ABSTRACT
A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to â¼30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.
Subject(s)
Fabaceae/genetics , Plant Proteins/genetics , Abscisic Acid/pharmacology , Amino Acid Sequence , Base Sequence , Chromatography, Gel , DNA, Complementary/genetics , DNA, Plant/metabolism , Desiccation , Electrophoretic Mobility Shift Assay , Fabaceae/drug effects , Fabaceae/metabolism , Fabaceae/ultrastructure , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Germination/drug effects , Germination/genetics , Molecular Sequence Data , Molecular Weight , Organ Specificity/drug effects , Organ Specificity/genetics , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Binding/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seeds/drug effects , Seeds/metabolism , Seeds/ultrastructure , Sodium Chloride/pharmacology , Stress, Physiological/drug effects , Stress, Physiological/geneticsABSTRACT
Although mungbean (Vigna radiata (L.) Wilczek) is commonly used as human food; the genomic resources of this species available in databases are limited. This study aims to develop expressed sequence tag (EST) resources for mungbean genes informative to early seedling development and chilling response. Two mungbean varieties that differ in disease resistance were found to also differ in their susceptibility to chilling temperatures. A total of 1,198 ESTs were obtained from one cDNA library and four PCR-select cDNA subtraction libraries; among these 523 were clustered into 136 contigs and 675 were singletons. The 811 non-redundant uniESTs were compared to GenBank using the Basic Local Alignment Search Tool (BLAST) and WU-BLAST algorithms, of these only 489 uniESTs had significant sequence homology, which may be involved in resuming the metabolic activity of seedlings, switching on photomorphogenesis, fuelling photosynthesis and/or initiating the unique developmental programs. Their encoded proteins may associate with regulatory proteins to trigger a direct stress response or participate in acclimation to environmental stressors. The uniEST platform reported will enrich the genomic resources of mungbean for functional genomic research on seedling development and chilling response of tropical crops and provide targets for improving the chilling tolerance of the tropical crops.
