Project description:Hairy vetch (Vicia villosa Roth) is recognized as a beneficial winter cover crop in the Midwestern U.S. DNA microarrays are used for assessing gene expression significance. The objective of the study was to identify a set of genes expressed in hairy vetch, that could be further analyzed for their potential in improving the crop. The RNA of four targets (soybean (Glycine max), hairy vetch, Vicia pannonica PI 170008, and Vicia pannonica PI 515988) were purified, labeled, and hybridized to 142 cDNA clones of biotic stress genes and gene sequences of soybean that were robotically spotted onto aminosilicated slides with duplicate spots and three arrays per slide. The microarray experiments were completed in a reference design experiment incorporating a two-dye system. The data were analyzed using the individual fluorescence intensities to fit two statistical models in a mixed model analysis of variance. In this analysis, systematic error effects were observed to account for 24% of the total variation. The use of a Bonferroni adjusted significance threshold allowed for adequate control over the number of falsely identified significant genes showing expression in the different target comparisons. We observed that 64 of the 142 gene sequences (45%) were differentially expressed in at least one of the target comparisons. Two of these expressed genes in hairy vetch encoded for a cold tolerance indicator, proline, and two other gene sequences encode for stress tolerance indicators, myo-inositol and calmodulin. A soybean cDNA microarray was used effectively to differentiate gene expression in hairy vetch. Keywords: comparative genomic hybridization (cross-species)
Project description:Nitrogen application to legume seeds regulates seed metabolism and composition. In order to improve nitrogen flux into the embryo, the Vicia faba amino acid permease VfAAP1 (Miranda et al. Amino acid permeases in developing seeds of Vicia faba L.: expression precedes storage protein synthesis and is regulated by amino acid supply. Plant J 2001 28: 61-72) was expressed in pea under control of the seed-specific LeB4 promotor (Bäumlein et al. Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within legumin box is essential for tissue-specific expression of a legumin gene. Plant J 1992 2: 233-239). Several transgenic lines have been generated. Mature seeds of the homozygous marker gene-free line AAP14/10 showed an increase in amino acid supply, seed nitrogen and protein content due to higher globulin fraction. The effect of VfAAP1 was tested under field conditions in two growing periods, 2005 and 2006, and the data could be confirmed. Over-expression of VfAAP1 interferes with storage protein metabolism and alters fluxes of nitrogen during seed growth. The influence of VfAAP1 on altered gene expression in developing cotyledons was analysed using a 6k-Oligo-microarray. Four developmental stages (18, 22, 26 and 30 DAP) from seeds, grown 2006, of the transgenic line AAP14/10 were hybridized against wildtype control.