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
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).
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
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.