Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada.
J Agric Food Chem. 2013 Jan 30;61(4):901-12. doi: 10.1021/jf304328n. Epub 2013 Jan 18.
Arabidopsis thaliana lines expressing only one cruciferin subunit type (double-knockout; CRUAbc, CRUaBc, or CRUabC) or devoid of cruciferin (triple-knockout; CRU-) or napin (napin-RNAi) were generated using combined T-DNA insertions or RNA interference approaches. Seeds of double-knockout lines accumulated homohexameric cruciferin and contained similar protein levels as the wild type (WT). Chemical imaging of WT and double-knockout seeds using synchrotron FT-IR spectromicroscopy (amide I band, 1650 cm(-1), νC═O) showed that proteins were concentrated in the cell center and protein storage vacuoles. Protein secondary structure features of the homohexameric cruciferin lines showed predominant β-sheet content. The napin-RNAi line had lower α-helix content than the WT. Lines entirely devoid of cruciferin had high α-helix and low β-sheet levels, indicating that structurally different proteins compensate for the loss of cruciferin. Lines producing homohexameric CRUC showed minimal changes in protein secondary structure after pepsin treatment, indicating low enzyme accessibility. The Synchrotron FT-IR technique provides information on protein secondary structure and changes to the structure within the cell.
利用 T-DNA 插入或 RNA 干扰方法，生成了仅表达一种芸薹素亚基类型（双敲除；CRUAbc、CRUaBc 或 CRUabC）或缺乏芸薹素（三敲除；CRU-）或 napin（napin-RNAi）的拟南芥品系。双敲除系的种子积累了同六聚体芸薹素，且蛋白水平与野生型（WT）相似。利用同步辐射 FT-IR 光谱显微镜（酰胺 I 带，1650 cm(-1)，νC═O）对 WT 和双敲除系种子进行化学成像显示，蛋白集中在细胞中心和蛋白储存液泡中。同六聚体芸薹素系的蛋白二级结构特征显示出主要的β-折叠含量。napin-RNAi 系的α-螺旋含量低于 WT。完全缺乏芸薹素的系具有较高的α-螺旋和较低的β-折叠水平，表明结构不同的蛋白可以补偿芸薹素的缺失。产生同六聚体 CRUC 的系在胃蛋白酶处理后，蛋白二级结构变化最小，表明酶的可及性较低。同步辐射 FT-IR 技术提供了关于蛋白二级结构及其在细胞内结构变化的信息。
