| Mercury is regarded as nonessential element in plant, and also it is one of the most hazardous pollutants of the environment throughout the world which seriously intimidates the ecosystem and human health. Rice (Oryza sativa L.) is not only the most important crop worldwide, but also the model plant for the plant research. When feeling the stress signals, rice regulates the related proteins expression to adjust their own forms and physiological process which helped them to adapt to adverse environment. With proteomics study playing an increasingly important role in the functional genomics era, rice proteomics is setting off an upsurge gradually and continuously which has been made great progress over the years.A Hg+-tolerant rice mutant and a Japonica rice(Oryza sativa cv. "Zhonghua 11") were used in the studies, and comparative proteomics study was done between the mutant and wild type rice roots after treated with 50μM HgCl2 for 3 hours. Through the heteroexpression of the functional genes of rice roots in Saccharomyces cerevisiae (BY4741), a preliminary study of the relationship between the aim genes and Hg2+-tolerant indicated that the aim genes were associated with Hg2+-tolerant in rice roots during Hg2+stress. The major findings were as follows:1. Through optimizing the extraction of rice roots protein, IEF and SDS-PAGE electrophoresis conditions, we obtained the high-resolution and reproducible gel maps based on the technical platform of a large-scale 2-DE system.2. A total of 29 different protein spots were changed obviously in the two-dimensional electrophoresis between the Hg2+-tolerant rice mutant and wild type after treated with 50μM HgCl2 for 3 hours. Among the 16 most interesting protein spots of the total 29 protein spots. Mass spectrometry analysis helped to identify as 27 different proteins which were involved in redox homeostasis, signal transduction, stress response, metabolism, cellular structure proteins, regulatory proteins and unknown proteins. To investigate the changes of gene expression at the mRNA level, we performed qRT-PCR analysis and found that mRNA level of the differently expressed proteins was not absolutely consistent with the protein level.3. Obtained the full-length sequences of the three rice genes (OsTCTP, Translationally-controlled tumor protein; OsMAPK, Mitogen-activated protein kinase 2; OsKNOLLE, Syntaxin-related protein KNOLLE) by RT-PCR technique. During the heterologous expression analysis in yeast, we found that the S.cerevisiae cells transformed with OsTCTP/OsMAPK/OsKNOLLE showed better survival ability than the empty vector transformed S.cerevisiae cells under Hg2+stress, so we suggest that OsTCTP/OsMAPK/OsKNOLLE may play crucial roles in the Hg2+tolerance of rice. Meanwhile, we also studied the relationship of the three proteins and other stress, there was a close relationship between OsTCTP protein and Cu2+; OsKNOLLE protein was related with salt, Cu2+, H2O2 and Cd2+ stress, in which the role made in Cd2+stress was more obvious; OsMAPK protein, there was no certain relevance with other stress.Comparative proteomic analysis was first applied to explore the protein expression in rice roots by a Hg2+-tolerant rice mutant. And also for the first time we identified OsTCTP and OsKNOLLE proteins associated with Hg2+-tolerant. To carry out this experiment, it will help to find out the Hg2+-tolerant-related genes (or proteins), clarify tolerance mechanism of the plants during Hg2+stress, cultivate the Hg2+-tolerant rice breeds, or improve the Hg2+tolerant in other crops through transgenic technology. In conclusion, this study has a very important significance on food security, ecological environment protection and sustainable development of agriculture in our country. |
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