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20. Gene analysis of new 57H mutant gene, glup6, in rice
  H.D. TIAN1 , T. KUMAMARU1 , Y. TAKEMOTO1 , M. OGAWA2 and H. SATOH1

1)Laboratory of Plant Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan
2)Department of Life Science, Yamaguchi Prefectural University, Yamaguchi, 753-8502 Japan

Rice storage proteins, prolamin and glutelin, are deposited into the separated protein bodies (PBs). Glutelin synthesized as 57 kD precursor on the ER, is transported via the Golgi body, cleaved into 40 kD alpha and 20 kD beta subunits and accumulated into PB II in the vacuole (Yamagata and Tanaka, 1986). 57H mutants with the high amount of 57 kD polypeptides and the low amount of two glutelin subunits are good materials for studying genetic mechanism controlling glutelin accumulation in rice. So far, 6 genes, esp2, Glup1, glup2, glup3, glup4 (Satoh et al. 1999) and Glup5 (not published) have been identified for 57H mutation. However, identification of new genetic materials is necessary to resolve overall the genetic mechanisms controlling glutelin accumulation. In this study, we identified a new 57H gene in a 57H mutant line, EM939, derived from the MNU treatment of rice cultivar Taichung65 (T65).

SDS-PAGE analysis of total seed proteins in EM939 showed the increased amount of 57 kD polypeptide, the decreased amount of the two glutelin subunits and the decreased amount of 26 kD globulin and 13 kD b prolamin (26L, 13bL) compared with T65 (Fig. 1A). Westernblot analysis revealed stronger reactivity of 57 kD polypeptides in EM939 with anti-glutelin antibody, confirming that the increase of 57 kD polypeptides in EM939 resulted from the high deposition of glutelin precursor (Fig. 1B).

F1 seeds obtained from the cross between EM939 and T65 showed normal type. In the F2 seeds (178 grains), the segregation mode of normal (135 grains) and 57H (43 grains) types fitted well the expected monogenic ratio of 3:1, indicating that 57H mutation of EM939 was controlled by a single recessive gene. Furthermore, we found that 26L and 13bL types co-segregated with 57H type, suggesting that the decrease of 26 kD globulin and 13 kD b


prolamin resulted from the pleiotropic effect of this gene.

Table 1 shows results of SDS-PAGE analysis of F1 and F2 seeds from crosses between EM939 and marker lines with individual registered 57H gene. The F1 seeds from the crosses with the individual marker lines showed normal type (AB) except for the F1 seeds from the crosses with Glup1 / Glup5 mutants which showed 57H types. The F2 seeds from the crosses with each of esp2 glup2 glup3 and glup4 mutants segregated into normal (AB), respective parent (aB, Ab) and double mutant (ab) types; the segregation fitted the expected ratio of 9:3:3:1. The F2 seeds from the cross with Glup1 mutant segregated into normal (Ab), respective parent (ab, AB) and double mutant (aB) types, the segregation fitted the expected ratio of 3:1:9:3. The double mutant types decreased highly the amount of glutelin subunits and increased highly the amount of glutelin precursor compared with parent types. In the F2 seeds from the cross with Glup5 (incomplete dominance) mutant, since the double mutant type could not be distinguished from the parent types by SDS-PAGE profiles, the segregation of normal



(Ab), parents / double mutant, and incomplete 57H (AInB) types fitted the expected ratio of 3:7:6. These indicate that 57H gene in EM939 differs from other six genes. Thus, it was named glup6.

Trisomic analysis of F2 seeds from crosses with Triplo 4, 5, 6, 7, 8, 9, 10, 11 or 12 plants showed that segregation of normal type and glup6 type fitted ratio of 3:1, suggesting that Glup6 was located on chromosome 1, 2 or 3 (Table 2).

Our results indicate that glup6 controlls the high accumulation of glutelin precursor independently of other registered 57H genes, and results in the mutation of decreased 26 kD globulin and 13 kD prolamin pleiotropicly.

References

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.

Yamagata, H. and K. Tanaka, 1986. The site of synthesis and accumulation of storage proteins. Plant Cell Physiol. 27: 135-145.


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