ABSTRACT
The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual-colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD-banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species.
Subject(s)
Chromosomes, Plant/genetics , Fabaceae/genetics , Genome, Plant/genetics , Base Sequence , DNA, Plant/chemistry , DNA, Plant/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Fabaceae/cytology , In Situ Hybridization, Fluorescence , Karyotype , Mitosis/genetics , Molecular Sequence Data , Phylogeny , Plant Leaves/cytology , Plant Leaves/genetics , Sequence Analysis, DNA , Species SpecificityABSTRACT
Complete coding DNA sequences of a closely related chlorogenic acid synthetase gene (LjCCoAOMT1) were isolated from Lonicera japonica Thunb. by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). LjCCoAOMT1 was subsequently overexpressed in Escherichia coli and a 25-kD protein was detected by electrophoresis and western blot analysis. High-performance liquid chromatography (HPLC) analysis showed that recombinant LjCCoAOMT1 methylates the caffeic acid substrate to generate ferulic acid. Further analysis showed that the chlorogenic acid content was significantly correlated with the expression level of LjCCoAOMT1 in various tissues of L. japonica Thunb. at different developmental stages. A plant expression vector containing LjCCoAOMT1 was constructed and Agrobacterium-mediated transgenic rice was successfully obtained. Light treatment analysis showed that LjCCoAOMT1 transgenic rice was more sensitive than wild-type rice in responding to the changes in lighting conditions. Although gibberellic acid (GA3) could promote the growth of both wild-type and LjCCoAOMT1 transgenic rice, LjCCoAOMT1 transgenic rice appeared to be more sensitive to GA3. Furthermore, high concentrations of GA3 significantly facilitated the growth of LjCCoAOMT1 transgenic rice.
Subject(s)
Acyltransferases/genetics , Cloning, Molecular , Gene Expression , Genes, Plant , Lonicera/genetics , Oryza/genetics , Acyltransferases/metabolism , DNA, Complementary/genetics , Flowers/genetics , Flowers/metabolism , Gibberellins/metabolism , Light , Oryza/metabolism , Oryza/radiation effects , Plants, Genetically ModifiedABSTRACT
Mitotic chromosome spreads of 16 plant species belonging to six families were analyzed using an improved combined PI and DAPI (CPD) staining procedure. Fluorescence in situ hybridization (FISH) with 45S rDNA probe was conducted sequentially on the same spreads to evaluate the efficiency and sensitivity of the technique. Fluorochrome staining with chromomycin A3 (CMA)-DAPI also was conducted to clarify the properties of the sequences involved in the CPD banded regions. Our results revealed that all of the NORs (rDNA sites) in the species tested were efficiently shown as red bands by CPD staining, and the number and position of the bands corresponded precisely to those of the 45S rDNA FISH signals, indicating that the detection sensitivity of CPD staining is similar to that of FISH. In 10 of the species tested including Aegilops squarrosa, Allium sativum, Oryza sativum ssp. indica, Oryza officinalis, Pisum sativum, Secale cereale, Setaria italica, Sorghum vulgare, Vicia faba and Zea mays, CPD bands were exhibited exclusively in their NORs, while in other six species including Hordeum vulgare, Allium cepa, Psophocarpus tetragonolobus, Arabidopsis thaliana, Brassica oleracea var. capitata and Lycopersicon esculentum, CPD bands appeared in chromosomal regions other than their NORs. The CPD bands were in accordance with the CMA bands in all species tested, indicating GC-rich sequences in the CPD bands and that the improved CPD staining procedure is specific for GC-rich regions in plant genomes. Our investigation not only elucidated the banding mechanisms of CPD, but also demonstrated that the CPD staining technique, which may be preferable to CMA staining, is an effective tool for detecting NORs and other GC-rich chromosomal regions in plants.
