3. Studies on subspecies differentiating protein markers in Oryza sativa by two-dimensional polyacrylamide gel electrophoresis

X.Z. BI, Y.H. XIAO and W.F. LIU

College of Life Sciences, Wuhan University, Wuhan, 430072 P. R. China

Cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. There are several differences between indica and japonica rices (Glaszmann 1987). But there is no single criterion that distinguishes clearly without exceptions between these subspecies (Oka 1991: Morishima and Oka 1981). Zhou et al. (1988) proposed that several criteria combined together could be used to distinguish between the subspecies.

Zhang et al. (1992) detected the differentiation of indica and japonica rice by RFLP analysis. Qian et al. ( 1994) further found that 24 among 68 DNA clones could be used as indica-japonica differentiating probes. Through RFLP analysis, they showed similar hybridization patterns within subspecies, but different patterns between the subspecies. They showed that RG358, G318 are indica specific clones. Proteins are steady products of gene expression, show a high resolution, and two-dimensional gel electrophoresis can directly identify the presence of genes and the differences between indica and japonica rice. So, protein markers for subspecies differentiation can represent the changes of cultivated rice proteins, and the differences of genetic background can be detected from protein pattern of 2-D PAGE.

Materials in this study were ten male-sterile rice lines including 3 indica cytoplasm male sterile (CMS) rices, 1 temperature-sensitive genie male-sterile rices (TGMR), 6 photoperiod-sensitive genic male-sterile rice (PGMR) and two normal fertile rice (Table 1). Proteins from seed embryos, endosperm, etiolated buds of these materials were analyzed by two dimensional electrophoresis according to the method of Yang et al. (1993), and the gels were scanned by LKB laser gel scan 2D system.

The results revealed that: about 500 components could be identified from all the embryos analyzed, the molecular weight (MW) and isoelectric point (pI) ranged from MW 12-95kD. pI 3.5-8.0. Water soluble protein components from 12 rice strains were 200, and etiolated buds could show 550 components, their MW and pI were 10-95kD and 3.5-8.8.

No special proteins which correlated significantly with male sterility were found in the tissues of different rice strains, and no large difference in the protein compositions existed between 12 rice strains. Protein [MW27.8kD, pI5.6] and [MW27.8kD, pI7.6] from seed embryos and endosperm showed differences between indica and japonica rice except 8912s which was developed from the cross of Shuang 8-14s (japonica) x IR8 (indica) (Table 1. Fig. 1 A-B); protein [MW35.5kD, pI7.5] and [MW40.7kD, pI7.4] from etiolated buds also showed differences between indica and japonica photoperiod-sensitive genic male-sterile rice (Table 1, Fig. 2 A-B). Therefore these four proteins might be regarded as biochemical markers for indica and japonica rice differentiation or indica and japonica male-sterile rice. These would be valuable in early identification of indica and japonica characteristics of a new PGMR in rice genetics and breeding studies.


Fig. 1. Parts of 2D pattens of proteins from seed embryo, showing two different protein spots between indica and japonica rice.

A. 3037 (indica); B. Nongken 58 (japonica)


Rice (Oryza sativa) seed storage proteins have been characterized and intensively studied (Yamagata et al. 1982: Yamagata and Tanaka 1986: Chen and Cheng 1986; Takaiwa et al. 1987: Komatsu et al. 1993). Chen and Chen (1989) indicated that most indica varieties developed in Taiwan have Band 57 (faster-migrating band) and japonica varieties have Band 56 (slower-migrating hand), and their molecular weigh) were about 27-28kD in most cultivars. Protein MW27.8kD in this report appears similar to their results. Variations in band intensity were frequently observed when the maternal genotype was different (Chen and Chen 1989). We also found that protein spot [MW35.5, pI7.5] in W6154 etiolated buds was weaker in staining intensity than that of others.

Saruyama and Shinbashi (1992, 1993) showed that 10 kinds of proteins (A-J) in rice embryos were different between different varieties by two-dimensional electrophoresis with silver staining. They found that protein E only existed in typical indica rice, and protein A and F were present only in typical japonica rice. They indicated that indica and japonica specific proteins of seed embryos could be identified, and the results could be used to classify the rice of unknown origin. Our results support this conclusion.



Fig. 2. Laser scaning of part of 2D gel proteins from Maxie A and Nongken 58s etiolated buds, showing protein 1 and 2.

A. Maxie A (indica); B. Nongken 58s (japonica)



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