47. Molecular cloning of OSDIM cDNA and its linkage analysis in rice
L. TAO1, N. KAMEYA1, Y. FUKUTA2 and I. NAKAMURA1
1) Iwate Biotech. Res. Cent.,22-174-4 Narita, Kitakami,
2) Hokuriku Natl. Agri. Exp. Stn., 1-2-1 Inada, Joh-etsu, 943-21 Japan.
The stature of higher plants is largely dependent upon cell division and elongation and expansion patterns. It is well known that light signals, environmental stresses and phytohormones trigger the progress of cell growth and development. And although cytoskeletons and microtubules such as actins and tubulins are major components of backbone to determine cell shape and play vital roles in many aspects of the cell division cycle, instead of a number of the ensuing works, the molecular mechanisms of cell elongation and expansion are largely unknown. Takahashi et al. (1995) isolated a recessive dwarf mutant, named dimunuto (dim), from T-DNA tagging lines of Arabidopsis thaliana. As the elongation of cells in every organ is greatly reduced in the dim mutant, the gene responsible for dim phenotype will be a good tool to study the molecular mechanisms controlling the elongation of plant cell and dwarfism found in rice and other plants.
We found a partial cDNA clone (SSU009) homologous to Arabidopsis DIM cDNA from saline cDNA library of rice cultured cells made by Umeda et al. (1994). Using SSU009 clone as a probe, thirty positive clones have been isolated after screening of 60,000 recombinants. One of the full length cDNA clones was sequenced and revealed an open reading frame of 1,683 bp encoded 561 amino acid residues and 5' and 3' flanking sequences were 155 bp and 314 bp in length, respectively. The nucleotide sequence of rice OSDIM cDNA was compared with those of Arabidopsis and pea DIM cDNAs (Shimizu and Mori 1996). Although no similarity was observed in the 3' and 5' untranslated regions of the cDNAs, in the coding region, rice OSDIM cDNA showed 74% and 73% homology with Arabidopsis and pea DIM cDNAs, respectively. The deduced amino acid sequence of OSDIM showed high homology with those of the Arabidopsis DIM (80%) and pea DIM (79%) and multiple alignment analysis revealed that they shared homology throughout a whole stretch of these proteins (Fig. 1).
For the linkage analysis of OSDIM gene, a recombinant inbred line from a cross between Milyang 23 and Akihikari was used. Genomic DNAs from 191 plantlets of F5 recombinant inbred line were extracted and digested with restriction enzyme. Linkage analysis of rice OSDIM gene was performed using 165 RFLP markers which were developed by Saito et al. ( 1991 ) and Kurata et al. (1994) in the F5 generation. OSDIM gene was mapped on rice chromosome 10 and showed linkage with RFLP marker loci, G1048, XNpb133, SSK33, XNpb37 and R1877 (Fig. 2). The most nearest DNA marker to OSDIM locus was found to be G1048, and the distance between them is estimated to be 8.2cM.
Dwarfness is a genetic character which is frequently found in rice and many other plant species. Generally dwarf phenotypes are governed by single or a few major genes and numerous studies have been carried out on the genetic identification of genes responsible for dwarfness. Over fifty kinds of dwarf genes were reported in rice and most of them have been already mapped on specific chromosomes. However, none of the dwarf gene has been mapped on chromosome 10 (Kinoshita and Shinbashi 1982). Thus, OSDIM appears to be a dwarf gene which has not been mapped on a rice chromosome as yet.
Kinoshira, T. and N. Shinbashi, 1982. Identification of dwarf genes and their character expression in the isogenic background. Japan. J. Breed. 32: 219-231.
Kurata, N.. Y. Nagamura, K. Yamamoto, K. Harushima, N. Sue, J.Wu. B.A. Antonio, A. shomura, T. Simizu, S.-Y. Lin, T. Inoue, A. Fukuda, T. Shimano, Y. Kuboki, T. Toyama, Y. Miyamoto. T. Krihara. K. Hayasaka, A. Miyao, L. Monna, H.S. Zhong, Y. Tamura, Z.-X. Wang. T. Momma, Y. Umehara, M. Yano. T. Sasaki and Y. Minobe, 1994. A 300 kilobase interval genetic map of rice including 883 expressed sequences. Nature Genet. 8:365-372.
Saito. A.. M. Yano, N. Kishimoto, M. Nakagahra, A. Yoshimura, K. Saito, S.Kuhara, Y. Ukai, M. Kawase, T. Nagamine, S. Yoshimura, O. Ideta, R. Ohsawa, Y. Hayano, N. Iwata and M. Sugiura, 1991. Linkage map of restriction fragment length polymorphism loci in rice. Japan. J. Breed. 41: 665-670.
Shimizu, S. and H. Mori, 1996. AcDNAfrom Pisum sativum encoding the DIMINUTO homologue. Plant Physiol. 112: 862.
Takahashi, T., A. Gasch, N. Nishizawa and N. Chua, 1995. The DIMINUTO gene of Arabidopsis is involved in regulating cell elongation. Gene & Development 9: 97-107.
Umeda. M., C. Hara. Y. Matsubayashi. H-H. Li. Q. Liu, F. Tadokoro, S. Aotsuka and H. Uchimiya. 1994. Ex-pressed sequence tags Irom cultured cells of rice ( oryza sativa L.) under stressed conditions: analysis of transcripts of genes engaged in ATP-generating pathways. Plant Mol.Biol. 25: 469-478.