| Proteomics is playing an increasingly important role in the functional genomics era. Two-dimensional electrophoresis (2-DE) is one of the key techniques in proteomics studies, which have many characteristics such as better repetition, high sensitivity and resolution. Potato (Solanum tuberosum L.) is the fourth most important food crop consumed worldwide. It can provide the theoretical basis for the cultivation, breeding and production of potato through study on the development mechanism of potato tubers. The development of potato tubers is a complex but regular process. There exsist a certain difference in the number, types and abundance of tuber protein during the different development stage. These proteins are related with the expression of some gene and the regulation of some important traits in potato tubers. Therefore, it has an important significance for enhancing potato tuber yield and qulity to study the growth and development mechanism of potato tubers using the method of proteomics.In order to study the discrepancy expression of potato tuber protein during the different development stage, 2-DE technique system of potato tuber protein needs to be established firstly. Sample preparation is a critical step in a 2-DE proteome approach and is absolutely essential for good results. Therefore, this study focused on the specific problems of protein extraction from potato tuber and evaluated these methods. In our study, potato tuber protein was extracted with four methods: trichloroacetic acid (TCA)/acetone precipitation, the improved TCA/acetone precipitation, phenol extraction methanol/ammonium acetate precipitation and two-step precipitation. The proteins concentration of each sample protein was determined by Bradford method, and the proteins were analyzed by 2-DE. Meanwhile, the yield and purity of protein and the resolution of 2-DE profile were compared. The results indicated that the higher yield and purity of protein and the better 2-DE profile of each sample were obtained by the two-step precipitation method than other methods. Therefore, the two-step precipitation method of potato tuber protein and the establishment of 2-DE technique provided a basis of work for study on the proteomics of potato tuber in our reserch.Potato tuber proteins of the four developmental stages (stolon initiation, stolon elongation growth, stolon diageotropic growth and tuber initiation) were extracted with two-step precipitation method, and the proteins were analysised by 2-DE. The results indicated that the molecular mass of potato tuber proteins ranged from 15.5 kD to 87.2 kD and the pI ranged from 3.8 to 7.2. Eight protein spots (107.8 kD, pI 4.9), (37.7 kD, pI 7.3), (42.8 kD, pI 6.2), (21.3 kD, pI 8.9), (37.7 kD, pI 6.0), (33.8 kD, pI 7.2), (23.7 kD, pI 5.1) and (77.3 kD, pI 6.4) expressed only in the first developmental stage and disappeared in the following developmental stages. At the stage of stolon elongation growth, there appeared thirteen new proteins. In these proteins, spots (25.2 kD, pI 8.6), (26.3 kD, pI 7.7) and (30.4 kD, pI 6.1) were disappeared in the following developmental stages; The abundance of three protein spots (51.3 kD, pI 5.8), (51.6 kD, pI 6.7) and (50.9 kD, pI 4.2) was obviously decreased. At the stage of stolon diageotropic growth, there appeared ten new proteins. In these proteins, spots (45.1 kD, pI 6.8), (45.3 kD, pI 4.8), (44.3 kD, pI 6.1), (44.7 kD, pI 5.2) and (42.9 kD, pI 7.3) were only specific expression in this stage. The abundance of fourteen proteins was increased. Spot (22.7 kD, pI 8.8) showed the highest abundance, but its abundance was decreased at the stage of tuber initiation. There also exsisted seventeen proteins whose abundance was decreased. In these proteins, the change of spot (61.3 kD, pI 6.8) and spot (58.5 kD, pI 6.4) were the most significant. At the last developmental stage (tuber initiation), there appeared twenty-two new proteins, which are distributed in the low molecular weight area (53 kD-23 kD). The abundance of nineteen proteins was obviously increased and the abundance of nine proteins was significantly decreased. The abundance of Patatin protein was gradually enhanced in all developmental stages. Therefore, those proteins were some differential expressed proteins at the specifically developmental stage during the development of potato tuber, and their appearance and disappearance may be correlated with accordingly developmental stage. |
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