Relationship Between Heat-stable Proteins And Indica-japonica Differentiation In Rice

Rice is one of major food crops of mankind, there are more than 70,000 accessions of rice germplasm resources preserved in the National Crop Genebank of China. The cultivated rice mainly differentiated into indica and japonica after the long domestication, and utilization of heterosis in rice is the main approach to raise rice yield; in addition, there is significant difference in storability of indica and japonica. So investigation of indica-japonica differentiation is of great importance to make use of heterosis in rice and preservation of rice germplasm resources. At present, the research of indica-japonica differentiation mostly focus on the level of morphology, biochemistry and DNA molecular, however, the study on the feature of protein is very little. In this study, 127 accessions of cultivated rice varieties and 16 accessions of common wild rice were used as materials, SDS-PAGE and western bloting were used to analyze the relationship between the expression differentiation of heat-stable proteins and indica-japonica differentiation, thereby offer theoretical and scientific basis for investigation of rice evolution model and efficient utilization and preservation of rice germplasm resources. Main results are as follows.1.Heat-stable proteins of 40 cultivated rice varieties were separated by SDS-PAGE. The result showed that heat-stable proteins were different between varieties: ten typical japonica all contained special proteins Bandâ… (45.2KDa) or Bandâ…¡(46.5KDa); but ten typical indica contained neither of the two special proteins bands at the same molecular weight; seven in ten Japonicaclinous contained Bandâ… or Bandâ…¡,three in ten Japonicaclinous contained neither of these two bands; six in ten Indicaclinous contained Bandâ… o r Bandâ…¡,four in ten Indicaclinous contained neither of these two bands. This indicated that there is indica-japonica differentiation in cultivated rice at protein level, and these two proteins could be used to study indica-japonica differentiation of cultivated rice. We concluded initially that Bandâ… and Bandâ…¡could be used as marker bands to study indica-japonica differentiation and distinguish cultivated rice, varieties containing Bandâ… or Bandâ…¡were Japonica, varieties containing neither of these two bands were Indica.2,Differential expression of marker bands in indica and japonica was tested by western blotting. Firstly, Bandâ… and Bandâ…¡are purified and identified through two-dimensional electrophoresis (2-DE) and Q-TOF. After blasting at Japonica protein data bank of NCBI, we found that Bandâ… and Bandâ…¡match to a protein Os03g0168100 whose gene number is GI: 115450973, consisting of 400 aa, and having three LEA₄ interpros (IPR004238), its function is unknown. One polypeptide (EKANEGYEKVKEKAKEK) of protein Os03g0168100 was selected to prepare antibody, and 127 cultivated rices were verified by using western blotting to confirm feasibility of rice classification by using these marker bands. The results showed that the antibody had specific immune responses with Bandâ… and Bandâ…¡, but the antibody had specific immune responses with another protein of japonica, named Bandâ…¢, which molecular weight is about 42.0KDa, blasting antigen peptide at indica protein data bank of NCBI, we found that the peptide match to a protein which name is OsI₁0172, GI:125542557, consisting of 380 aa, and having two LEA₄ interpros (IPR004238), its function is also unknown. Although the antibody was not specific to japonica protein, but the molecular weight of Bandâ… ,â…¡,â…¢were different, so varieties containing Bandâ… or Bandâ…¡could be classified to Japonica, varieties containing Bandâ…¢could be classified to indica, and on this base we could study indica-japonica differentiation of cultivated rice and classify them. 127 cultivated rice were classified by methods of marker bands and Cheng's index, the results indicated that the correlativity of the two methods in identifying typical indica and japonica was 81.1% and 90.9%, which showed that Bandâ… ,â…¡,â…¢could be used as classification markers of typical indica and japonica, and this method is visualized and convenient.3,After analysis of heat-stable proteins of common wild rice, we found that heat-stable proteins Bandâ… ,â…¡,â…¢were expressed in common wild rice materials, the band expression was more abundant than which in cultivated rice, including three forms: only containing one of Bandâ… ,â…¡,â…¢, containing both Bandâ… a nd Bandâ…¡, and containing both Bandâ…¡and Bandâ…¢. This indicated that there are differentiation at protein level in common wild rice and high diversity of protein was detected. Combining of heat-stable protein differentiation of cultivated rice, author is inclined to support the independent origin hypothesis of cultivated rice, which is that indica evolved from indica common wild rice, and japonica evolved from japonica common wild rice.