visualize promoter activities, protein localizations, and protein-protein interactions. Genes of interest are assembled with promoters and termination signals in Escherichia coli by means of GATEWAY-derived binary vectors that represent the current versatile cloning tools. Finally, future promising developments in transgene technology are considered.