Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

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.
Keywords: oilseeds • seed oil content • DIGE • quantitative proteomics • statistical proteomics

本研究以种子干重油脂含量分别为25%（自交系RHA280）与48%（RHA801）的近等基因系向日葵为材料，于开花后18天采用二维（涵盖pI 3−10与pI 4−7范围）差异凝胶电泳（Difference Gel Electrophoresis, DIGE）技术，以3次生物学重复开展对比分析。此外，油酸含量存在差异的两个自交系HA89（油酸含量18%）与HA341（油酸含量89%）也采用相同方式进行了分析。
针对上述向日葵品系的统计分析流程如下：首先对每个蛋白斑点的光密度体积进行对数转换，并拟合混合效应线性模型以校正凝胶电泳间的系统误差；随后对不同品种的标准化光密度斑点体积均值进行显著性检验。研究利用斑点分析得到的P值，以10%的错误发现率（false-discovery rate, FDR）作为阈值筛选差异表达蛋白斑点。
对油脂含量组与油酸组成组的比较分析分别筛选得到77个和42个符合10%FDR阈值的蛋白斑点，随后将其判定为差异表达斑点。对上述差异斑点进行液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）分析后，分别成功为44个和17个斑点提供了蛋白鉴定结果。
功能注释结果显示：果糖激酶、质体磷酸甘油酸激酶与烯醇化酶在高油品系中呈上调表达；而磷酸果糖激酶、胞质磷酸葡萄糖变位酶以及胞质磷酸甘油酸激酶在低油品种中上调。此外，除12S贮藏蛋白与一类类豆球蛋白贮藏蛋白外，低油品种中4种参与氨基酸合成的酶活性也呈上调趋势。值得注意的是，两个被鉴定为14-3-3蛋白的二维电泳斑点在高油酸品种中呈现上调表达。
糖酵解与氨基酸生物合成相关酶类以及贮藏蛋白水平的改变表明，种子油脂含量与碳水化合物代谢及蛋白质合成之间存在紧密且复杂的调控关联。
关键词：油料种子 • 种子油脂含量 • 二维差异凝胶电泳（DIGE） • 定量蛋白质组学 • 统计蛋白质组学