29. Diversity of granule-bound starch synthase (GBSS) level in Bangladesh rice cultivars
  M.S. JAHAN1, T. KUMAMARU1, A. HAMID2 and H. SATOH1

1) Plant Genetics Laboratrory, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan
2) Department of Agronomy, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1703, Bangladesh

The waxy gene of rice encodes starch granule-bound starch synthase (GBSS, or more commonly, Wx protein with molecular mass of 60 kD) which is responsible for the synthesis of amylose in rice endosperm. Two wild-type alleles, Wxa and Wxb, predominate at the Waxy (Wx) locus of cultivated rice. The expression level of Wxa is 10-fold higher than that of Wxb at the RNA and protein levels. High expression of Wxa results in high amylose content, which makes cooked rice discrete; lower expression of Wxb results in stickier rice. Wxa has a wider geographical distribution than Wxb. The geographical distribution of these two alleles reflects local preference for stickiness. In this article, we report the variations of Wx protein expression levels observed in Bangladesh rice cultivars and the relationship between Wx protein and apparent amylose content.

Five hundred and seventy five Bangladesh rice cultivars were used in the study as well as three rice cultivars Kinmaze, EM21 and IR36 as controls. Crude proteins were extracted from 20 mg rice powder with buffer solution containing 0.125 M Tris, 4% SDS, 5% 2-Mercaptoethanol and 8 M Urea (pH 6.8). Waxy proteins were extracted from 20 mg fine rice powder with 1.2 ml solution A (0.055 M Tris, 2.5% SDS, 5% 2-Mercaptoethanol, pH 6.8) and dissolved in SDSPAGE sample buffer solution containing 8 M Urea. SDS-PAGE was carried out on a gel containing 10% (w/v) acrylamide and 0.035% (w/v) bisacrylamide according to the method of Laemmli (1970). The proteins were transferred electrophoretically to a nitrocellulose membrane (Amersham Pharmacia Biotech). Bands were detected using horseradish peroxidaselabelled goat anti-rabbit IgG (Bio-Rad) and an ECL detection reagent (Amersham Pharmacia Biotech). The apparent amylose content of endosperm starches from brown rice grains was estimated on a single grain basis according to the method of Satoh et al. (1990), with a slight modification.

Kinmaze showed a low level of GBSS (Wxb), while IR36 had a high intensity of GBSS (Wxa). GBSS was absent in EM21 (wx). Three GBSS levels; absent (EM21 type), intermediate (intermediate between Kinmaze and IR36) and high (IR36 type) were observed in Bangladesh rice cultivars tested (Fig. 1). The genotype of intermediate GBSS cultivars was named Wxin. High level of GBSS was found in a maximum number of cultivars, suggesting that nonsticky rices are grown widely in Bangladesh. None of the cultivars had low level (Kinmaze type) of GBSS. Sano et al. (1991) also detected intermediate alleles in rice cultivars from Nepal and India, where no Wxb has been detected. This is the first report on GBSS level variation from Bangladesh rice cultivars. Results indicate a high genetic diversity among these cultivars.

Apparent amylose content of Bangladesh rice cultivars varied from 5% to 30% (Table 1). The rice cultivars tested were divided into five groups based on apparent amylose content; very low (5~10%), low (10~15%), intermediate (15~20%), high (20~25%) and very high

(25~30%). Intermediate and high amylose contents were equally distributed among the cultivars analyzed, whereas few cultivars had very low to low amount of apparent amylose. These results suggest that non-sticky rice cultivars with intermediate to high amylose are preferred in Bangladesh.

The relationship between GBSS levels and apparent amylose content in Bangladesh rice cultivars is shown (Table 2). Cultivars with the same level of GBSS contained different amount of apparent amylose, suggesting differential activity of the GBSS enzyme. Cultivars with no GBSS showed up to 7.22% apparent amylose content, indicating that factors other than GBSS are also involved in the determination of apparent amylose in Bangladesh rice cultivars. The distribution of intermediate GBSS in different cultivars indicate that more than 82% of the cultivars with intermediate GBSS possessed intermediate (15~20%) apparent amylose (Table 2). This observation is in accordance with the finding that intermediate amylose content is simply inherited (Kumar and Khush 1987). Cultivars with high amount of GBSS showed a wide range of apparent amylose, from low to very high, indicating differential Wxa activity in these cultivars. These results also suggest that the inheritance of amylose content is rather complicated in non-waxy rice cultivars, as reported by Sano (1984).

References

Kumar, I. and G. S. Khush, 1987. Genetic analysis of different amylose levels in rice. Crop Sci. 27: 1167-1172.

Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

Sano, Y., 1984. Differential regulation of waxy gene expression in rice endosperm. Theor. Appl. Genet. 68: 467-473.

Sano, Y., H. Y. Hirano and M. Nishimura, 1991. Evolutionary significance of differential regulation at the wx locus of rice. Rice Genetics II: 11-20.

Satoh, H., H. M. Ching'ang'a, D. Ilaila and T. C. Katayama, 1990. On amylose content of cultivated rice collected in Tanzania, 1988. Kagoshima Univ. Res. Center South Paci., Occa. Papers No. 18: 92-100.