Prolamins are accumulated in the protein body (PB) I derived from endoplasmic reticulum (ER), while glutelins are accumulated in the PBII derived from vacuole.

Genetic analysis of glup (glutelin precursor) mutants have been carried out (Kumamaru et al. 1988, Satoh et al. 1994,1999, Tian et al. 2001) isolated from MNU induced rice mutant. Satoh et al. (1999) decided the location site on rice genome as demonstrated that Glup1, glup2, glup3 and glup4 genes were located on Chr.s 9, 9, 4 and 12, respectively. These glup mutants except for glup3 are characterized by the presence of mutant type PB derived from ER, with high amount of 57kDa glutelins precursor polypeptides. In Glup1 and glup2, ER derived prolamin containing PB with various sizes are observed under TEM. Immuno-gold labeling observation demonstrated that glutelin precursor polypeptides co-localized with prolamin polypeptide in the unique ER-PB in both mutants. However key genes of Glup1 and glup2 mutants have been unknown. Although it has been clear that esp2 mutant with greatly accumulated glutelin precursor was deficient in the endosperm specific protein disulfide isomerase, and resulted in default of PB I formation (Takemoto 2002).

Satoh et al. (1999) reported that glup4 is independent of esp2, Glup1, glup2 and glup3, and glup4 shows the additive effect with these four genes. Immuno-gold labeling observation of glup4 mutant demonstrated that glutelin precursor polypeptides co-loccalized with prolamin polypeptide in the mutant type PB (data not shown). This result suggested that function of key gene in glup4 mutation participates in the sorting out of glutelin precursor and prolamins within ER lumen as well as Glup1 and glup2 genes.

In this study, to identify and isolate the key gene involved in glup4 mutation, we constructed the linkage map of Glup4 gene on chromosome 12. A glup4 mutant line, EM956, induced from a japonica rice variety Taichung65, was crossed with an indica rice variety Kasalath. F₁ plants were grown and self-pollinated. F₂ seeds derived from the cross between them were cut into half. Total proteins extracted from half cutting seeds without

embryo were subjected to SDS-PAGE and homozygous glup4 seeds showing the enriched 57 kD polypeptide were selected. glup4 homozygous plants from embryo half seed were cultivated and the genome DNA was extracted from the seedling of them. Linkage analysis using 58 glup4 homozygous F₂ plants showed that the glup4 gene was mapped within 5.9 cM region between PCR markers, L714 and C901 on Chromosome 12 (Fig. 1).

This study was partly supported by Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.

References

Kumamaru T., H. Satoh, N. Iwata, T. Omura, M. Ogawa and K. Tanaka, 1988. Mutants for rice storage proteins 1. Screening of mutants for rice storage proteins of protein bodies in the starchy endosperm. Theor. Appl. Genet. 76: 11-16.

Satoh, H., W.X. Li, Y. Takemoto, T. Ito, T. Kumamaru, L. Q. Qu and M. Ogawa, 1999. glup 4 gene controlling a 57H character was located on chromosome 12 in rice. RGN 16: 98-100.

Satoh, H., T. Kumamaru, S.Yoshimura, M. Ogawa, 1994. New 57 kDa glutelin genes on chromosome 9 in rice. RGN 11: 158-161.

Takemoto, Y., Sean J. Coughlan, T. W. Okita, H. Satoh, M. Ogawa, and T Kumamaru, 2002. The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase. Plant Cell Physiol. 128: 1212-1222.

Tian H.D., T. Kumamaru, Y. Takemoto, M. Ogawa and H. Satoh, , 2001. Gene analysis of new 57H mutant gene, glup6, in rice. RGN 18: 48-50.