Abstract
A differential display of proteins with a two-dimensional polyacrylamide gel electrophoresis approach was used to analyze protein expression changes during development of the basal region in rice seedlings (Oryza sativa L. cv. Nipponbare). The proteins were detected as 700 Coomassie Brilliant Blue-stained spots with pI values from around 3.5 to 9.0. A proteome reference map was established for the basal region of two-week-old seedlings. The basal region proteome map was used to analyze quantitative variations in the tissue during development from 2-, 4-, 6-, 8-, and 10-week-old seedlings. During development, 31 proteins were up-regulated, and 30 proteins were down-regulated compared with the 2-week-old basal region proteome map. The main functions of these proteins were primary metabolism and protein synthesis or maintenance. Calreticulin precursor, enolase, and voltage-dependent anion channel were identified among the up- and down-regulated proteins. The twin spots of calreticulin precursor and enolase with different pI values are possibly due to post-translational modifications such as phosphorylation. In addition, seven proteins showed developmental stage-specific expression. All of the developmentally regulated proteins of the basal region were clustered by the S-system, a differential equation that fit to time course of cluster and analyzed for cluster relationships. Proteins with unknown functions were tentatively assigned to functional groups based on cluster relationships. Basal region development proteome data will be valuable for resolving questions in functional genomics. In addition, cluster analysis of the basal region proteome during development will be useful for the assessment of functional proteins.
Highlights
A differential display of proteins with a two-dimensional polyacrylamide gel electrophoresis approach was used to analyze protein expression changes during development of the basal region in rice seedlings
The 2D map of proteins was reproducibly observed in several independent experiments with the high and low pI ranges overlapping at around pI 5.6 (Fig. 2A)
Using only the IEF gel for first dimension separation, 352 protein spots were detected by 2D-PAGE and Coomassie Brilliant Blue (CBB) staining [16], whereas using the IEF and IPG gels, 431 proteins were detected [19, 25]
Summary
Protein Extraction—Two grams of basal regions of fresh seedlings of each developmental stages were homogenized with 4 ml of a lysis buffer [33] containing 8 M urea, 2% Nonidet P-40, 0.8% Ampholine (pH 3.5–10 and pH 5– 8, Amersham Biosciences), 5% 2-mercapthoethanol, and 5% polyvinyl pyrrolidone using a glass mortar and pestle on ice. Homogenates were centrifuged twice at 15,000 rpm in a RA-50 JS rotor (Kubota, Tokyo, Japan) for 5 min each. Two-dimensional Polyacrylamide Gel Electrophoresis—Samples were separated by 2D-PAGE in the first dimension by IEF for low pI range (around pI 3.5– 8.0) or IPG tube gels (Daiichi Kagaku, Tokyo, Japan) for high pI range (pI 6.0 –10.0) and in the second dimension by SDS-PAGE.
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