46. Meiosis-specific gene RiLIM15 expressed in rice cultured cells

      J. Shimazu 1, M. Niizeki 1, C. Matsukurs 2, S. Tabata3, R. Ishikawa1, T. Harada 1, M. Senda 4 and

1) Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, 036-8561 Japan

      Genetic variations which have often arisen through cell cultures in many species of plants have become known as somaclonal variation (Larkin and Scowcroft 1981). However the mechanism of somaclonal variation is as yet unclear from many perspectives. In order to clarify the mechanism, we focused on the mitotic homologous recombination (mitotic crossing over) in the cultured cells in vitro.

     A gene, RiLIM15, in rice was isolated from a commercial genomic library of a strain 1R36 by Sato eta!. (personal communication). The gene was highly homologous to a lily meiotic specific gene, LIM1S (Kobayashi et al. 1993). Amino acid sequences predicted from the DNA sequences of the genes of rice and lily were very similar to that of a yeast (Saccharomyces cerevisiae) gene, DMC1, which is specifically expressed for the DNA homologous recombination during meiosis (Bishop eta!. 1992). Therefore, the RiLIM15 is presumed to be an important gene in regards to the meiotic homologous recombination of rice. The expression of RiLJM15 was analyzed in rice cultured cells, young panicles at the stage of meiosis, mature leaves and roots. The strain 1R36 is indica type and the cell culture of this type is not so easy. So we used a japonica type of a strain A58 in this experiment. The reverse-transcription (RT)-PCR revealed that the transcription of RiLIM15 not only occurred specifically in the meiosis of young panicles, but also in the somatic cultured cells. On the other hand, no cDNA of the RiLIM15 was obtained from any mature leaves and roots. DNA sequence analysis of the RiLIM15 cDNAs derived from cultured cells and young panicles indicated that in some of the cDNA clones the specific regions are deleted and these clones can be classified into four types (Fig. 1 and

2). Type 1 cDNA had no deleted regions and was isolated from both cultured cells and young panicles. Type 2 was isolated from only young panicles and was characterized by the deletion of exon 5 of the RiLIM15. Type 3 was isolated from only cultured cells and was characterized by the deletions of exon 5 and 29 bp of exon 8. Type 4 was isolated from only cultured cells and was characterized by the deletions of exon 10 and 11. These results showed that the deleted regions of RiLIM15 cDNA are different between cultured cells and young panicles.

     In order to obtain the genomic clones of RiLIMI1, all regions from exon 1 to exon 14 was amplified by PCR using total DNA of mature leaves of the strain A58. It was found that the obtained clones were classified into two kinds by the determination of DNA sequences. Both clones were the same size of about 3.8 kb. Homology of the sequence of one clone to that of RiLJM15 genomic clone isolated from IR36 was very high. With respect to the intron, the DNA sequence of the other clone was quite different from that of IR36. These results suggested that the RiLIM15 of A58 may be a multi gene family consisting of at least two genes. Furthermore, the deletion of exons found in cDNA of the cultured and meiotic cells may be caused by an alternative splicing because no deleted regions were detected in both genomic clones. At present time, the reason for the different sequences of introns of IR36 and A58 is unclear. for plant improvement. Theor. Appl. Genet. 60: 197-214.