Hajduch Martin, Casteel Jill E, Tang Shinxue, Hearne Leonard B, Knapp Steve, Thelen Jay J
Department of Biochemistry, 109 Christopher S. Bond Life Sciences Center, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
J Proteome Res. 2007 Aug;6(8):3232-41. doi: 10.1021/pr070149a. Epub 2007 Jun 20.
Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.
对种子干重含油量分别为25%（自交系RHA280）和48%（RHA801）的近等基因向日葵品系，在开花后18天进行了三次生物学重复实验，采用二维（pH 3 - 10和4 - 7）差异凝胶电泳进行比较分析。此外，还以同样方式分析了油酸含量不同的两个自交系，HA89（油酸含量18%）和HA341（油酸含量89%）。对这些向日葵品系进行统计分析时，首先对每个点的对数转换后的光密度值拟合混合效应线性模型，以考虑凝胶差异，然后检验品种间平均转换后的光点光密度值的显著性。然后，利用10%的错误发现率（FDR），通过斑点分析程序得到的p值来确定差异表达的临界值。油含量和油酸组成品系的比较显示，分别有77个和42个蛋白质点低于10% FDR临界值，因此被判定为差异表达。对这些蛋白质点分别进行液相色谱 - 串联质谱分析，结果分别鉴定出44个和17个点。已确定果糖激酶、质体磷酸甘油酸激酶和烯醇酶蛋白在高油品系中上调，而磷酸果糖激酶、胞质磷酸葡萄糖变位酶和胞质磷酸甘油酸激酶在低油品种中上调。此外，除了12S贮藏蛋白和一种类似于豆球蛋白贮藏蛋白的蛋白质外，低油品种中参与氨基酸合成的四种活性也上调。有趣的是，在高油酸品种中发现两个被鉴定为14 - 三体 - 3蛋白的二维斑点上调。糖酵解和氨基酸生物合成酶以及贮藏蛋白水平的改变表明，种子油含量与碳水化合物代谢和蛋白质合成以复杂的方式紧密相关。
