프로테옴 분석법에 의한 식물의 저온 스트레스 반응 단백질의 동정 : 李東起 [저]
프로테옴 분석법 식물 저온스트레스반응 단백질동정;
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Proteomics is the large-scale study of protein, usually by biochemical methods. The word proteomics has been associated traditionally with displaying a large number of proteins from a given cell line or organism on two-dimensional polyacrylamide gels. In this sense proteomics already dates back to the late 1970s when researchers started to build databases of proteins using the then newly developed technique of two-dimensional gel electrophoresis (2-DE). This resulted in extensive catalog using of spots from two-dimensional gels to create databases of all expressed proteins. Two-dimensional electrophoresis is known as the most effective as well as one of the simplest methods for separating proteins. The high resolution of 2-DE is useful for separating complex protein mixtures. Due to its high-resolving power, the technique has been employed in studying alterations in proteins expression during development and differentiation or in response to environmental changes. 2-DE analysis has been used to establish master maps for entire organism, tissue types, and developmental model organism or to examine altered gene expression patterns under diverse biological processes. However, a few hundred plant leaf proteins out of thousands visualized on a 2-DE gel can be identified by chemical analysis due to the presence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) that limits protein loading. Plants respond to conditions of severe environmental changes or stresses, such as drought, low temperature, or high salt, with a number of physiological and development changes. Plants have a remarkable ability to cope with highly variable environmental stresses. Nevertheless, these stresses together represent the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50%. Low temperature is an important environmental factor that greatly influences the growth, development, survival and distribution of plants. To identify cold-regulated protein in rice, seedling was subjected to cold treatment. Total soluble proteins were extracted from rice leaf and stem tissue that had been treated at 10℃ or 15℃ for 0,1, 3, and 5 days, respectively. The soluble proteins were fractionated with 15% PEG. Protein profiles from rice after cold treatment were analyzed by two-dimensional gel electrophoresis. In the first dimension, isoelectric focusing was performed in the pH range 4.5∼7.5, and 10.5 or 11% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in the second dimension. Protein spots were visualized by staining with silver nitrate. Differential expressed spots were identified by peptide mass fingerprinting using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After cold treatment, out of 96 spots changes investigated, 51 spots were identified. This enabled identification of 51 protein spots representing 31 different proteins. The identified proteins were classified into stress-responsive, signal transduction, carbohydrate and amino acid metabolism-related proteins. The prominent proteins up-regulated by cold treatment were Thioredoxin H-type, Cu-Zn superoxide dismutase (Cu-Zn SOD), MADS-box protein, chloroplast translational elongation factor Tu, Ran and enolase. These results suggest a possibility that the above proteins have protective function from cold stress or regulatory function in controlling gene expression under cold stress.