The major storage proteins of rice consist of 40 kDa acidic and 20 kDa basic glutelin subunits, 26 kDa globulin polypeptide, and 16 kDa, 14 kDa, 13 kDa and 10 kDa prolamin polypeptides (Tanaka et al. 1980). Seed protein extracted from glup3 mutant lines, which were introduced from North Asian countries and preserved in Kyushu University, contain higher quantities of 57 kDa polypeptides and lower levels of 40 kDa acidic and 20 kDa basic glutelin subunits compared to Taichung 65 (Satoh et al. 1995).

Glutelins are initially synthesized as a 57 kDa precursor and transported to the vacuole where they are proteolytically processed into acidic and basic subunits (Yamagata et al. 1982). The 57 kDa polypeptide accumulated in glup3 mutants is the proglutelin precursor. The glup3 mutation is not a glutelin structural gene but a gene that likely affects the efficient processing of proglutelin precursor to mature glutelin subunits. The proglutelin precursor is accumulated in PBII, with the mature glutelin polypeptide in glup3 mutant (unpublished data). Based on these, the analysis of glup3 mutant, and the isolation and characterization of Glup3 gene may lead to an understanding of the mechanism of glutelin accumulation.

To investigate the key gene of glup3 mutant, we constructed the high-resolution linkage map of Glup3 gene on chromosome 4. A glup3 mutant line, EM856, was used. EM856 was induced from japonica rice variety Taichung 65. Linkage analysis was conducted using 50 homozygous plants for glup3 from F2, a cross between EM856 and indica rice variety

Kasalath. The locus of glup3 was mapped within 5.4 cM region, between RFLP markers R374 (81.9 cM position from short arm end) and R1783 (87.3cM). For high-resolution mapping, about 2,200 glup3 homozygous plants from F2 were selected. The genome DNA was extracted from the seedling of glup3 homozygous plants. Using CAPS markers C377 (74.5cM) and R3261 (87.3cM), 45 recombinants in the side of short arm and 88 in the side of long arm were selected. From the above recombinants, 2 between glup3 and RFLP marker L1091 and 8 between glup3 and R2783 were selected. The results indicate that Glup3 locus is located within 0.5cM region, between L1091 and R2783. The search of BAC clone database revealed that L1091 and R2783 corresponded to BAC clones OSJNBa0091D06 and OSJNBa0011L07, respectively. Both clones overlapped. This shows that Glup3 gene sequence is contained within these BAC clones. To shorten the region containing Glup3 gene, CAPS markers from the sequence of OSJNBa0091D06 were constructed and used for further mapping of Glup3 gene. As a result, we obtained several recombinant plants (Fig. 1). These results showed that Glup3 gene was contained in 95kb, from L1091 to long arm side. The high homologous sequence with vacuolar processing enzyme in castor bean, soybean and Arabidopsis, which is related to maturation of the protein in the vacuole (Hara-Nishimura et al. 1993, Kinoshita et al. 1995, Shimada et al. 1994), was detected. The sequence has been considered as the candidate of Glup3 gene. The complementation test by transformation with Glup3 candidate gene is currently underway.

References

Hara-Nishimura, I., Y. Takeuchi and M. Nishimura, 1993. Molecular characterization of a vacuolar processing enzyme related to a putative proteinase of Schistosoma mansoni. Plant Cell 5: 1651-1659.

Kinoshita, T., M. Nishimura and I. Hara-Nishimura, 1995. Homologous of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana. Plant Mol. Biol. 29: 81-89.

Satoh, H., T. Kumamaru, M. Ogawa, M. Shiraishi, B.G. Im, M.Y. Son and Y. Takemoto, 1995. Spontaneous 57 kDa mutant in rice. RGN 12: 194-196.

Shimada, T., N. Hirai, M. Nishimura and I. Hara-Nishimura, 1994. Vacuolar processing enzyme of soybean that converts proproteins to the corresponding mature forms. Plant Cell Physiol. 35: 713-718.

Tanaka, K., T. Sugimoto, M. Ogawa and Z. Kasai, 1980. Isolation and characterization of two types of protein bodies in rice endosperm. Agric. Biol. Chem. 44: 1633-1639.

Yamagata, H., T. Sugimoto, K. Tanaka, and Z. Kasai, 1982. Biosynthesis of storage proteins in developing rice seeds. Plant Physiol. 70: 1094-1100.