| The biotic and abiotic stresses of rice during its survival can cause the accumulation of reactive oxygen species and adversely affect its growth and yield.The Super Oxide Dismutase(SOD)as a repressor in response to reactive oxygen species plays an important role in rice growth and development,but its regulatory studies focus on the detection of transcription level and enzyme function level.For the preparation of antibodies that recognize the Cu/Zn SODs in rice,a series of related studies have been carried out around bioinformatics analysis,antibody preparation and functional identification.The results were as follows:Firstly,the related SOD genes of rice sub-species Japonica Rice and Indica Rice were analyzed by bioinformatics to obtain the characteristic sequence of Cu/Zn SOD in rice chloroplast,which can be used as antigen immunized to animal in order to obtain the polyclonal antibody with the Cu/Zn SOD recognisation ability;Secondly,based on the results of analysis,the prokaryotic expression plasmid chloroplast Cu/Zn SOD in rice was constructed,and the recombinant protein with enzyme activity was purified.After immunizing New Zealand White Rabbit,affinity chromatography was used to obtain the rabbit polyclonal antibody of the Os Cu/Zn SODs;Nextly,the application of this antibody were detected by Immunoblotting,Immunoprec-ipitation,ELISA,and Immunofluorescence.The results showed the rabbit polyclonal antibady of Os Cu/Zn SOD can effectively identify plant Cu/Zn SOD both in the denatural condition and natural condition.The Cu/Zn SOD in human Hela cell lines was identified on Immunofluorescence method using thise antibody.The content of chloroplast Cu/Zn SOD in rice were quantified by indirectly competing ELISA method based on this antibody;Finally,the analysis of Cu/Zn SOD protein expression in four rice seedlings under high temperature stress using this antibody showed that this Cu/Zn SOD antibody could be a good tool used to analyze the changes of Cu/Zn SOD translation level in rice.A rabbit polyclonal antibody of Os Cu/Zn SODs was successfully prepared in this paper,and several immune detection systems based on this antibody were established.The specific bands were obtained in both Japonica Rice and Indica Rice by immunoblotting with this antibody,in order to realise the multi-level analysis of the gene regulation network response in transcription level,translation level and enzyme function level of Cu/Zn SODs,which provides a good research tool for further revealing the molecular mechanism of Cu/Zn SOD genes expression regulation. |
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