RESUMO
COPII vesicles mediate protein transport from ER to Golgi. Sec13 makes up lattice structure with Sec31 to form COPII vesicles. We analyzed expression of two Arabidopsis thaliana Sec13 homologs, AtSec13A and AtSec13B. AtSec13A was expressed in most parts of seedlings, while AtSec13B was partially expressed. Interaction of AtSec13A or AtSec13B with Sec31 homolog was demonstrated by bimolecular fluorescence complementation (BiFC).
Assuntos
Arabidopsis/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Complexo de Golgi/metabolismo , Proteínas de Plantas/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Células Cultivadas , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/genéticaRESUMO
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.
Assuntos
Fusão Gênica Artificial , Vetores Genéticos/genética , Plantas/genética , Proteínas Recombinantes de Fusão/genética , Transformação Genética , Sequência de Aminoácidos , Sequência de Bases , Caulimovirus/genética , Linhagem Celular , Clonagem Molecular/métodos , Fusão Gênica , Genes Reporter , Glucuronidase/análise , Glucuronidase/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas/química , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/análiseRESUMO
We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP, were employed. Some vectors carry the 2x35S-Omega promoter for higher-level expression. The kanamycin- and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.