47. Polymorphism of prolamine polypeptides in rice cultivars from Bangladesh, Nepal and Bhutan, a preliminary study

Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fakuoka, 812 Japan

Prolamin or alcohol-soluble proteins of rice consists of three major polypeptide groups with different "apparent molecular masses" (Yamagata and Tanaka 1986), which were each compsed of several components with different isoelectric points (Hibino et al. 1989). Du Cros et al. (1979) demonstrated that differences in electrophoretic patterns of prolamin fractionation by SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and IEF (Isoelectris focusing gel electrophoresis) were useful for variety identification. Satoh et al. (1990a,b) reported that the number and staining intensity of prolamin polypeptide bonds differed greatly among cultivars from Tanzania and Madagascar. On the other hand, Japanese varieties preserved in Kyushu University showed a much narrower range of variation in prolamins. The present investigation was carried out to examine the prlamin polypeptide variation among rice cultivars from Bangladesh, Nepal and Bhutan, an area neighboring the supposed tract of origin of cultivated rice.

A total of 82 rice cultivars collected from Bangladesh, Nepal and Bhutan were studied. Prolamins extracted from one mature seed per variety were analyzed by SDS-PAGE and IEF methods. For SDS-PAGE analysis, proteins were extracted with sample buffer containing 4M urea, 0.125M Tris, 5% 2-mercaptoethanol and 4% SDS, and electrophoresis was carried out by the discontinuous buffer system of Leammli (1970) on a slab gel.

The result showed that prolamins were separable into 10, 13 and 16 kDa polypeptide groups. Kumamaru et al. (1988) pointed out that the 13 kDa group consisted of the 13a and 13b polypeptides exhibiting two bands on the gel. A large variation was observed among the cultivars used in the presence or absence and the stianing intensity of bands 13a and 13b. Furthermore, a new band was found between 13a and 13b, which was designated as 13c. Based on the SDS-PAGE profiles, the cultivars tested were classified into seven types, as shown in Fig. 1.

Generally, the cultivars tested showed a wide range of variation in the 13 kDa polypeptides as compared with Japanese cultivars. Frequencies of the seven types of prolamine found in cultivars from the three countries are given in Table 1. The pattern of distribution differed among the countries. The cultivars from Bangladesh showed the widest range of variation, covering six of the seven types.

For IEF analysis, prolamins extracted with 5% 2-mercaptoethanol and 60% n-propanol were suspended in sample buffer containing 8.5M urea, 2% (v/v) Nonidet P-40 and 5%(v/v) 2-mercaptoethanol. IEF was done by polyacrylamide slab gel electrophoresis. The slab gel contained 4%(w/v) acrylamide, 0.2%(w/

Fig. 1. Seven types of 13 kDa polypeptide.

These types are characterized by relative intensities of the three bands as
follows:

Type  Band 13a      Band 13b      Band 13c
1     high          very low      none
2     moderate      moderate      none
3     high          low           none
4     low           high          none
5     moderate      none          moderate
6     low           low           high
7     high          high          low

Table 1.  Number of cultivars belonging to the seven types of 13 kDa
          polypeptides in Bangladesh, Nepal and Bhutan materials
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Country/Type:          1   2  3   4  5  6  7  Total
_______________________________________________________________________________
Bangladesh             4   1  4   9  1  6        25
Nepal                     11  5  22        1     39
Bhutan                     1  2  13  1  1        18
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A wide range of variations in prolamin was also detected by IEF analysis. The number and staining intensity of bands differed among the cultivars tested. The number of bands varied from 6 to 15. The IEF profiles showed that the cultivars tested can be classified into more than 15 types. The range of variations among the materials tested, particularly that from Bangladesh, was much wider than that of Japanese cultivars. The IEF analysis seems to be quite effective in detecting prolamin variations, although this work is still under way.

References

Du Cros, D. L., C. W. Wrigley and A. B. Blakeney, 1979. Fractionation of rice-grain proteins bv gradient gel electrophoresis and gel isoelectric focusing. Characterization of rice genotypes. Riso 28: 275-284.

Hibino, T., K. Kidzu, T. Masumura, K. Ohtsuki, K. Tanaka, M. Kawabata and S. Fujii, 1989. Amino acid composition of rice prolamin polypeptides. Agric. Biol. Chem. 53(2): 513-518.

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

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

Satoh, H., H. M. Ching'ang'a, D. Ilaila and T. C. Katayama, 1990a. SDS-PAGE analysis of storage proteins of cultivated rice collected in Tanzania, 1988. Kagoshima Univ. Res. Center S. Pac., Occ. Papers. 18: 114-126.

Satoh, H., X. R. Ratokonjanahary and T. C. Katayama, 1990b. SDS-PAGE analysis of storage proteins of cultivated rice collected in Madagascar, 1988. Kagoshima Univ. Res. Center S. Pac., Occ. Papers. 18: 101-113.

Yamagata, H. and K. Tanaka, 1986. The site of synthesis and accumulation of rice storage proteins. Plant Cell Physiol. 27(1): 135-145.