We investigated molecular basis of the RFLPs between normal and deletion lines of Chinese Spring wheat that were found during deletion mapping of a cytochrome P450 gene (TaBx3) to sub-arm regions of chromosome 5A. We identified a TAC clone carrying the TaBx3-A1 allele and designed PCR primers to specifically amplify sequences surrounding the TaBx3-A1 allele. Comparing the nucleotide sequences, we revealed that the RFLP detected in Dral digestion was caused by a nucleotide substitution, and that nucleotide substitutions were widespread throughout the 58 kb interval. Sequence comparisons of the TaBx3 genes ruled out the possibilities of homoeologous recombination between group-5 chromosomes and introgression from Aegilops cylindrica whose gametocidal chromosome 2C was used for the production of the deletion lines.
P-9 K. Kawaura1, K. Mochida2, Y. Yamazaki3,
Y. Ogihara1 (1Kyoto Prefectural University 2Nagahama
Institute of Bio-Science and Technology 3National Institute of Genetics)
Global analysis of gene expression patterns in response to salt stress using
wheat 22k cDNA oligo-microarray
For investigating the transcriptome of hexaploid wheat, we have designed cDNA oligo-microarray including 21,939 genes. From 148,676 ESTs, 34,064 contigs were grouped by the phrap method. The sense strand of each contig was predicted and the 60 mers of probes were designed (Agilent Co Ltd). ThenEQ we conducted pilot experiments to validate by using common wheat treated with salt. Every experiment revealed that most probes were detectable and reproducible for signal intensities. We selected the 1,811 genes whose expressions were two-fold changed by salt-treatment. These genes were classified according to the hierarchical clustering method and functions of these groups were inferred with the gene ontology. By applying these tools, functions of wheat genes, if not all, might be possibly estimated.
P-10 M. Yamamoto1, N. Fujiwara2, G.
Suzuki2, Y. Mukai2 (1Department of Nutritional
Sciences for Well-being, Faculty of Health Sciences for Welfare, Kansai University
of Welfare Sciences, 2Laboratory of Plant Molecular Genetics, Division
of Natural Science, Osaka Kyoiku University)
Physical mapping of LMW glutenin genes in wheat by FISH
In order to analyze the over-all organization of LMW glutenin gene region, we visualized the overlapping region among clones and distribution of the genes by fluorescence in situ hybridization (FISH). We used three BAC clones, I3, D8, and D9, with the large DNA fragments containing the LMW glutenin gene of Aegilops squarrosa (2n=14, DD). FISH data showed that the length of the overlapping region between I3 and D8 was 10.5 kb, and 5.4 kb between D8 and D9. But, no overlapping region between I3 and D9 was observed. Three clones were located in order of I3, D8, and D9, spanning 279.1 kb in the LMW glutenin gene region. The FISH experiment using subclones of the gene as probes, visualized that I3, D8, and D9 clones include two, four, and four loci, respectively.
P-11 C. Shimamura, R. Ohno, C. Nakamura, S. Takumi (Faculty
of Agriculture, Kobe University)
Improvement of freezing tolerance in transgenic tobacco with wheat cold-responEQsive
genes
A wheat cold-responsive gene Wcor15 encodes a cbloroplast-targeted COR protein. Three cold-responsive elements (CRTs) were found in the 5' upstream region of Wcor15. The CRT motifs are putatively recognized by a transcription factor WCBF2 in wheat cells. To investigate functions of the Wcor15 and Wcbf2 genes under low temperature conditions, we produced transgenic tobacco plants with each of the genes under control either of the CaMV35S promoter or the 5' upstream region of Wcor15. Transgenic plants overexpressing the introduced chimeric genes dramatically increased freezing tolerance, clearly indicating their positive contribution in the development of freezing tolerance in plant cells. The WCOR15 protein fused to GFP was targeted into the stroma of tobacco chloroplasts.