Proteome Approach to the Characterisation of Protein Composition in the Developing and Mature Wheat-grain Endosperm
Proteome Approach to the Characterisation of Protein Composition in the Developing and Mature Wheat-grain Endosperm
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DOI：
10.1006/jcrs.2000.0321
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发表时间：
2000-09
影响因子：
3.8
通讯作者：
D. Skylas;J. Mackintosh;S. Cordwell;D. Basseal;B. Walsh;J. Harry;C. Blumenthal;L. Copeland;C. Wrigley;W. Rathmell
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文献类型：
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作者：
D. Skylas;J. Mackintosh;S. Cordwell;D. Basseal;B. Walsh;J. Harry;C. Blumenthal;L. Copeland;C. Wrigley;W. Rathmell
摘要利用蛋白质组学方法对小麦品种Wyuna在开花后17天的胚乳蛋白进行了分析和表征。这涉及提取，溶解和随后的二维分离的总小麦籽粒胚乳蛋白。约1300个多肽被解析。在两个pH范围内使用等电聚焦进行第一维分离：pH 4·0-7·0和pH 6·0-11·0。将蛋白质印迹到PVDF上，切除并使用常规N -末端Edman降解微测序进行表征。通过FASTA算法将序列提交到SWISS-PROT和TrEMBL数据库。总共提交了321种蛋白质进行分离后表征;从数据库匹配中鉴定了177种（55%）蛋白质，55种（17%）蛋白质不匹配，89种（28%）蛋白质未产生任何N -末端序列数据。然后使用Melanie II 2-D PAGE图像分析软件比较发育（开花后17天）和成熟（开花后45天）生长阶段胚乳内的蛋白质表达。这种方法提供了一个深入了解胚乳蛋白质异质性的复杂性质，并为今后的研究环境变化对蛋白质组成的影响提供了基础。
Abstract A proteomic approach was used for the analysis and characterisation of wheat-grain endosperm proteins at a developmental stage (17 days post-anthesis) of the wheat cultivar Wyuna. This involved the extraction, solubilisation and subsequent two-dimensional separation of total wheat-grain endosperm proteins. About 1300 polypeptides were resolved. Separation in the first dimension was performed using isoelectric focusing across two pH ranges: pH 4·0–7·0 and pH 6·0–11·0. Proteins were blotted to PVDF, excised and characterised using conventional N -terminal Edman degradation microsequencing. Sequences were submitted to SWISS-PROT and TrEMBL databases via FASTA algorithm. In total, 321 proteins were submitted for post-separation characterisation; 177 (55%) proteins were identified from database matches, 55 (17%) proteins were not matched and 89 (28%) proteins did not yield any N -terminal sequence data. Protein expression within the endosperm at both the developmental (17 days post-anthesis) and mature (45 days post-anthesis) stages of growth were then compared using Melanie II 2-D PAGE image analysis software. This approach has provided an insight into the complex nature of endosperm protein heterogeneity and provides a basis for the future examination of the effects of environmental variation on protein composition.