| Widely distributed in soil, arbuscular mycorrhizal fungi (AM) fungi who are kinds of beneficial plant endophytic fungi can establish endosymbiotic mutualism with the vast majority of plants in nature. They promote plant growth and enhance plant tolerance to all kinds of biotic (pathogens and herbivores) and abiotic (e.g., drought) stresses. It is commonly observed that AM fungi can colonize the roots of woody leguminosae plant, Amorpha fruticosa, and form symbiotic tissues. The occurrence of AM fungi colonizing on A. fruticosa roots necessarily induces a series of physiological and biochemical as well as observable morphological changes in roots, in consistent with which their protein expression patterns alter correspondingly. The application of differential proteomics method to explore the variation of distinctive induced proteins during the formation process of symbiosis between AM fungi and A. fruticosa will contribute a lot theoretically to reveal partly the molecular mechanisms of mycorrhiza formation.The changes of colonization percentage over plant growing time and the growth promoting response of AM fungi on A. fruticosa were measured in this paper. Two-dimensional electrophoresis (2-DE) coupling mass spectrometry (MS), as a frequently used method in differential proteomics, was employed to establish a set of suitable system for separating and identifying A. fruticosa root proteins. The extraction method, loading amounts and staining method were optimized in this study in order to obtain a good result. Subsequently, the optimized 2-DE system was applied to separate A. fruticosa mycorrhizal proteins derived from signal recognition and mature symbiotic period, and then the separated differential proteins were sequenced by nano ESI MS/MS and bioinformatics analysis was carried out to analyze the determined sequences. Through comprehensive analysis on the correlation between colonization percentage and plant growing indexes, significantly growth promoting of AM fungi onA. fruticosa could be concluded and with infection intensity increase, this promoting effect showed more evident, which could be easily observed from signal recognition stage and mature symbiotic stage. TCA/acetone method and improved phenol extraction method were used to extract A. fruticosa root proteins and compared, and comparison of different loading amounts of protein sample and different staining methods were conducted. Using improved phenol extraction method, with sample of 1000 ug as loading amounts and coomassie brilliant blue R-350 as staining agent, good reproducible 2-DE images with high resolution could be obtained, which paved the subsequent research on AM fungi-A. fruticosa symbiosis.The optimized 2-DE system was used to analyze the change of protein level in signal recognition stage and mature symbiotic stage. The results showed at signal recognition stage the expression of twelve proteins changed, among which nine up-regulated, two down-regulated, one novel protein, and at mature symbiotic stage, total 32 proteins were induced to change their expression, of which fourteen up-regulated, three down-regulated, and fifteen novel proteins.Based on the detected results that changed in abundance, the total 44 symbiosis-related protein spots from signal recognition and mature symbiotic stage were sequenced by MS, and then the sequences were queried on green plant database and fungal protein database via MASCOT search engine in Swiss-Prot. Total 37 spots were successful identified, ten from signal transduction stage,27 sequences from mature stage. Function classification was carried out on the identified proteins and they were attributed to nine classes including metabolism related, energy related, DNA regulation related, DNA synthesis related, signal transduction proteins, protein synthesis related, cellular structure proteins, defence proteins and unknown proteins. |
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