RESUMEN
We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.
Asunto(s)
Fusión Artificial Génica , Vectores Genéticos/genética , Plantas/genética , Proteínas Recombinantes de Fusión/genética , Transformación Genética , Secuencia de Aminoácidos , Secuencia de Bases , Caulimovirus/genética , Línea Celular , Clonación Molecular/métodos , Fusión Génica , Genes Reporteros , Glucuronidasa/análisis , Glucuronidasa/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Plantas/química , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/análisisRESUMEN
The PHT1 promoter::GUS fusion gene was constructed and introduced into Arabidopsis and rice by Agrobacterium-mediated transformation. Strong beta-glucuronidase (GUS) activity was detected in roots and showed phosphate starvation induction both in Arabidopsis and rice. In contrast, GUS activity in aerial tissues such as those of the leaf and stem was low. In situ GUS staining of root tissue indicated that PHT1 was expressed in root hairs and the outer layer of the main roots, but not in root tips. The PHT1 promoter has a desirable character for biotechnological transgene expression in monocot rice plants.